Effect of Ultrasonication on Self-Assembled Nanostructures Formed by

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Article Cite This: ACS Omega 2019, 4, 4540−4552

http://pubs.acs.org/journal/acsodf

Effect of Ultrasonication on Self-Assembled Nanostructures Formed by Amphiphilic Positive-Charged Copolymers and NegativeCharged Drug Le Hang Dang,†,⊥ Minh Thanh Vu,‡ Jun Chen,§ Cuu Khoa Nguyen,∥,⊥ Long Giang Bach,*,# Ngoc Quyen Tran,*,∥,⊥ and Van Thu Le*,∥,⊥ †

School of Biotechnology, International University, National Universities in Hochiminh, Ho Chi Minh City 70000, Vietnam Institute of Chemistry and Materials, 17 Hoang Sam, CauGiay, Hanoi 400000, Vietnam § Department of Orthopedic Sports Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China ∥ Graduate University of Science and Technology and ⊥Institute of Applied Materials Science, Vietnam Academy of Science and Technology, Ho Chi Minh City 700000, Vietnam # NTT Hi-Tech Institute, Nguyen Tat Thanh University, 300A Nguyen Tat Thanh, Ward 13, district 4, Ho Chi Minh City 700000, Vietnam

Downloaded via 95.85.70.220 on March 4, 2019 at 19:16:01 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Self-assembly of the amphiphilic copolymer into core−shell-like nanoparticles is the new tactic to tailor carriers toward rationalization in the field of drug-delivery systems. Herein, a facile route for examining how the entrapment of a hydrophobic and negative-charge drug affects the micellar structure of a positive-charged copolymer and its biological behavior was developed. In this study, Pluronic F127-grafted chitosan (CF127) was utilized as a positivecharged copolymer for in situ loading of nanocurcumin in a cosolvent condition. Ultrasonication was found to be an effective method to control the self-assembly of phosphocasein and its interaction with curcumin. The superstructure of the incorporated nanoparticles was fabricated in the medium under unimolecular micelles as vesicular structure (SV) at lower ultrasonic condition while large complex micelles (multimicelle aggregates, LCMs) at higher ultrasonic power density. According to transmission electron microscopy, variable UV−visible spectrophotometry, as well as fluorescence spectroscopy, nanocurcumin was not only incorporated into the hydrophobic micelle cores via hydrophobic interaction but also underwent electrostatic interaction with amine groups on chitosan backbone, resulting in micellar aggregation and finally turning in LCMs. Furthermore, regarding dynamic light scattering measurements, correlation coefficients of SV, as well as LCMs, were higher than 0.9, which means that all nanostructures were homogeneous in size under precise control by ultrasonication. Cell-culture studies showed that both unique morphologies endowed fibroblast cell development. Interestingly, the more complex structure as LCM exhibited as a potential candidate in cancer therapy. These corollaries suggest that the morphology of micelles based on cationic amphiphilic block copolymer can be modulated by adding negative-charged/hydrophobic molecules under varying condition of ultrasonication that reinforces their prospective applications as nanocarriers for drug-delivery systems.

1. INTRODUCTION

application of self-assembly. Moreover, many novel nanoscale materials with highly ordered ensembles of molecules have been employed in diverse applications, especially in drugdelivery systems.10−12 In this case, core−shell structures are erected amphiphilic block copolymers, which is well-known via their excellent and efficient way to construct all kinds of delicate complex self-assemblies.10,12−14 In other words, selfassemblies are the cornerstone for fabricating well-defined

Self-assembly is a useful and general strategy for designing various delicate supramolecular structures that attracted a great deal of attention of biologists, chemists, and physicists.1−5 Based on the explanation of the formation of protein in living organisms, the self-assembly process guides progressively manifold hierarchical structures for each kind of protein to precise their functions, which can adapt to response the stimuli from environments resulting in the correctly functioning proteins or enzyme complexes.6 In addition, various regular material structures such as molecular crystals,7 semicrystalline structures,8 or liquid crystal9 are the best examples of the © 2019 American Chemical Society

Received: November 29, 2018 Accepted: February 18, 2019 Published: March 1, 2019 4540

DOI: 10.1021/acsomega.8b03346 ACS Omega 2019, 4, 4540−4552

ACS Omega

Article

The pluronic, water-soluble form of A−B−A is known as a triblock copolymer of poly(ethylene oxide)−poly(propylene oxide)−poly(ethylene oxide) (PEO−PPO−PEO), which shows the best self-assembly behavior into a micellar structure expressed by a hydrophobic core of PPO and a shell of the solvated PEO.31 However, the micelle system formed by pluronic exhibits the inability to provide sustained drug delivery over more than just a few days.12,32,33 As a first step to overcome this problem, the modified pluronic was grafted to amine groups on chitosan backbone to obtain a cationic and thermoresponsive CS-g-F127 (CF127) copolymer.34 Herein, we tried to encapsulate nanocurcumin, which was aggregated in the cationic grafting copolymer-removed organic solvent, to generate well-defined core−shell structures such as unimolecular micelles (vesicular structure, SV) or multimicelle aggregate (large complex micelle, LCM) nanoparticles under support of hydrophobic and electrostatic interactions as well as the ultrasonic process. Ultrasonic power density was evaluated for its effect on these superstructural morphologies of the SV and LCM nanoparticles.

core−shell structures, which show many exploitable characteristics in drug-delivery systems. These are tractable configuration in both watery phase and organic phase to concomitant in the loading and release of drugs, excellent adaptation in various milieu, modified surface with multifunctional groups to interact favorably with cells, and specific tolerance to various stimuli.15 However, the standard preparation protocols are facing an intractable problem when synthesizing these kinds of nanostructures because of the nonequilibrium structures originated from the poorly water-soluble nature and the high glass-transition temperature of the hydrophobic segments of a polymer.16,17 Core−shell structures composed of a water-soluble shell and a discriminative hydrophobic core are novel, unusual morphologies for application in a variety of fields, including medicine, pharmacology, and biotechnology.14,15 So far, the preparation and control of the formation of core−shell structure systems have been shown to follow the coassembly strategies or environmental stimuli-responsive copolymers through an intermediary of subunits preassembling.12,18,19 Step-growth polymerization is shown to be the significant force to develop core−shell-like nanoparticles in almost all reports; however, it does not take payload into account. Moreover, even though core−shell structures are based on synthetic thermal block amphiphilic polymers, these micelles after micellar solubilization of drug are in random morphologies leading to uncontrolled size distribution, which are not precisely predictable.20,21 Recently, through the guidance of multiphasic liquid mixtures, different various assembling hierarchies of nanoscale particles to tailor polymer molecules have been exploited to refine their aggregation into the desired shape.21,22 Two distinct routes inspire a strategy as they guide the particle assembly by dint of structure-directing agents of both emulsions and bubbles.21−23 In the first route, the assembly is guided by expelling the dispersed phase toward the aggregation of the particles in this phase to sort out into multiple structures, while through another route, particles assemble because of the propensity of the particle to firmly adsorb at fluid−liquid interfaces. It is believed that the ultrasonication emulsification process is a superior approach of self-assembly, which can be well addressed via both required routes. Moreover, liquid-phase self-assembly is continuously combined with a steady unsettling of colloids. To control the assembly process for designing nanoparticle shapes, the critical requirement is kinetic control of the propensity of aggregation in the liquid by taking advantage of ultrasonication.24,25 Ultrasonication provides the acoustic wave that can quickly generate nucleation and lead to perfect control of the growth and collapse of bubbles resulting in the formation of single nanoparticles as well as the guidance of complex nanoparticles with narrow size range and excellent yield.26−28 Ultrasonication is also utilized to support the self-assembly process of polymeric nanoparticles with high homogeneity.29,30 Indeed, even the organization of aggregated micelles into complex structures can be bolstered by ultrasonication. Although selfassembly by ultrasonication has been emphatically developed in recent decades, this method is not the best strategy in the field of polymeric material. Ultrasonication seldom approves of an ordered assembly of polymeric nanoparticles because of their low crystallinity. Moreover, to date, there is a lack of studies investigating the effect of drug loading (DL) on the polymeric self-assembly.

2. MATERIALS AND METHODS 2.1. Materials. Curcumin was acquired from Merck, Singapore (CAS 458-37-7). CF127 was prepared following the previous report,33,34 presented in Section S1. All of the solutions and chemical agents used in cell culture were purchased from Grand Island Biological Company. Staining agents, including trypan blue, acridine orange (AO), sulforhodamine B (SRB), and ethidium bromide (EB), were obtained from Sigma-Aldrich (Singapore). Other reagents used in this study were supplied by Merck (Singapore), Promega, Scharlau (Spain), and Fisher. 2.2. Examination of Critical Micelle Concentration (CMC). Chemical characteristics of CF127 are presented in the Supporting Information (Section S1). CF127 was evaluated at a point of the critical micelle concentration (CMC) at fluorescence intensities of 334 nm (I1) and 337 nm (I3) in the pyrene emission spectra as a function of the concentration of CF127 in aqueous solutions. CMC values were identified as the crossing points of two linear lines of regression generated by a piecewise fitting function. 2.3. Preparation of Molecular Assemblies. Various concentrations of curcumin were prepared in cosolvent (dichloromethane (DCM) and ethanol). The effect of different ratios of DCM and ethanol on the dynamic size, ζ-potential value, and the physical performance of micelles was examined. Based on several screening experiments, the perfect ratio of DCM and ethanol was selected for further study. CF127 was dissolved in 50 mL of deionized (DI) water and ultrasonication was started with UIP1000, Hielscher instrument, at 50−200 W/mL of solution. To assist in the formation of thermodynamically stable self-assembled nanoparticles, curcumin solution was added dropwise during the sonication of the CP solution (the temperature was maintained below 45 °C). Then, vacuum evaporation was used to obtain the products following resuspension in DI water and freeze drying for further examination. 2.4. Characterization of Self-Assembly Nanoparticles. Morphology of assembly particles was investigated by transmission electron microscopy (TEM, JEOL-JEM-1400) at an accelerating voltage of 100 keV without staining. Dynamic light scattering (DLS) was conducted on SZ-100 Nanopartica Series Instruments (Horiba). DLS experiments 4541


ACS Omega

Article

CO2. After 3−4 days, the fibroblast outgrowth was checked by a light microscope. The fresh medium was replaced every 3−4 days, taking care not to agitate the coverslip. When the fibroblast cells begun to round up during examination by the optimal volume of trypsin/ethylenediaminetetraacetic acid, about 1 mL of complete growth medium (ice-cold) was provided to deactivated trypsin and then the fibroblast cells were harvested by gentle pipetting. The collected fibroblast suspensions undergoing centrifugation (1500 rpm for 10 min at 4 °C) were resuspended in 150 μL of fresh ice-cold complete growth medium. Trypan blue/PBS (10 μL, 0.4%) was then added into 10 μL of this suspension before counting by a cell counter machine (Bio-Rad TC10). 2.5.2. Preparation of Cancer Cells. Human breast cancer cell lines, including MCF7 cells (HTB-22) and Hela cells (CCL-2), were purchased from the American Type Culture Collection (Manassas, Rockville). The cell lines were cultured in accordance with the literature.37 2.6. In Vitro Cytotoxicity. 2.6.1. Sulforhodamine B (SRB) Assay. This assay was performed following the protocol of Nguyen et al.37 to investigate the toxicity of each nanoformulation based on the quantity of cellular protein. Briefly, various cells (fibroblast cells, MCF7 cells, and Hela cells) were seeded in 96-well plates at a density of 10 000 cells/well in 24 h. Then, the medium containing each variation (water as control, CP copolymer, SV, and LCMs) at different concentrations was substituted for the older one. After 48 h culture, 10% (w/v) trichloroacetic acid (Merck) was gently added into each well and incubated for 1−3 h under cold condition to fix cells before staining with SRB (0.2% w/v, Sigma-Aldrich) for 60 min. Then, 1% acetic acid (Merck) was used to remove the unbound dye, and 150 μL (10 Mm) of Tris base solution (Promega) was then put on these wells and incubated for 30 min. Optical density (OD) was read at 492 and 620 nm (for background) using a 96-well microtiter plate reader (Synergy HT, BioTek Instruments). The percentage of growth inhibition (% I) was calculated using the following formula

were carried out at a scattering angle of 90°, where the influence of dust was minimized, to obtain the average hydrodynamic radius and polydispersity as well as the correlation function of the scattered electric field. The temperature of the sample cell was controlled at 25 °C using a temperature controller. All dry samples were dispersed in DI water and kept at room temperature for 24 h before measuring. The optimal concentration (5 ppm) was selected from the size measurement of a series of suspensions. ζ-Potential measurement was conducted on SZ-100 Nanopartica Series Instruments (Horiba). All samples were dispersed in DI water, and ζvalues were measured at 25 °C. To investigate the self-assembly formation, UV−vis and fluorescence intensity was proposed with the comparison of behaviors of curcumin in raw form, SV form, and LCM form. Ethanol was used to dissolve raw curcumin before diluting with phosphate-buffered saline (PBS) 7.4 to reach 5 ppm concentration. SV and LCM samples were dissolved in PBS 7.4 to get the same concentration (5 ppm). The UV absorbance was recorded from 250 to 700 nm using a UV spectrophotometer (Shimadzu UV-1800, Japan). The fluorescence behaviors of curcumin in various tests were obtained using a fluorescence spectrophotometer (Hitachi F7000, Japan). Emission spectra were recorded in the wavelength range of 400−700 nm at two excitation wavelengths (425 and 355 nm). To measure the drug loading (DL) and entrapment efficiency (EE) of nanoformulation, high-performance liquid chromatography (HPLC) measurements were performed in C18 column (Agilent) eluting with 45:55 acetonitrile/acid water (pH = 5.0) in volume after dissolving the desired amount of dried nanoformulation in the same volume of solvent. The measurements were made in triplicate, and the DL (%) and EE (%) were calculated according to a standard curve. The standard curve was obtained through a concentration gradient of curcumin ethanol solutions. All of the preparations followed the protocol of Ankrum et al.35 Direct dispersion method as explained in our previous report34 was used to investigate the release behavior of curcumin from the nanoformulation. Briefly, samples were immersed in 10 mL of PBS (0.05 M) at two different pH values (7.4 and 5.5). The test tubes were continuously shaken at 150 rpm and maintained at 37 °C. At definite time intervals, 0.1 mL of solution was taken out and 100 μL of fresh PBS was added into each tube. The curcumin releasing in the media was calculated referring to the standard curve built by the same HPLC method as in the previous description. 2.5. Cellular Behaviors of Nanoparticles. 2.5.1. Preparation of Human Fibroblasts. Human fibroblasts were isolated from donor foreskins under ethical guidelines (No. 1387/QĐ-ĐHQG) of Vietnam National University, Ho Chi Minh City. After obtaining signed patient consent, the foreskins were cleaned by 70% ethanol and then cut into small rectangular specimens (2−3 mm × 5 mm) before stored in PBS 1× (P5493). After washing with PBS, the small specimens were set in the center of sterile 60 mm glass Petri dishes covered by a sterile glass coverslip, in which 0.1% gelatin was added at the bottom. All of the dishes were cultured with complete growth medium (Dulbecco’s modified Eagle’s medium, fetal bovine serum, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution 1 M, 100× nonessential amino acid mixture, 100× L-glutamine, 100× penicillin/ streptomycin, and 100× sodium pyruvate) at 37 °C and 5%

%I = 100% −

ODt × 100% ODc

where ODt and ODc are the optical density values of the test sample and control sample, respectively. Camptothecin (Calbiochem) was used as a positive control. All trials were repeated three times. 2.6.2. Acridine Orange/Ethidium Bromide (AO/EB) Dual Staining. Both cell lines with a density of 2 × 105 cells/well were incubated at 37 °C and 5% CO2 for 24 h. The medium was replaced by the fresh one supplementing with LCM (at the IC50 value that was calculated via SRB assay). At the desired time (24, 48, 72, and 80 h), after washing with PBS 1×, 1 μL of dual AO/EB was added into each well. Cell morphology was observed by fluorescence microscopy. 2.7. Statistical Analysis. Data performances were analyzed using ORIGIN 8.5.1 (Northampton, MA). The data are presented as average ± standard error (SE) of the mean of at least three independent determinations on one sample for each measurement. Moreover, one-way analysis of variance was used to determine the relationship between the ratio of mixture solvents (DCM/Eth) and the hydrodynamic size of the selected samples, ζ-potential, as well as 4542


ACS Omega

Article

Figure 1. Physicochemical characteristics of CF127 micelles: CMC value of the micelles (a), TEM morphology image, (b) size distribution (c), and its autocorrelation function (d) measured at the scattering angle of 90° and room temperature.

and single population of particles with a Z-average size of 20.9 nm and a polydispersity index (PDI) of 0.21. No sedimentation was detected in solution, which is similar to its normalized intensity autocorrelation function. The fitting curve (solid line) and scattering data (circle point) show the perfect association along with the decay time distribution and the agreement with the zero baselines at high relaxation time (Figure 1d). Altogether with the TEM image (Figure 1b), it can be concluded that CF127 undeformed nanomicelles were well dispersed as individual micelles at room temperature. 3.2. Behavior of Nanocurcumin Loading CF127 Micelles in Mixtures of Solvent. Despite the potential protocol of utilizing micelles for encapsulation of lipophilic bioactive compounds, the influence of the dispersion methods of loading agents on the properties of micelles is not well understood. For encapsulating nanocurcumin, it is critical to select a mixture of the organic solvent for dispersion of curcumin and partial compatibility with CF127 in further studies. Ethanol is one of the popular solvents using to dissolve curcumin, in which two forms of curcumin (keto−enol) are in fair proportions.41 Dichloromethane (DCM), however, is one of the best solvents used in extraction as well as nanocurcumin processing.42,43 Thus, in this study, the cosolvents (ethanol and DCM) were used to produce nanocurcumin CF127 solution under ultrasonic-assisted condition. In spectrophotometry, the results are similar to other reports related to conformational equilibria of enol−keto.42−44 Following the increase of ethanol from 0 to 30% (vol/vol), the weak shoulder at 368.5 nm is presented together with a major peak at 429 nm, owing to the presentation of enol and mono-anion forms (data not shown).

polydispersity index (PDI) values. Statistical significance was set at p < 0.05.

3. RESULTS AND DISCUSSION 3.1. Characterization of CF127 Micelles. The amphiphilic CF127 copolymers consisting of the hydrophilic (PEO and CS) segments and hydrophobic (PPO) segments can show their self-assembling phenomena in aqueous solutions. To examine this behavior, pyrene was used as fluorescence probe, which is capable of providing microscopic information relating to the micellization of amphiphilic CF127 in aqueous solution. Regarding the nature of pyren,38−40 pyren molecules tend to encapsulate into the hydrophobic segments of CF127; consequently, the proportion of the intensities of the first to the third (I1/I3) vibronic components in the pyrene fluorescence emission spectrum decreases (Figure 1a). The threshold aggregate concentration or the critical micelle concentration (CMC) is determined by the intersection points of two linear regression functions. It is found that the CMC value of CF127 (0.35 mg/mL) is higher than that of F127 alone (0.035 mg/mL38,39). In addition, in comparison with F127 grafting on a negative-charged polymer such as heparin, the CMC value of CF127 is also an order of magnitude larger.40 The reason may be the miscibility between positivecharge CS backbones, and as the result, the electrostatic repulsion causes the lower aggregation, leading to the micellization of CF127 at higher concentration. Moreover, CF27 results in the formation of the monomodal distribution of aggregation in both TEM and DLS data. As shown in Figure 1c, the size distribution of CF127 is in narrow 4543


ACS Omega

Article

Figure 2. Effect of proportional cosolvent (DCM/Eth) on the CF127 loading amount of curcumin (50 mg) with respect to Z-average size distribution, PDI values (a) and ζ-potential (b). The error bars are standard error of the mean, SE (n = 3).

Figure 3. Typical TEM images of CF127 encapsulating curcumin at 1 W/mL (a) and 3 W/mL (b). Andor Dragonfly confocal microscope was used to identify the morphology of the self-assembly of cationic amphiphilic grafted copolymer CF127/curcumin in the form of (c) SV and (d) LCMs under 100× objective.

However, when the percentage of ethanol is up to 100%, the weak shoulder at 368.5 nm completely disappeared, whereas the prominent peak at 429 is well defined with the highest intensity. These observations reveal that curcumin in enol form is predominant with the increase in ethanol concentration in the cosolvents. The variation of F127 particles loading curcumin in size distribution, PDI values, as well as ζ-potential with respect to different ratios of cosolvents was determined,

and the results are presented in Figure 2. There was a significant difference (p < 0.05) between the hydrodynamic size of selected samples, ζ-potential, and PDI values. The use of a single solvent has been reported to result in a higher Zaverage size with higher PDI value as well as lower ζ-potential. Interestingly, the nanoparticles prepared by cosolvent methods (DCM/Eth) have had crucially smaller particle size range than those prepared using single solvents like ethanol or DCM. 4544


ACS Omega

Article

Figure 4. (a) Photographic images of curcumin in water, curcumin in cosolvent DCM/ethanol, curcumin in CF127 without ultrasonication, curcumin/CF127 in SV form, and curcumin in LCM form (from left to right, label as 1 → 5). UV−visible absorption spectra of curcumin and (b) fluorescence spectra of curcumin in SV form and LCM form at two different excited wavelengths: (c) 425 nm and (d) 355 nm.

Meanwhile, the ratios 30:70 and 70:30 exhibit significantly higher ζ-potential values, indicating the excellent stability of these nanoparticles. Furthermore, the lowest PDI values are obtained in the samples prepared with cosolvents. This propensity also occurred for all three curcumin-loading percentages (50, 75, and 100 mg). These remarkable points altogether support that the combination of DCM and ethanol leads to the formation of optimal curcumin-loaded micelles with smaller size and narrower size distribution. 3.3. Effect of Nanocurcumin on the Suprastructure of CF127 Micelles. In particular, the self-assembly into micelles of the thermal responsive copolymer in an aqueous solution is naturally based on the critical micelle concentration as well as solvent.36 However, the conventional self-assembly concerns some disadvantages related to the purification process and risk for the residual organic solvent in the assembly. In this study, we want to investigate the morphology of nanocurcumin selfassembly (hydrophobic and negative-charged form) into micelles based on the cationic and amphiphilic CF127 copolymer under additional ultrasonication. Overall, the results of this study show that applying ultrasonic waves to the process of curcumin-loading CF127 could tend to self-assemble into spherical micelles with such a core packing motif or multimicelle aggregates that further fuse together to form larger complex micelles (LCMs). For DLS as well as TEM protocol, 1 ppm of each sample in DI water was prepared. Figure 2a shows that the core region of the assemblies was loaded by curcumin, presenting core−shell structure in the size range of 5−10 nm by TEM. More accurate

evidence for the location of curcumin was obtained from fluorescence techniques. All samples were prepared in microscope glass slides without any label agents, capped with coverslips. Figure 3c,d shows fluorescent images of the samples at room temperature with and without filter. The particles demonstrate a core−shell structure in which curcumin seems to concentrate near the shell (Figure 3c) in the case of SV and contribute on the multicore packing in LCMs (Figure 3d). Specifically, TEM images expose the evolution of curcuminloading CF127 micelles to vesicular morphology (SV) and further the self-assembly into multicore packing motif with the unique nanostructure, which is known as LCM (Figure 3b), with the increase of ultrasonic power density via the comparison of particles size. The size of SV (Figure 3a) is about 15.2 ± 0.12 nm, quite similar to the size of single-core motif in LCMs (average size ∼ 14.5 ± 0.56 nm). In other words, SV particles are aggregated and become the building unit if higher ultrasonic power density is applied; consequently, the close-packed spherical cluster of SV is constructed. In particular, the micellar morphology of LCMs appeared to be more interesting. The formation of SV or LCM structure can be explained through facile self-assembly of the cationic amphiphilic grafted copolymer (CF127) inclusive of interactions with payloads (hydrophobic and negative-charged curcumin compound) under ultrasonic condition in aqueous solution. The ζ-potential measurement of colloidal curcumin solution showed a value close to −25.9 mV, whereas CF127 was found to be nearly +36.6 mV in water (Supporting Information, Section S2). This suggests that CF127 is in the 4545


ACS Omega

Article

Figure 5. TEM image combined with DLS results (a, c, e, and g) as well as autocorrelation function (b, d, f, and h) of curcumin/CF127 nanoplex at different power densities of 1−4 W/mL. Other conditions such as time, final volume of reaction, concentration of CF127, concentration of curcumin, and the Eth/DCM ratio were kept constant.

organic-soluble, organic nanocurcumin occurs in hydrophobic segments on CF127 backbone. When the solvent polarity increases, the hydrophobic interaction between curcumin and among PPO units becomes dominant. Some aggregation can be observed when lower ultrasonic power density is applied; however, the lack of energy causes the reduction of randomly aggregated curcumin/CF127 guiding the emergence of core packing motif. In the case of higher ultrasonic power density, because curcumin is more solvated by the organic solvent

cationic form in solution while the negative form of curcumin is dominated in a mixture of the solution. As gradually removed organic solvents, the mixture of CF127 copolymer and the nanocurcumin form self-assembled complexes through hydrophobic interaction and electrostatic, which leads to the variation of morphology from spherical micelles to core−shell structure, and further orientate into multimicelle aggregates along with the increase of ultrasonic power intensity. Under this condition, because curcumin is not water-soluble but 4546


ACS Omega

Article

Figure 6. TEM image and size distribution as well as its autocorrelation function of various morphologies of curcumin/CF127 nanocomplex at different sonication times and a power intensity of 3 W/mL: 5 min (a, b), 10 min (c, d), and 15 min (e, f).

π−π* transitions in the enolic form of curcumin, at both different concentrations (1 and 5 ppm) in PBS medium (pH 7.4), which is in good agreement with other reports.47−49 In the case of LCMs, a dominant band at 420 nm is observed along with the band at 368 nm, that is, the presentation of diketo form of curcumin (a higher hydrophobic form).50,51 These results are in agreement with our hypothesis. At a lower ultrasonic power density, the hydrophobic interaction between the CF127 and nanocurcumin (in enolic form) was predominant, leading to the orientation of curcumin in the polar environment or the core of the micellar structure. In other words, when the CF127 micelles form in polar solution, the standard interface via hydrophobic−hydrophobic interaction between curcumin and PPO segments is proposed to inhibit the contact between curcumin and water, resulting in the formation of SV forms. At higher power density, the more the contact with amide group on chitosan backbone, the more curcumin tends to contact with water; thus, the equilibrium shifts toward ketone form.

(DCM/ethanol), curcumin molecules tend to interact with the copolymer because of their opposite electric charges. As soon as these interactions are introduced, the reduction in the interaction with the polar solvent system of CS fragments on CF127 increased when the charge on CS becomes neutral. Thus, they tend to aggregate again.30 Through ultrasonic waves at higher power density, aggregation and deaggregation occurred with higher velocity, inducing the order of aggregation into LCM structure. To confirm the aggregation of the SV to become the LCM structure, behaviors of curcumin in SV, as well as in LCMs, were compared to those of raw curcumin in ethanol using UV−vis and fluorescence intensities. Figure 4b testifies shift in the absorption of curcumin in various forms (raw, SV, and LCM) at 25 °C.45 The absorbance of curcumin in ethanol shows a clear maximum at 420 nm, in the same manner as reported by Gupta.46 There is a significant difference in curcumin’s absorbance between vesicles and LCM-core micelles in the recording of the absorbance of curcumin. SVs exhibit an absorbance peak at ∼420 nm conforming to the 4547


ACS Omega

Article

Figure 7. DLS data (a, b) versus curcumin content for curcumin dissolved directly in Eth/DCM (70:30), which was then dropwise added into CF127 aqueous solution under ultrasonication (3 W/mL and 5 min). The nanoparticulation efficiency of different amounts of curcumin loading (c) was evaluated by HPLC. The error bars are standard error of the mean, SE (n = 3).

3.4. Effect of Ultrasonic Conditions on the Morphology of Molecular Assemblies. The change in morphology of vesicle nanoparticles to LCMs is observed following the increase of ultrasonic power intensity (Figure 5), while other factors show the effect the particle size only. Powerdependence TEM observation was performed to investigate the evolution of morphological self-assembly in constant applied time and reagents (curcumin and CF127). The TEM images of the sample synthesized by applying 3 W/mL (Figure 5c,d) show the generation of LCM morphology. The outermost shell of the particle becomes the loose structure in the wall of the nanocomplex, indicating the interaction of curcumin and chitosan causing lack of contact in CF127 chain compare to self-assembly of this copolymer with 1 W/mL (Figure 5a) and 2 W/mL (Figure 5c). The best-fit curve with the autocorrelation data as well as the baseline signal reveal that the high size homogeneity with further increase of power intensity to 3 W/mL (Figure 5e,f). With 4 W/mL, the clusters are observed. However, the core packing inside nanocomplex is hardly identified except for the edge region because of the overlapping of the clusters, although the DLS results (Figure 5g) indicate the high homogeneity of these self-assembled nanoparticles with the relaxation model (Figure 5h). Sonication time is also played a critical role in the growth of cluster into LCMs. At 5 min, the resulting products are LCM spheres with diameter 40−50 nm (Figure 6a) in TEM while 69.3 nm in the measurement of DLS. By increasing time to 10 min, LCMs with more fused micelles are obtained (Figure 6c). The diameter of these spheres is increased to 80 nm. While the synthesis time was increased to 15 min, more aggregation

Also, the emission behavior of curcumin in different morphologies (SV, LCMs, and raw form) was also observed by exciting curcumin molecules at 425 nm (for enol form) and 355 nm (for β-diketone form) (Figure 4c,d). The emission peak of raw curcumin (in 3% ethanol) is at 570 nm with very weak fluorescence intensity at both excitation wavelengths, which is analogous to the fluorescence aspects of curcumin in aqueous buffers.51−53 For SV and LCMs, however, the hypsochromic shift of the emission peak of curcumin is detected at both excitations. Excited at 425 nm, the emission peak is at 560 nm (∼10 nm) and 530 nm (∼40 nm) in SV and LCMs, respectively, compared to raw curcumin. Upon curcumin excitation at 355 nm, a well-defined peak in SV is observed at 560 nm (∼10 nm), whereas the peak is at 524 nm (∼46 nm) in the case of LCMs.54 These results suggest that the translocation of curcumin from the polar environment to a nonpolar environment is the major reason for the significant blue-shifting of the fluorescence peak of curcumin from raw form to SV and LCMs. This is consistent with that obtained by other studies. For example, the hypsochromic shift in curcumin fluorescence spectra from 540 to 500 nm51 was observed when curcumin was encapsulated in bovine casein micelles, confirming the interaction between the hydrophobic domains of casein protein and curcumin. Moreover, shifting to the blue spectrum was also reported in the case of curcumin with other surfaces such as bovine serum albumin,55 Tween 80,56 or cetyltrimethylammonium bromide.52 Therefore, the massive blue shift in the emission spectra of curcumin in the LCM sample exposes that this self-assembly morphology can provide a stronger binding between curcumin and CF127. 4548


ACS Omega

Article

It shows that burst release is detected in the acidic medium, approximately 65% which is about 3.4 times higher in comparison to the physiological medium in the first 24 h. After that, the release rate at both conditions increased gradually. Up to 72 h, 78% curcumin is released under acidic medium, whereas the cumulative curcumin release is just 20%. Based on the release profile of LCMs in two different pH conditions, it is believed that LCMs show pH-responsive behavior. In acidic medium, the CS domain on the surface of LCMs first absorbs H+, leading to the disruption of LCM structure that further causes the burst release of curcumin from this system. However, protonation of the amine group on CS, which led to the stronger cationic property of CF127, provides more electrostatic interactions with curcumin molecules; hence, the sustained release is expected in the second time interval. Under physiological environment, CS is deprotonated so that the electrostatic interactions between CS and curcumin are static. At this time, the hydrophobic interaction and the hydrogen bonding between the curcumin and another part of CF127 are the dominant forces that help to maintain the stability of LCMs. Thus, the main curcumin release mechanism is due to the larger swollen LCMs following the disruption of their infrastructure, which can be used to explain the lower release rate of LCMs in physiological than acidic medium. 3.6. In Vitro Anticancer Activity. Normal somatic cells (fibroblast cells isolated from foreskins) and cancer cell lines (MCF7 and Hela cells) were selected to assess the anticancer activity as well as the in vitro cytotoxicity of CF127/curcumin in the form of SV and LCMs compared to the control groups (blank CF127, raw curcumin, and camptothecin). The sample SV was prepared with 50 W/mL for 10 min, whereas the sample LCMs was treated with 150 W/mL for 10 min. In the concentration range of 2 ppm to above 20 ppm, SRB assay result of all cancer cell lines displays the typical dose−response model in the treatment of LCMs as well as camptothecin, except with blank CF127, SV, and raw curcumin. The value of IC50, which is the markable point for cell toxicity induced by treatment, is undetermined in blank CF127 when its concentration is up to 200 ppm, which is used to confirm the excellent biocompatibility of CF127. Free curcumin (1% dimethyl sulfoxide (DMSO)) is nontoxic to both cancer cell lines in the testing concentration range. This was proven in various studies.38,44,46,48,50 The IC50 value of curcumin is very high (greater than 25 ppm) due to its poor solubility, quick hydrolysis in physiological medium, and low cellular uptake. No significant difference is detected in Hela cells and MCF7 cells. The IC50, which is the markable point for cell toxicity induced by treatment, fall in the range of 2−4 ppm (Figure 9a,b). Surprisingly, SV groups show a deficient percentage of inhibition. Based on this data, it can be concluded that SV has no impact on anticancer activity. At the same concentration (14 ppm), the anticancer activity of LCMs is higher than that of SV, about 2.5 times (Figure 9c). This result tends to provide stronger evidence of the acid-responsive behavior of LCMs compared to SV forms. The critical aim of the drug-delivery system is to reduce the effect on the nontarget cells.10 As shown in Figure 9c, nontoxicity to normal somatic cells is observed in all groups (SV, LCMs, raw curcumin, and CF27 blank). The significantly higher toxicity to cancer cells than somatic cells reveals that LCMs have a significant influence on cellular internalization of cancer cells. This finding makes LCMs a promising candidate for cancer theranostics.

propensities with 146.4 nm (Figure 6e) in dimension are observed. The micelle clusters are constructed by several small unilamellar micelles as the building unit. Moreover, g2(τ) is presented as a single sharp peak in all samples (Figure 6b,d,f), indicating the homogeneous dispersion of these nanoformulations in aqueous solutions without any aggregation. The growth of LCMs followed by further enhancing of sonication time could be elucidated regarding the hydrophobicity of the templates following the increase of temperature. With increase of sonication time, the temperature of the solution also increased. The thermal property of CF127 becomes prominent, leading to the stronger hydrophobicity of this system; hence, aggregation and fusion of micelles increased along with condensation, leading to the incensement of size. The effect of increasing initial curcumin concentration was also examined with the constant power density and treatment time. As shown in Figure 7a, the initial concentration of curcumin affects the particles size as well as the stability. The Z-average size of the particles shows gradual increase, whereas the PDI values fluctuate when the initial loading of curcumin increases from 50 to 300 mg. Also, the reduction of ζ-potential from 20.3 ± 1.7 to 9.23 ± 1.24 mV (Figure 7b) explores the lower orientation of the aggregation in CF127 micelles upon the addition of curcumin. In fact, because of the incorporation of curcumin in the hydrophobic domains of CF127, larger particles are expected. However, in this case, the increase of hydrodynamic size is followed by the increase in the number of core packing motif rather than the expansion of the hydrophobic region, which can be proved by the tendency of drug-loading efficacy (Figure 7c). This would designate a higher aggregation of CF127 loading curcumin due to enhancement of interaction between curcumin and CS domain. 3.5. Release Behavior of Curcumin Nanoparticles. One of the most critical features of the drug-delivery system is the sustainable release. According to previous studies, the intracellular environment of cancer is acidic with a pH of 5.5, while the pH value in the normal cell is about 7.4, except some cells at the stomach or bladder.10,12 In this study, two different in vitro conditions were performed by using PBS as the medium. There is a significantly different phenomenon of LCMs in two distinct pH media over a 72 h study (Figure 8).

Figure 8. Release profile of curcumin encapsulated in LCM nanoparticles under two physiological conditions: PBS at pH 7.4 and PBS at 5.5, at 37 °C. The data points are exhibited as mean ± SE (n = 3). 4549


ACS Omega

Article

Figure 10. Visualization of apoptosis in MCF7 and Hela cells by AO/ EB dual staining over time under treatment with LCMs and nontreatment (control). Magnification = 200×.

stage of apoptosis due to the added diffusion of EB. The color changed to red-orange when more EB is concentrated in the nucleus, which is the signal of late apoptotic cells. For necrotic cells, the cell volume increased and the nucleus exhibits uneven orange-red fluorescence and an unapparent outline, indicating that it is disintegrating. As shown in Figure 10, both Hela cells and MCF7 cells treated with LCMs shows yellow-orange granules in the cytoplasm after 48 h. Moreover, in both cell lines treated with LCMs, at all culture times, abundant cells showed bright green patches or fragments and perinuclear chromatin condensation, but neither red-orange nor yelloworange fluorescence was observed. These cells were in an early apoptotic stage, which have intact membrane, but DNA fragmentation was initiated; thus, EB cannot enter these cells. At 72 h culture, the yellow-orange granules gradually reduced over time following the increase of red-orange fluorescence, indicating the expansion of cell in late apoptotic cells in the presence of LCM nanoparticles. Over 80 h culture, most of the Hela cells and MCF7 cells have condensed nucleus and a large number of cells with red-orange color in LCM groups, while nothing in the control sample. This suggests that after 80 h culture, LCMs induce Hela cells and MCF7 cells into the late apoptotic stage. It is evident that LCM nanoparticles promote cell apoptosis or programmed cell death. Therefore, LCMs are successful in interference with the proliferation of Hela cell lines as well as MCF7 cell lines, and the effect is comparable.

Figure 9. Cytotoxicity of LCMs on cancer cells MCF7 (a) and Hela cells (b) as a function of concentration (2−14 ppm). (c) Comparison of the percentage of dead cells when normal cells (fibroblast) and cancer cells, MCF7 and Hela, were treated with free curcumin (in DMSO), CF127 (in water), SV, and LCMs at the same concentration (14 ppm).

4. CONCLUSIONS The use of ultrasonication together with the properties of the loading agent and copolymer can exemplify a quick route to control nanoscale micellar morphologies, which contributes to better drug-delivery system. The morphology of nanocurcumin-encapsulated CF127 relies on the regulation of ultrasonic power density. The ultrasonic-assisted self-assembly of curcumin-loaded CF127 provided uniform core−shell-type polymeric micelles as vesicular structure (SV) having a diameter of 32.6 nm. As the ultrasonic power density increased, more effective aggregations to form the LCMs with the finely regulated morphology were observed ranging from 80 to 146 nm in diameter. The LCMs are also generated by using another cationic gelatin-g-Pluronic F127 with the same method as curcumin (Section S3). Furthermore, the

In this study, we aimed to advance the in vitro release profile of curcumin from LCM sample through a cellular uptake study by human cancer cells (Hela and MCF7 cells) after varying time interval (Figure 10). This involved treating Hela (cervical cancer) and MCF7 (breast adenocarcinoma) cells with both water as the control and the curcumin nanoparticles encapsulated with CF127 or LCMs sample. To assess the apoptosis of LCM assemblies, they were examined using dual AO/EB staining assay. The EB/AO method is based on the action of AO, which can diffuse into all cells leading to green color, while EB is only passed across a cell membrane if it is impermeable due to necrosis or apoptosis, resulting in a redorange color. For nonapoptotic cells or live cells, only AO staining produces green fluorescence. Yellow-green fluorescence is presented in the nucleus if the cells are in the early 4550


ACS Omega

Article

(6) Bai, Y.; Luo, Q.; Liu, J. Protein self-assembly via supramolecular strategies. Chem. Soc. Rev. 2016, 45, 2756−2767. (7) Steed, J. W.; Atwood, J. L.; Gale, P. A. Definition and emergence of supramolecular chemistry. Supramol. Chem. 2012, DOI: 10.1002/ 9780470661345.smc002. (8) Miao, J.; Cui, L.; Lau, H. P.; Mather, P. T.; Zhu, L. Self-assembly and chain-folding in hybrid coil− coil− cube triblock oligomers of polyethylene-b-poly (ethylene oxide)-b-polyhedral oligomeric silsesquioxane. Macromolecules 2007, 40, 5460−5470. (9) Choi, J. H.; Joung, Y. K.; Bae, J. W.; Choi, J. W.; Quyen, T. N.; Park, K. D. Self-assembled nanogel of pluronic-conjugated heparin as a versatile drug nanocarrier. Macromol. Res. 2011, 19, 180−188. (10) Le, P. N.; Nguyen, N. H.; Nguyen, C. K.; Tran, N. Q. Smart dendrimer-based nanogel for enhancing 5-fluorouracil loading efficiency against MCF7 cancer cell growth. Bull. Mater. Sci. 2016, 39, 1493−1500. (11) Li, X.; Chen, G. Glycopolymer-based nanoparticles: synthesis and application. Polym. Chem. 2015, 6, 1417−1430. (12) Tong, N.-A. N.; Nguyen, T. P.; Cuu Khoa, N.; Tran, N. Q. Aquated cisplatin and heparin-pluronic nanocomplexes exhibiting sustainable release of active platinum compound and NCI-h460 lung cancer cell anti-proliferation. J. Biomater. Sci., Polym. Ed. 2016, 27, 709−720. (13) Dehvari, K.; Lin, K. S.; Hammouda, B. Small-angle neutron scattering studies of microenvironmental and structural changes of Pluronic micelles upon encapsulation of paclitaxel. J. Taiwan Inst. Chem. Eng. 2017, 71, 405−413. (14) Talelli, M.; Barz, M.; Rijcken, C. J.; Kiessling, F.; Hennink, W. E.; Lammers, T. Core-Crosslinked Polymeric Micelles: Principles, Preparation, Biomedical Applications and Clinical Translation. Nano Today 2015, 10, 93−117. (15) Zhang, Y.; Zhou, J.; Yang, C.; Wang, W.; Chu, L.; Huang, F.; Liu, Q.; Deng, L.; Kong, D.; Liu, J.; Liu, J. Folic acid-targeted disulfide-based cross-linking micelle for enhanced drug encapsulation stability and site-specific drug delivery against tumors. Int. J. Nanomed. 2016, 11, 1119−1130. (16) Salim, N. V.; Hanley, T. L.; Waddington, L.; Hartley, P. G.; Guo, Q. A simple and effective approach to vesicles and large compound vesicles via complexation of amphiphilic block copolymer with polyelectrolyte in water. Macromol. Rapid Commun. 2012, 33, 401−406. (17) Attia, A. B. E.; Ong, Z. Y.; Hedrick, J. L.; Lee, P. P.; Ee, P. L. R.; Hammond, P. T.; Yang, Y. Y. Mixed micelles self-assembled from block copolymers for drug delivery. Curr. Opin. Curr. Opin. Colloid Interface Sci. 2011, 16, 182−194. (18) Wang, Z.; Sun, S.; Li, C.; Hu, S.; Faller, R. Controllable multicompartment morphologies from cooperative self-assembly of copolymer−copolymer blends. Soft Matter. 2017, 13, 5877−5887. (19) Milani, R.; Houbenov, N.; Fernandez-Palacio, F.; Cavallo, G.; Luzio, A.; Haataja, J.; Giancane, G.; Saccone, M.; Priimagi, A.; Metrangolo, P.; Ikkala, O. Hierarchical Self-Assembly of HalogenBonded Block Copolymer Complexes into Upright Cylindrical Domains. Chem 2017, 2, 417−426. (20) Lock, L. L.; LaComb, M.; Schwarz, K.; Cheetham, A. G.; Lin, Y. A.; Zhang, P.; Cui, H. Self-assembly of natural and synthetic drug amphiphiles into discrete supramolecular nanostructures. Faraday Discuss. 2013, 166, 285−301. (21) Hasobe, T.; Oki, H.; Sandanayaka, A. S. D.; Murata, H. Sonication-assisted supramolecular nanorods of meso-diaryl-substituted porphyrins. Chem. Commun. 2008, 6, 724−726. (22) Brugarolas, T.; Tu, F.; Lee, D. Directed assembly of particles using microfluidic droplets and bubbles. Soft Matter 2013, 9, 9046− 9058. (23) (a) Prestidge, C. A.; Simovic, S. Nanoparticle encapsulation of emulsion droplets. Int. J. Pharm. 2006, 324, 92−100. (b) Hohl, L.; Röhl, S.; Stehl, D.; von Klitzing, R.; Kraume, M. Influence of Nanoparticles and Drop Size Distributions on the Rheology of w/o Pickering Emulsions. Chem. Ing. Tech. 2016, 88, 1815−1826.

biological evaluation of various forms of CF127 with Hela cells and MCF7 cells depicts a considerable cellular internalization and cytotoxicity that promote the promising features of LCMs nanosystems. Interestingly, LCM nanoparticles are nontoxic to healthy cells. Despite the lack of validation from in vivo testing, we firmly believe that the method could be used to extensively study other negative-charged drugs or bioactive compounds for application in cancer chemotherapy.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03346. Preparation and characterization of CF127 copolymer, ζ-potential of CF127 as well as curcumin, and the formation of other cationic amphiphilic polymers following the same protocol investigated in the manuscript (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected], [email protected] (N.Q.T.). *E-mail: [email protected]. Phone: +84.856785648 (V.T.L.). *E-mail: [email protected]. Phone: +84.969294297 (B.L.G.). ORCID

Jun Chen: 0000-0003-4573-801X Ngoc Quyen Tran: 0000-0003-2899-5452 Funding

This work was funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.02-2017.60. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors acknowledge Department of Genetics, Faculty of Biology, University of Science, for providing cancer cell line (MCF7 cells and Hela cells). They thank some donor patients from Binh Dan Hospital in Vietnam. A written informed consent was obtained from the donors donating tissue. This study was approved by the Ethical Committee for Biomedical Research of the Vietnam National University, Ho Chi Minh City (ref 1387 QĐ-ĐHQG).

■

REFERENCES

(1) Ree, B. J.; Satoh, Y.; Jin, K. S.; Isono, T.; Kim, W. J.; Kakuchi, T.; Satoh, T.; Ree, M. Well-defined and stable nanomicelles selfassembled from brush cyclic and tadpole copolymer amphiphiles: a versatile smart carrier platform. NPG Asia Mater. 2017, 9, No. e453. (2) Wang, L.; Lin, J.; Zhang, X. Hierarchical microstructures selfassembled from polymer systems. Polymer 2013, 54, 3427−3442. (3) Cheng, C. C.; Lee, D. J.; Chen, J. K. Self-assembled supramolecular polymers with tailorable properties that enhance cell attachment and proliferation. Acta Biomater. 2017, 50, 476−483. (4) Bertula, K.; Myllymäki, T. T. T.; Yang, H.; Zhu, X. X.; et al. Hierarchical self-assembly from nanometric micelles to colloidal spherical supperstructures. Polymer 2017, 126, 177−187. (5) Dhotel, A.; Chen, Z.; Delbreilh, L.; Youssef, B.; Saiter, J. M.; Tan, L. Molecular Motions in Functional Self-Assembled Nanostructures. Int. J. Mol. Sci. 2013, 14, 2303−2333. 4551


ACS Omega

Article

(24) Bang, J. H.; Suslick, K. S. Applications of ultrasound to the synthesis of nanostructured materials. Adv. Mater. 2010, 22, 1039− 1059. (25) Gröschel, A. H.; Müller, A. H. Self-assembly concepts for multicompartment nanostructures. Nanoscale 2015, 7, 11841−11876. (26) Grzelczak, M.; Vermant, J.; Furst, E. M.; Liz-Marzán, L. M. Directed Self-Assembly of Nanoparticles. ACS Nano 2010, 4, 3591− 3605. (27) Santos, A. C.; Pattekari, P.; Jesus, S.; Veiga, F.; Lvov, Y.; Ribeiro, A. J. Sonication-Assisted Layer-by-Layer Assembly for Low Solubility Drug Nanoformulation. ACS Appl. Mater. Interfaces 2015, 7, 11972−11983. (28) Bondy, C.; Söllner, K. On the mechanism of emulsification by ultrasonic waves. Trans. Faraday Soc. 1935, 31, 835−843. (29) Guédra, M.; Valier-Brasier, T.; Conoir, J. M.; Coulouvrat, F.; Astafyeva, K.; Thomas, J. L. Influence of shell compressibility on the ultrasonic properties of polydispersed suspensions of nanometric encapsulated droplets. J. Acoust. Soc. Am. 2014, 135, 1044−1055. (30) Kim, B. G.; Kim, M. S.; Kim, J. Ultrasonic-assisted nanodimensional self-assembly of poly-3-hexylthiophene for organic photovoltaic cells. ACS Nano 2010, 4, 2160−2166. (31) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. Pluronic block copolymers for overcoming drug resistance in cancer. Adv. Drug Delivery Rev. 2002, 54, 759−779. (32) Choi, J. H.; Joung, Y. K.; Bae, J. W.; Choi, J. W.; Quyen, T. N.; Park, K. D. Self-assembled nanogel of pluronic-conjugated heparin as a versatile drug nanocarrier. Macromol. Res. 2011, 19, 180−188. (33) Dang, L. H.; Nguyen, T. H.; Tran, H. L. B.; Doan, V. N.; Tran, N. Q. Injectable Nanocurcumin−Formulated Chitosan-g-Pluronic Hydrogel Exhibiting a Great Potential for Burn Treatment. J. Healthcare Eng. 2018, 2018, No. 5754890. (34) Nguyen, T. B. T.; Dang, L. H.; Nguyen, T. T. T.; Tran, D. L.; Nguyen, D. H.; Nguyen, V. T.; Nguyen, C. K.; Nguyen, T. H.; Le, V. T.; Tran, N. Q. Green processing of thermosensitive nanocurcuminencapsulated chitosan hydrogel towards biomedical application. Green Process. Synth. 2016, 5, 511−520. (35) Ankrum, J. A.; Miranda, O. R.; Ng, K. S.; Sarkar, D.; Xu, C.; Karp, J. M. Engineering cells with intracellular agent−loaded microparticles to control cell phenotype. Nat. Protoc. 2014, 9, 233− 245. (36) Blanazs, A.; Armes, S. P.; Ryan, A. J. Self-assembled block copolymer aggregates: from micelles to vesicles and their biological applications. Macromol. Rapid Commun. 2009, 30, 267−277. (37) Nguyen, M. N. T.; Ho-Huynh, T. D. Selective cytotoxicity of a Vietnamese traditional formula, Nam Dia long, against MCF-7 cells by synergistic effects. BMC Complementary Altern. Med. 2016, 16, 220. (38) Priyadarsini, K. I. Photophysics, photochemistry and photobiology of curcumin: Studies from organic solutions, bio-mimetics and living cells. J. Photochem. Photobiol., C 2009, 10, 81−95. (39) Batrakova, E.; Lee, S.; Li, S.; Venne, A.; Alakhov, V.; Kabanov, A. Fundamental relationships between the composition of pluronic block copolymers and their hypersensitization effect in MDR cancer cells. Pharm. Res. 1999, 16, 1373−1379. (40) Kozlov, M. Y.; Melik-Nubarov, N. S.; Batrakova, E. V.; Kabanov, A. V. Relationship between pluronic block copolymer structure, critical micellization concentration and partitioning coefficients of low molecular mass solutes. Macromolecules 2000, 33, 3305−3313. (41) Ma, D.; Zhang, H. B.; Tu, K.; Zhang, L. M. Novel supramolecular hydrogel/micelle composite for co-delivery of anticancer drug and growth factor. Soft Matter 2012, 8, 3665−3672. (42) Mondal, S.; Ghosh, S.; Moulik, S. P. Stability of curcumin in different solvent and solution media: UV−visible and steady-state fluorescence spectral study. J. Photochem. Photobiol., B 2016, 158, 212−218. (43) Harigae, T.; Nakagawa, K.; Miyazawa, T.; Inoue, N.; Kimura, F.; Ikeda, I.; Miyazawa, T. Metabolic fate of poly-(lactic-co-glycolic acid)-based curcumin nanoparticles following oral administration. Int. J. Nanomed. 2016, 11, 3009.

(44) Dende, C.; Meena, J.; Nagarajan, P.; Nagaraj, V. A.; Panda, A. K.; Padmanaban, G. Nanocurcumin is superior to native curcumin in preventing degenerative changes in Experimental Cerebral Malaria. Sci. Rep. 2017, 7, No. 10062. (45) Bhatia, N. K.; Kishor, S.; Katyal, N.; Gogoi, P.; Narang, P.; Deep, S. Effect of pH and temperature on conformational equilibria and aggregation behaviour of curcumin in aqueous binary mixtures of ethanol. RSC Adv. 2016, 6, No. 103275. (46) Aggarwal, B. B.; Yuan, W.; Li, S.; Gupta, S. C. Curcumin-free turmeric exhibits anti-inflammatory and anticancer activities: Identification of novel components of turmeric. Mol. Nutr. Food Res. 2013, 57, 1529−1542. (47) Ghosh, M.; Singh, A. T.; Xu, W.; Sulchek, T.; Gordon, L. I.; Ryan, R. O. Curcumin nanodisks: formulation and characterization. Nanomedicine 2011, 7, 162−167. (48) Muthoosamy, K.; Abubakar, I. B.; Bai, R. G.; Loh, H. S.; Manickam, S. Exceedingly higher co-loading of curcumin and paclitaxel onto polymer-functionalized reduced graphene oxide for highly potent synergistic anticancer treatment. Sci. Rep. 2016, 6, No. 32808. (49) Erez, Y.; Simkovitch, R.; Shomer, S.; Gepshtein, R.; Huppert, D. Effect of acid on the ultraviolet−visible absorption and emission properties of curcumin. J. Phys. Chem. A 2014, 118, 872−884. (50) Bai, F.; Diao, J.; Wang, Y.; Sun, S.; Zhang, H.; Liu, Y.; Wang, Y.; Cao, J. A New Water-Soluble Nanomicelle Formed through SelfAssembly of Pectin−Curcumin Conjugates: Preparation, Characterization, and Anticancer Activity Evaluation. J. Agric. Food Chem. 2017, 65, 6840−6847. (51) Ke, D.; Wang, X.; Yang, Q.; Niu, Y.; Chai, S.; Chen, Z.; An, X.; Shen, W. Spectrometric study on the interaction of dodecyltrimethylammonium bromide with curcumin. Langmuir 2011, 27, 14112− 14117. (52) Dutta, A.; Boruah, B.; Saikia, P. M.; Dutta, R. K. Stabilization of diketo tautomer of curcumin by premicellar cationic surfactants: a spectroscopic, tensiometric and TD-DFT study. J. Mol. Liq. 2013, 187, 350−358. (53) Boruah, B.; Saikia, P. M.; Dutta, R. K. Binding and stabilization of curcumin by mixed chitosan−surfactant systems: A spectroscopic study. J. Photochem. Photobiol., A 2012, 245, 18−27. (54) Sahu, A.; Kasoju, N.; Bora, U. Fluorescence study of the curcumin-casein micelle complexation and its application as a drug nanocarrier to cancer cells. Biomacromolecules 2008, 9, 2905−2912. (55) Leung, M. H.; Kee, T. W. Effective stabilization of curcumin by association to plasma proteins: human serum albumin and fibrinogen. Langmuir 2009, 25, 5773−5777. (56) Ghosh, S.; Mondal, S. Spectroscopic study on the interaction of medicinal pigment, curcumin with various surfactants: An overview. J. Surf. Sci. Technol. 2012, 28, 179−195.

4552


Effect of Ultrasonication on Self-Assembled Nanostructures Formed by

Recommend Documents