Novel naproxen-peptide-conjugated amphiphilic dendrimer self-assembly micelles for targeting drug delivery to osteosarcoma cells

Abstract

The aim of the current study was to synthesize and prepare novel self-assembly micelles loaded with curcumin (Cur) based on naproxen (Nap)-conjugated amphiphilic dendrimers. The apoptosis-inducing capacity of Nap-conjugated dendrimers and curcumin, the efficiency of uptake and the potential molecular mechanism on human osteosarcoma cells were investigated. The Nap-conjugated amphiphilic dendrimers were successfully synthesized, and the corresponding Cur-loaded micelles were conventionally prepared via self-assembly. These micelles showed good drug-encapsulating capacity, physiochemical properties, and drug-release profiles. The cellular proliferation and uptake assay suggested that the Cur-M-Nap induced more apoptosis of MG-63 human osteosarcoma cells. The western blot results suggested that these Nap-modified micelles could enhance the Cur-inducing apoptosis, mainly via intrinsic pathways and inhibition of the inflammation pathway. In summary, the Cur-loaded amphiphilic dendrimer-based micelles were successfully developed and they enhanced drug delivery to MG-63 cells. Mechanistic experiments suggested that Cur loaded into amphiphilic dendrimer-based micelles had a higher proliferation-inhibiting ability than free Cur, and could induce more apoptosis.

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Introduction

As repetitively branched synthetic macromolecules, dendrimers possess homogeneous structures from their interior layers to their surface layers, especially in their branching units. It is well established that dendrimers bear a series of unique intrinsic properties, such as monodisperse molecular weight, good biocompatibility, and spherical three-dimensional structures with well-defined surface functionalities.^1–6 Dendrimers have exhibited great potential for application in many fields, including drug/gene delivery, as catalysts, and in bioimaging. Among these, applications to the co-delivery of drugs and/or genes using dendrimers stand out and have been a key focus of research in nanotechnology and nanobiology.^7,8 Because bioactive compounds could be trapped into the interior cavities of dendrimers via hydrophobic or electrostatic interactions, dendrimers could act as efficient nanocarriers of bioactive compounds.^9–12 On the other hand, the targeted capacity of dendrimers predominantly depends on their exterior layer groups. Moreover, dendrimers could be condensed into nanoscale dendritic boxes, which are characterized by the high encapsulation efficiency of guest molecules, regular shape, nanosize, and the formation of functional surfaces.^6,7,13–17

Naproxen (Nap, see Fig. 1) is a member of the class of non-steroidal anti-inflammatory drugs (NSAIDs). As one of the most commonly used cyclooxygenase (COX) inhibitors, it has been used for the treatment of many inflammation-associated conditions, e.g. arthritis, gout, tendinitis, and bursitis.^18–20 Although some COX-2 inhibitors were demonstrated to be related to increases in the risk of cardiovascular events and gastrointestinal adverse effects.,²¹ an emerging body of data suggested that Nap rarely increases the risk of adverse cardiovascular events compared to other NSAIDs. Moreover, the development of urinary bladder cancer has been demonstrated to be inhibited by Nap treatment, but the detailed molecular mechanism as well as the binding targets remained unclear.^22–29 Recently, we designed and synthesized several types of Nap-modified peptide dendrimers with good drug-delivery properties.^30–35 In addition, the peptide dendrimers constituted by glutamic acid or aspartic acid exhibited potent selectivity to bone tissues.^31,36–40 Herein we have further designed and developed Nap and oligo-aspartic acid modified dendrimers as probable targeted carriers of osteosarcoma.

Fig. 1 Molecular structures of naproxen and curcumin.

Curcumin (alias diferuloylmethane, see Fig. 1) is a natural product extracted from the herb Curcuma longa (also called turmeric). It has been reported that Cur has therapeutic effects, such as anti-inflammation, antioxidant, and anticancer activities.^41–45 It should be noted that Cur can efficiently suppress a number of types of cancer, including breast cancer, colon cancer and osteosarcoma.^46,47 Moreover, Cur has regulated transcription factors, adhesion molecules, growth factors, and apoptotic and autophagy-related proteins.^42,48–55 We have also demonstrated the synergistic antitumor effects of curcumin and an H₂S release prodrug of NSAIDs.⁵⁶ In addition, the phase-2 clinical trial of the preventative effect of Cur for colorectal cancer treatment has achieved exciting results. However, its water insolubility, instability under illumination, heat and alkaline conditions obviously limits the further clinical application of Cur in cancer therapy. The encapsulation of curcumin into nano-formulations has been reported to render Cur dispersible into aqueous media and these nano-formulations have displayed prior drug release behaviour and enhanced stability.^{45,47,49,53,56,57}

Using mPEG–PLGA nanoparticles, the potential synergistic effect of curcumin and an H₂S release prodrug of aspirin was observed in ovarian cancer cells. However, the effective therapeutic concentration of curcumin is far lower than that of NSAIDs, which makes it difficult to ensure the homogeneity and stability of the co-delivery system. In this study, we have designed, synthesized and characterized a series of naproxen-conjugated amphiphilic dendrimers bearing an oligo-aspartic acid tailor. The dendrimers are capable of rapidly transferring into micelles via self-assembly and efficiently encapsulating curcumin with a satisfactory loading amount. These Cur-loaded micelles displayed apoptosis-inducing capacity inhibition on MG-63 osteosarcoma cells. The mechanistic studies suggested that inflammation and apoptosis-related proteins were involved in the active mechanism of Cur-loaded micelles.

Materials and methods

Materials and reagents

All reagents were obtained at analysis grade from commercial sources, and directly used unless specially noted. The intermediates and compounds were purified by flash chromatography using a 200–300 mesh silica gel. ¹H NMR spectra were recorded on a Bruker Avance III-400 spectrometer at 400 MHz. The chemical shifts (δ) of NMR relative to an internal control, tetramethylsilane (TMS), were recorded in units of parts per million (ppm). The mass spectrometry was performed on a Waters Bio-QTOF instrument. The purities of all compounds were above 95.0%, as determined by the HPLC normalization method on a Shimadzu LC-20A instrument with a Diamonsil C18 column (5.0 μm, 4.6 × 250 mm, 5 μm, Dikma Technologies). The mobile phase was a mixture of water and methanol and an elute rate of 1.0 mL min⁻¹ was used. Naproxen and Cur were obtained from Energy Chemical Co., Ltd.

The L-aspartic acid, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenltetrazolium bromide (MTT), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and o-benzotriazole-N,N,N,N-tetramethyl-uronium-hexafluorophosphate (HBTU) were purchased from GL biochem. Co., Ltd. (Shanghai, China). The N-protected amino acids were purchased from Chengdu Kaijie Co. Ltd. (Chengdu, China). The ultrapure water was prepared with a Milli-Q water system without specification. The MG-63 human osteosarcoma cell lines were obtained from the ATCC (American Type Culture Collection, Rockville, MD, USA). MG-63 cells were incubated in DMEM (Dulbecco's modified eagle medium, Gibco, USA) and supplemented with 10% fetal bovine serum (FBS, Gibco, USA). All cells were maintained at 37 °C in a humidified incubator containing 5% CO₂.

Synthesis and characterization of Nap-conjugated dendrimers

The synthesis processing of Nap and oligo-aspartic acid modified dendrimers followed our previously reported method with some modifications. The synthetic route is shown in Scheme 1. As shown in Scheme 1A, the benzyl acrylate was coupled with di-ethanol amine via a Michael addition reaction to form intermediate 1. Then the two hydroxyl groups were esterified by the carboxyl group of naproxen to form compound 2, under catalysis with EDCI and 4-N,N-dimethylaminopyrimidine. After hydrogenation catalysis by 10% Pd–C, the benzyl of 2 was removed to give intermediate 3. Similar coupling and deprotection reactions were performed on compounds 1 and 3 to form intermediate 4. The structures of intermediates 1 to 4 were determined by ¹H NMR. The oligo-aspartic acid peptide and its Nap-dendrimer conjugates 5, 6 and 7 were synthesized by standard solid phase peptide synthesis (SPPS) protocols on a CSBio 136XT peptide synthesis instrument. The peptide was purified by high-performance liquid chromatography (HPLC) to 95% purity.

Scheme 1 Synthetic route for the production of Nap-conjugated dendrimers (A), and the solid phase synthetic route of Nap-conjugated oligo-aspartic acid peptide dendrimers (B).

Determination of the critical micelle concentration (CMC)

The CMC of compound 6 was determined according to the method described in our previous reports and literature.^56,58,59 Briefly, the so-called pyrene probe method was used for the determination of this important parameter in micelle systems. Fluorescence spectra were recorded on a PerkinElmer LS55 fluorescence spectrophotometer, and the pyrene was used as a fluorescence probe. Samples for fluorescence measurement were prepared, and the concentrations of dendrimer 6 were 0.0001, 0.0005, 0.001, 0.002, 0.005, 0.01, 0.025, 0.05, 0.10, 0.20, 0.50 and 1.0 mg mL⁻¹. The pyrene concentration in the aqueous solutions was 6.0 × 10⁻⁷ mol L⁻¹. For the measurement of pyrene excitation and emission spectra, the slit width for excitation spectra was maintained at 8 nm and the slit width for emission sides was 2.5 nm, and the emission spectra were recorded between 373 nm and 384 nm with an excitation wavelength of 334 nm. The intensity ratios of the first (374 nm) to the third (384 nm) vibronic peaks (I₃/I₁) were plotted as a function of the dendrimer 6 concentration.

Preparation and characterization of Cur-loaded micelles

The three Nap and oligo-aspartic acid heptapeptide conjugated dendrimers were synthesized and tested as self-assembling micelles. However, compound 7 was insoluble in both PBS buffer and most organic solutions, and only slightly soluble in DMSO. Compound 5 exhibited good water-solubility, but poor solubility in organic solvents. Therefore, only compound 6 was chosen for subsequent investigations. The curcumin-loaded micelles of Nap-conjugated dendrimers (Cur-M-Nap) and Cur-free micelles of Nap-conjugated dendrimers (M-Nap) were prepared as follows: curcumin and dendrimer (1 : 19, w/w) were fully dissolved in acetone. Then the solution was removed by vacuum evaporation. The Nap-conjugated amphiphilic dendrimers were re-dissolved into normal saline to form M-Nap. For the preparation of Cur-M-Nap, the curcumin was dissolved in acetone with Nap-conjugated dendrimers, and similar procedures were performed to prepare M-Nap. The obtained micelles were filtered and lyophilized into powder before use. The particle size and zeta potential distribution of Cur-M-Nap were determined by a Malvern ZS-90 laser particle size analyzer (Worcestershire, UK.). The microscopic morphological characterization of Cur-M-Nap was performed on a transmission electron microscope (TEM, H-6009IV, Hitachi, Tokyo, Japan) after negative staining by phosphotungstic acid. The drug loading (DL) and encapsulation efficiency (EE) were determined by the high-performance liquid chromatography (HPLC) method on a Shimadzu HPLC instrument (Shimadzu LC-20A, Milford, MA, Japan) equipped with a PDA (Photo-Diode Array) detector and a reversed-phase C₁₈ column (4.6 × 150 mm, 5 μm, Inertsil/WondaSil, Japan). The DL and EE of Cur-M-Nap were calculated according to the following equations:

DL = drug/(drug + polymer) × 100% (1)

EE = drug in micelles/drug in feed × 100% (2)

The release profiles of curcumin and naproxen from Cur-M-Nap or free curcumin were investigated by the dialysis method. In brief, 1 mL of free curcumin solution or Cur-M-Nap were placed into dialysis bags (molecular weight cut-off = 3500), then incubated at 37 °C with gentle shaking (100 rpm) in 50 mL of neutral phosphate buffered solution (PBS) containing Tween80 (0.5 wt%). After given time intervals, the dialysis medium was withdrawn and replaced with the same volume of fresh buffer. The cumulative amounts of released curcumin or naproxen were analyzed and quantified by HPLC. All the results are the mean value of three test runs and all data are shown as the mean ± SD.

Cellular proliferation inhibition and uptake of the micelles

The cellular proliferation inhibition capacity of Cur-M-Nap, M-Nap and free Cur were performed in MG-63 cells by MTT assay. In brief, the MG-63 cells were cultured in 96-well plates, treated with Cur-M-Nap, M-Nap and free Cur under different concentration gradients for 48 h. The mean inhibition percentages of cell proliferation relative to that of the control groups were collected from data of three individual experiments, and all the data are expressed as mean ± SD. In the cellular uptake experiment, MG-63 cells were suspended and seeded in a six-well plate (5 × 10⁴ cells per well), and incubated at 37 °C in a humidified incubator containing 5% CO₂ for a further 24 hours. Then the Cur-M-Nap or free Cur were added into each well. The cultures of MG-63 cells were further incubated for one hour and digested by trypsin, re-suspended and washed with cold PBS twice. Finally, each sample was examined by fluorescent microscopy and flow cytometry to quantify the fluoresce strength of the intracellular curcumin of each group.

Apoptosis assay by flow cytometry

The activities of Cur-M-Nap, M-Nap and free Cur to induce apoptosis in MG-63 cells were studied by flow cytometry. In brief, MG-63 cells were seeded in 6-well plates and incubated for 24–48 hours. Cells in different groups were exposed to a DMEM culture containing Cur-M-Nap, M-Nap or free Cur for an additional 48 hours. Then the cells were re-suspended by trypsin digestion, washed with PBS and stained with Annexin V/Propidium Iodide (BD PharMingen, San Diego, CA, USA) kit. The cell counts of early apoptotic (Annexin V+/PI−) and late apoptotic (Annexin V+/PI+) were quantified with a flow cytometry apparatus.

Western blot analysis

Cells were treated with Cur-M-Nap, M-Nap or free Cur and their total proteins were extracted with a protein extract kit buffer. The supernatants of cell lysates were collected after frozen centrifugation at 12 000 rpm for 10 min (Pierce, Waltham, MA, USA). Before SDS-PAGE gel electrophoresis, the BCA protein assay kit was used to quantify protein concentration, then the total proteins were separated and transferred into PVDF membranes. The primary antibodies were added and incubated with the PVDF membranes overnight at 4 °C or for two hours at room temperature. To avoid non-specific interactions, the PVDF membrane was incubated with skimmed milk or bovine serum at room temperature, washed with PBS several times and then incubated with HRP-conjugated IgG (Abmart, Shanghai, China) for several hours. The target proteins were visualized using enhanced chemiluminescence (Millipore, Billerica, MA, USA) according to the manufacturer's protocols.

Results

Characterization of Nap-conjugated dendrimer

The syntheses of Nap and oligo-aspartic acid modified dendrimers were partially referenced in our previous reports, but we have made some modifications.^22,28 In Fig. 1 the ¹H nuclear magnetic resonance (¹H NMR) spectra of intermediates 1, 3 and dendrimer 6, 7 are represented. As shown in Fig. 2, the signals of phenyl hydrogen atoms, benzyl group hydrogen atoms and the other methylene group hydrogen atoms in intermediate 1 were found to be as expected. The aromatic hydrogen signals of intermediate 3, including the single peak of methyloxyl hydrogen atoms and the double peaks of methyl hydrogen atoms, suggested that the naproxens were successfully conjugated to the dendrimer scaffold. Intermediate 3 was coupled with oligo-aspartic acid heptapeptides to afford compound 6. Compared to the spectra of intermediate 3, the multiple signals of aspartic acid β-methylene group hydrogens of compound 6 were found at δ2.8–3.0, and the aspartic acid α-hydrogen signals at around δ4.5. As shown in Fig. 1, compounds 6 and 7 have a similar structure, so there is something in common in their ¹H-NMR spectra, which are now described in detail. The multiplet signals at δ7.07–7.67 were attributed to the aromatic ring from naproxen. And the multiple signals at δ3.98–4.06 came from the methylene (–CH₂–O–) connected to oxygen atom. The strong single peak at δ3.89 was assigned to the methyl (CH₃–O–) linked to the oxygen atom. The weak peaks at δ3.75–3.80 belonged to the methyne in naproxen. The strong peaks at δ1.52–1.54 came from the proton of the methyl of naproxen. The weak peaks at δ2.79–2.94 in compound 6 belonged to the methylene (–CH₂–N–) connected to the nitrogen atom, while the same protons in compound 7 migrated to δ2.5. That is a normal phenomenon in ¹H-NMR spectra. Because compound 6 was insoluble in CDCl₃, it was dissolved in D₂O, so the solvent peak shifted from 7.25 to 4.7. These data confirmed the chemical structure of compound 6. For the NMR spectra of compound 7, DMSO-d₆ was used as solvent, because it was insoluble in both CDCl₃ and D₂O. Its peaks were similar to those of compound 6, and the relative integral areas of naproxen to oligo-aspartic acid heptapeptides were about twice those of compound 6. The high resolution mass spectrum results of compounds 6 and 7 were 1405.5 (M − H, calc. 1405.4) and 2147.7 (M − H, calc. 2147.8), respectively.

Fig. 2 The ¹H NMR spectrum of intermediates and Nap-conjugated dendrimers.

Preparation and characterization of Cur-loaded micelles

In general, Cur-M-Nap was prepared by following a self-assembly method, and the mean size of the curcumin-loaded micelles was about 35 ± 6 nm (Fig. 3A), with the optimum drug/polymer (w/w) ratio, the optimal drug loading (DL) and entrapped efficiency (EE) at 1 : 19, 4.6% ± 0.3% and 91.9% ± 3.1%, respectively. Furthermore, the polydisperse index (PDI), and zeta potential of the obtained curcumin-loaded Cur-M-nAP micelles were 0.24 ± 0.03, and −34.6 ± 7.2 mV, respectively (Fig. 3B). The atomic force microscopy (AFM) image of Cur-M-Nap is shown in Fig. 3C, and it suggests that the Cur-M-Nap was nearly spherical in shape, with a uniform diameter of 30–40 nm. The analysis of particle size and microscopic structure of micelles performed by AFM proved that the homogenous and stable Cur-M-Nap aqueous disperse could be achieved by loading curcumin into Nap-conjugated dendrimer-based micelles.

Fig. 3 Characterization of Cur-M-Nap. (A) Particle size distribution of Cur-M-Nap; (B) zeta potential of Cur-M-Nap; (C) AFM image of Cur-M-Nap; (D) the TEM image of Cur-M-Nap; (E) the determination of the CMC of dendrimer 6.

As shown in Fig. 3E, with an increase in concentration of dendrimer 6, the intensity ratio exhibited a substantial increase at a certain concentration, suggesting that pyrene probes were incorporated into the hydrophobic core upon micelle formation. The critical micelle concentrations (CMC) were therefore determined from the crossover point in the low concentration range. It is well known that plots of the pyrene I₃/I₁ ratio as a function of the total surfactant concentration show, a typical sigmoidal decrease around the CMC. Below the CMC, the pyrene I₃/I₁ ratio value corresponding to a polar environment, on the other hand, when the surfactant concentration increased, the pyrene I₃/I₁ ratio declined rapidly, indicating that the pyrene was encapsulated into a more hydrophobic environment. When the concentration was greater than the CMC, the pyrene I₃/I₁ ratio reached a roughly constant value because of the incorporation of the probe into the hydrophobic region of the micelles. According to Fig. 3E, the CMC of dendrimer 6 was around 10.6 μg mL⁻¹, which suggested that dendrimer 6 could be self-assembled into micelles at a therapeutic concentration.

The in vitro release profiles of curcumin and naproxen from Cur-M-Nap were studied at 37 °C and pH 7.4 and pH 6.5. These results suggested that curcumin can be well encapsulated in Cur-M-Nap, and released over an extended period. As shown in Fig. 4, approximately 52% of the total curcumin had been released after 24 h, followed by release of 81% after 96 h, and there were no significant differences between pH 6.5 and 7.4. There was a rapid release of free curcumin with over 90% of the drug being released within two hours. And the naproxen was released from the micelles in a slow manner. This cumulative drug release suggests the potential applicability of these micelles as a drug delivery system with a satisfactory pharmacokinetic profile in vivo and reduction of exposure in healthy tissues.

Fig. 4 Time course of curcumin and naproxen release from Cur-M-Nap at 37 °C and pH 7.4 (A) and pH 6.5 (B). Released curcumin and naproxen were separated by dialysis and quantified using HPLC.

Enhanced uptake of Cur after encapsulation in the micelles

The MG-63 osteosarcoma cells were treated with Cur-M-Nap with or without oligo-aspartic acid heptapeptides, to determine whether there was increased drug uptake of Cur-M-Nap into MG-63 cells. After incubation of free curcumin, Cur-M-Nap and Cur-M-Nap with oligo-aspartic acid heptapeptides, they were washed twice with PBS. Then the cells were collected and the relative curcumin-derived fluorescence was analyzed by fluorescent microscopy. The fluorescent microscopic images (Fig. 5A–D) showed the difference in fluorescent intensity in MG-63 cells treated with free curcumin, Cur-M-Nap and Cur-M-Nap with oligo-aspartic acid heptapeptides, respectively. The mean fluorescence intensity of the Cur-M-Nap group was 7.2 times stronger (mean fluorescence intensity = 4.5 vs. 32.5, p < 0.01) than the free curcumin group on MG-63 cells. This result suggested that the encapsulation into micelles increased the uptake of the curcumin into MG-63 osteosarcoma cells. Moreover, the increase in uptake of the Cur-M-Nap group was reduced (mean fluorescence intensity = 32.5 vs. 17.8, p < 0.05), when the oligo-aspartic acid heptapeptides were added into the culture. This suggests that the binding of Cur-M-Nap to MG-63 is competitive.

Fig. 5 Different cellular uptake of free curcumin and Cur-M-Nap on MG-63 cells. (A) Phase microscopic and fluorescent microscopic images of PBS group (control); (B) phase microscopic and fluorescent microscopic images of MG-63 cells treated by free curcumin (free curcumin); (C) phase microscopic and fluorescent microscopic images of MG-63 cells treated by Cur-M-Nap (Cur-M-Nap); (D) phase microscopic and fluorescent microscopic images of MG-63 cells treated by Cur-M-Nap and oligo-aspartic acid heptapeptide (Cur-M-Nap + Asp heptapeptide); and (E) the quantification of the mean fluorescence intensities of each group.

In vitro cytotoxicity and apoptosis assay of the micelles

In vitro cellular proliferation assays were performed by the MTT method in MG-63 cells. After incubation for 48 hours with the free curcumin, M-Nap or Cur-M-Nap, the cell survival ratios were measured following the absorbance of the degraded MTT at 570 nm. The cell survival ratios decreased according to the increase in curcumin concentration, and there were significant differences between the Cur-M-Nap and free curcumin groups as well as for the combination of free curcumin and naproxen (see Fig. 6). Moreover, there was no significant cell proliferation inhibition observed even in an M-Nap concentration up to 1.0 mg mL⁻¹. As shown in Fig. 6, the median inhibitory concentrations (IC₅₀) of curcumin in Cur-M-Nap were about 21.2 μg mL⁻¹, which was less than that of free curcumin (57.6 μg mL⁻¹).

Fig. 6 The in vitro cytotoxicity of the free curcumin, free curcumin combination with naproxen, M-Nap, and Cur-M-Nap on MG-63 cells. The percentage of viable cells was quantified using the methylthiazoletetrazolium (MTT) method.

In vitro apoptosis assays were performed using flow cytometry with Annexin V/PI staining to investigate the apoptosis-inducing potency of Cur-M-Nap, M-Nap and free curcumin. Both early apoptosis (Annexin V+/PI−) and late apoptosis (Annexin V+/PI+) cells were included. According to Fig. 7, the percentage of apoptotic cells in the Cur-M-Nap group was 78.0% ± 7.2%, which was significantly higher than that of apoptotic cells in the free curcumin (47.6% ± 4.1%), M-Nap (16.5% ± 1.2%) and control (0.39% ± 0.12%) groups. It was notable that M-Nap only slightly induced early apoptosis in MG-63 cells. There were significant differences between the Cur-M-Nap and free curcumin groups. There were more late apoptosis cells in the Cur-M-Nap (58.9% ± 6.7%) than in the free curcumin (4.49% ± 1.07%) group. However, there were more early apoptotic cells in the free curcumin group (43.1% ± 3.8%) than in the Cur-M-Nap group (19.1% ± 2.0%). The results of the FCM assay demonstrated that Cur-M-Nap could induce strong cellular apoptosis, probably due to the synergistic effects of curcumin and M-Nap.

Fig. 7 Flow cytometric analysis of MG-63 cells stained with Annexin V-FITC/PI after treatment with control group (A), 10 μM of M-Nap (B), free curcumin (C) or Cur-M-Nap (D).

Potential molecular mechanism of Cur-M-Nap

The expression levels of apoptosis-related proteins in MG-63 cells treated by Cur-M-Nap and free curcumin were evaluated by western blot assays. Among the tested proteins, were apoptotic proteins which use the mitochondrial pathway. Generally, apoptotic cell death is usually triggered by an intrinsic pathway, i.e. the mitochondria pathway or the extrinsic pathway involving death receptors. The death receptor pathway could be activated by the ligands of a tumor necrosis factor super-family, such as Fas, TRAIL, IFN-γ or TNF-α. The mitochondrial pathway was involved in many apoptotic proteins such as cytochrome c, apoptosis inducing factor (AIF), caspase-3, caspase-9, and Bcl-2 family members. According to reports in the literature, curcumin mainly induces apoptosis by a mitochondrial pathway. The initial mechanistic studies showed that Cur-M-Nap and free curcumin increased the levels of Bax and decreased the levels of Bcl-2 (Fig. 8). Interestingly, the alterations in Cur-M-Nap groups were more apparent than in the free curcumin group, which indicated free curcumin and Cur-M-Nap induced apoptosis took place mainly on the mitochondrial pathway. Because mitochondria apoptosis could activate the proapoptotic caspase cascade, we next investigated whether caspases 3 and 9 were activated. Treatment of MG-63 cells with curcumin or Cur-M-Nap could efficiently increase the expression levels of the cleaved forms of caspases 3 and 9, and eliminate the levels of original caspase 9 proteins (Fig. 8). These results provided additional evidence that curcumin-induced apoptosis in the mitochondrial pathway and the Nap-conjugated dendrimer could enhance the apoptosis-inducing capacity of curcumin on MG-63 human osteosarcoma cells.

Fig. 8 Free curcumin and Cur-M-Nap induced apoptosis via mitochondrial pathways in MG-63 human osteosarcoma cells, as detected by western blot analysis, with GAPDH (glyceraldehyde phosphate dehydrogenase) serving as a loading control.

Discussion

Curcumin is a promising proliferation inhibitor and apoptosis inducer in MG-63 osteosarcoma cells, but its poor water solubility limits its further development and application in cancer therapy. In recent years, numbers of nano-formulations have provided effective methods to improve the aqueous solubility of hydrophobic drugs. Besides, as a classical COX-2 inhibitor and NSAID, naproxen could mediate breast cancer cell death via inducing early apoptosis followed by activation of intrinsic caspase-cascade. Furthermore, naproxen could inhibit synthesis of PGE-2 and suppress cancer cell migration in vitro. Therefore, a combination of curcumin and naproxen might improve their therapeutic activities synergistically. In our previous study, H₂S release prodrugs of NSAID and Cur were encapsulated into mPEG–PLGA nanoparticles and it was shown that there were synergistic anticancer effects. However, the dosage of NSAID was higher than that of curcumin, and the NSAID and curcumin were hardly encapsulated into a nano-formulation simultaneously and homogenously at the appropriate ratios.⁶⁰ In the current study, we have reported the synthesis, characterization and in vitro evaluation of curcumin-loaded Nap-conjugated peptide dendrimer-based micelles through a modified self-assembling method. The prepared micelles had a mean particle size of 35 ± 6 nm and were monodispersed (PDI = 0.24 ± 0.03) in PBS. The obtained Cur-M-Nap was electro negative because of the oligo-aspartic acid heptapeptides on the shell of the micelles. The stability of the nanoparticles was increased by the negative zeta potential. Hence, the Cur-M-Nap could form a stable nano-formulation without mPEG segments or other steric stabilizers. In the Cur-M-Nap, the encapsulation efficiency of curcumin was 91.9% ± 3.1%, with a DL rate of 4.6% ± 0.3%. After encapsulation, curcumin was molecularly dispersed in the naproxen core of dendrimer-based micelles, and they could be steadily released from the micelles in vitro. In the cytotoxicity and apoptosis assays, the Cur-M-Nap retained the cytotoxicity of Cur on the MG-63 human osteosarcoma cells in vitro, which shows its better apoptotic-inducing ability than that of either M-Nap or free curcumin alone. Our results further suggested that the Cur-M-Nap induced more late apoptosis in MG-63 cells by triggering the mitochondrial signaling pathway.

Nanotechnology has provided alternative strategies to solve the issue of poor water solubility of hydrophobic drugs.^60–64 Moreover, there has been great interest in multidrug co-delivery that could encapsulate and deliver two or more types of therapeutics simultaneously.^65,66 Although conventional polymeric nanoparticles have many advantages, such as reduced nonspecific cellular uptake and a long circulation lifetime, there were still difficulties in the precise dosage control of multidrug encapsulation for combinatorial treatment. In the present study, we applied a combination therapy to human osteosarcoma cells with Cur-M-Nap. Naproxen acted as a hydrophobic core of the micelles, and the curcumin was encapsulated in the micelles with high entrapment efficiency. Compared to free curcumin, the micelles not only retained the cytotoxicity and apoptosis-inducing properties of curcumin in the MG-63 cells, but also showed better apoptotic-inducing capacity. Furthermore, the relative fluorescence of curcumin absorbed into MG-63 decreased significantly with the addition of the free heptapeptide into the culture, which suggested that the oligo-aspartic acid heptapeptide might play an uptake-enhancing role for the Cur-M-Nap. Thus, the results indicated that the co-delivery of naproxen and curcumin via a nanocarrier may provide a new vision in cancer therapy. The combination of curcumin and naproxen resulted in increased late apoptosis in MG-63 cells. The results of western blotting suggested that the Cur-M-Nap mainly triggered the mitochondrial-based intrinsic apoptosis pathway by activating the cascade signaling pathway of caspase-3 and caspase-9. In summary, our developed Cur-M-Nap is a potential candidate nanomedicine for the treatment of bone neoplasm, and provides a novel platform for the future design and discovery of bone-targeted drug delivery systems.

Conclusion

In conclusion, Nap-dendrimer micelles loaded with curcumin were prepared for human osteosarcoma chemotherapy. Their surfaces were modified with oligo-aspartic acid heptapeptides. The prepared Cur-M-Nap possessed both nanoscale particle size and potent negative zeta potential. In in vitro experiments, the drug-free M-Nap did not inhibit MG-63 cell proliferation and only slightly induced apoptosis. Cur-M-Nap had higher cytotoxicity than that of free curcumin in the cell proliferation assays, and induced more mitochondrial apoptosis. Cellular uptake analysis showed that the encapsulation of the micelles promoted the selective uptake of curcumin by MG-63 cells.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (81402500 and 21472130), China Postdoctoral Science Foundation Funded Project (2014M560720), and Support Foundation of Science and Technology Department of Sichuan Province (2014FZ0039).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra15022e

‡ These authors contributed equally to this work.

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