Combined Modality Therapy Based on Hybrid Gold Nanostars Coated with Temperature Sensitive Liposomes to Overcome Paclitaxel-Resistance in Hepatic Carcinoma

In this study, we prepared gold nanostar (GNS) composite nanoparticles containing siRNA of cyclooxygenase-2(siCOX-2) that were modified by tumor targeting ligand 2-deoxyglucose (DG) and transmembrane peptide 9-poly-D-arginine (9R) to form siCOX-2(9R/DG-GNS). Paclitaxel loaded temperature sensitive liposomes (PTX-TSL) were surface-modified to produce PTX-TSL-siCOX-2(9R/DG-GNS) displaying homogeneous star-shaped structures of suitable size (293.93 nm ± 3.21) and zeta potentials (2.47 mV ± 0.22). PTX-TSL-siCOX-2(9R/DG-GNS) had a high thermal conversion efficiency under 808 nm laser radiation and a superior transfection efficiency, which may be related to the targeting effects of DG and increased heat induced membrane permeability. COX-2 expression in HepG2/PTX cells was significantly suppressed by PTX-TSL-siCOX-2(9R/DG-GNS) in high temperatures. The co-delivery system inhibited drug-resistant cell growth rates by ≥77% and increased the cell apoptosis rate about 47% at elevated temperatures. PTX-TSL and siCOX-2 loaded gold nanostar particles, therefore, show promise for overcoming tumor resistance.


Introduction
Paclitaxel is widely administered to patients with breast and ovarian cancer and is beneficial to the treatment of lung and neck cancer [1,2]. Although paclitaxel is an effective chemotherapeutic agent, several mechanisms of paclitaxel resistance that impede its clinical efficacy have been reported [3][4][5][6], including the overexpression of P-glycoprotein (P-gp), resulting in increased drug efflux from tumour cells [7][8][9], microtubule mutations, and the overexpression of anti-and pro-apoptotic members of the B-cell lymphoma-2 (Bcl-2) family [10][11][12]. Due to drug resistance, epothilones (paclitaxel analogues) have replaced paclitaxel for cancer therapy, but their clinical efficacy is limited. In addition, new cytotoxic drugs have been developed for paclitaxel resistant ovarian cancer, but remission rates remain UGA CAC GUU CGG AGA A-3 ) were purchased from Biomics Biotechnologies (Shanghai, China). The primary antibodies including anti-COX-2 from Cell Signaling Technology (Danvers, MA, USA) and anti-GAPDH from KANCHEN (Shanghai, China). The secondary antibodies included IRDye 680-labeled secondary antibodies from Santa Cruz Biotech (Santa Cruz, CA, USA). Other experimental reagents were purchased from the Shanghai Chemical Reagent Company (Shanghai, China).

DG-PEG Synthesis
The first step was to protect the amino group of NH 2 -PEG-COOH by di-tert-butyl pyrocarbonate ((Boc) 2 O). NH 2 -PEG-COOH was dissolved in Dichloromethane and the catalyst 4-dimethylaminopyridine (DMAP) was added. After stirring for 5 min, (Boc) 2 O was added dropwise under ice bath conditions and stirred for 30 min. The solution was purified by dialysis (MW 2000) after stirring overnight at room temperature. The ninhydrin reaction was used to verify the successful protection by (Boc) 2 O.
Step two was the carboxyl group of NH 2 -PEG-COOH with Boc-protected amine reacting with DG. Firstly, the carboxyl group of NH 2 -PEG-COOH was catalytically activated by a DCC/NHS system (molar ratio of PEG:DCC:NHS = 1:1.8:2.25) in anhydrous dimethylformamide (DMF) solution with continuous stirring overnight at room temperature. Centrifugation was performed to remove byproducts before DG·HCl was dissolved in the activated PEG solution with stable stirring for 4 h (molar ratio of PEG:DG = 1:0.8). The obtained DG-PEG complex was then dissolved in a mixed solution of dichloromethane and trifluoroacetic acid with constant agitation (volume ratio: 7:3) to remove the Boc-protection on amino group of NH 2 -PEG-COOH. The DG-PEG complex was then purified by dialysis (MW 2000) and obtained by lyophilization.

Synthesis of DG-PEG-Cys Complex
The amine group of L-Cysteine (Cys) was protected by (Boc) 2 O, in a similar fashion to the amino group of NH 2 -PEG-COOH by (Boc) 2 O. The carboxyl group of Cys was activated by a DCC/NHS catalyst system (molar ratio of PEG:DCC:NHS = 1:1.8:2.25). The prepared DG-PEG complex was reacted with the activated solution with stable stirring for 4 h. The products were dissolved in a mixed solution of dichloromethane and trifluoroacetic acid (volume ratio of 7:3). Finally, DG-PEG-Cys was purified by dialysis (MW 2000) and obtained via rotary evaporation. NH 2 -PEG-OCH 3 without DG modifications was used for the reaction with the Cys to obtain the Cys-PEG-OCH 3 as a control.

Synthesis of the DG-PEG-Cys-9R Complex
EDC and NHS (molar ratio of 9R:EDC:NHS = 1:1.2:1.5) was used to activate the carboxy group of 9R (the terminal amine group protected by 9-fluorenylmethyloxycarbony (Fmoc)) for 4 h at room temperature. Then, the activated carboxy group of 9R was allowed to bond with the amine group of DG-PEG-Cys with constant stirring for 4 h at room temperature. The synthetic products were dissolved in a mixed solution of dimethyformamide (DMF) solution and 20% piperidine (volume ratio of 7:3) to divest Fmoc. Finally, the DG-PEG-Cys-9R complex was obtained (molar ratio of 9R:DG-PEG-Cys = 1:1.5). The experimental control group H 3 CO-PEG-Cys-9R was the same as above.

Characterization of DG-PEG-Cys-9R Complex
The synthesis of DG, HOOC-PEG-NH 2 , DG-PEG-Cys, H 3 CO-PEG-NH 2 and H 3 CO-PEG-Cys was identified by 1 H nuclear magnetic resonance ( 1 H NMR) spectra. Using deuterium dimethyl sulfoxide (DMSO) as the solvent DG, HOOC-PEG-NH 2 , DG-PEG-Cys, H 3 CO-PEG-NH 2 and H 3 CO-PEG-Cys with a mass concentration of 2 mg/mL was prepared for the assessment of its nuclear magnetic spectrum.
The hydrodynamic diameter, PDI, and zeta potential of hybrid GNS were measured using a Malvern Zetasizer Nano ZS90 (Malvern Instrument Ltd., Worcestershire, UK). The morphologies of the nanoparticles were imaged via JEM-1011transmission electron microscope (TEM) (JEOL, Tokyo, Japan). UV-VIS-NIR spectra were obtained on a 754 PC spectrometer (Jinghua Instruments, Shanghai, China).
The photothermal potential of prepared PTX-TSL-siCOX-2(9R/DG-GNS) was investigated by NIR laser irradiation (808 nm) (LEO-Photoelectric, Shenzhen, China). The temperature was monitored with a thermometer every 30 s for 10 min. The photothermal performances of PTX-TSL-siCOX-2(9R/DG-GNS) under various laser intensities and different concentrations of samples were investigated respectively. In addition, GNS and GNS composite carriers were irradiated for 10 min and photothermal performances were monitored. We also investigated the photothermal performances of 9R, DG, HOOC-PEG-NH 2 , 9R/DG and 9R/DG-GNS alone, which served as negative controls. To further demonstrate the photothermal stability of PTX-TSL-siCOX-2(9R/DG-GNS), the photothermal efficiency was tested for five cycles of NIR laser irradiation and cooling down.
To examine the drug-loading properties of temperature sensitive liposomes in detail, high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) was used to detect the PTX content of PTX-TSL-siCOX-2(9R/DG-GNS). The chromatographic conditions were as follows: detection wavelength: 227 nm; column: C18; mobile phase: water-acetonitrile-methanol (38:40:22); injection volume: 20 µL; flow rate: 1 mL/min, column temperature: 25 • C. A PTX standard curve was constructed by dissolving the temperature-sensitive liposome solution containing PTX and filtering through 0.45 µm membranes. Drug loading and encapsulation efficiency were calculated under the above liquid phase conditions and estimated from the following formula: Drug loading efficiency% = weight of drug in liposomes weight of liposomes
The phase change temperature of the temperature sensitive liposome were measured by a differential scanning calorimeter (DSC) (Netzsch, Selb, Bavaria, Germany).
The dialysis method was used to detect the release properties of temperature-sensitive liposomes under different temperature conditions. PTX-TSL and PTX-TSL-siCOX-2(9R/DG-GNS) were added to dialysis bags and 250 mL of 30% ethanol in PBS was used as the dialysis medium. The dialysis media (1 mL) was collected at 0, 1, 5, 10, 20, 40, 60 min and added to an equal volume of PBS. The amount of PTX released in the dialysis medium was determined by HPLC. The release percentage of PTX was calculated as the formula of weight (cumulative release of PTX)/weight (total PTX).
To investigate the stability of the co-delivery system, the particle sizes and PDI of PTX-TSL-siCOX-2(9R/DG-GNS) were detected at different weeks: 1, 2, and 3 at 4 • C.

Cell Biocompatibility
MTT assay was performed to assess the cytotoxicity of free GNS and GNS composite carriers. Briefly, Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in RPMI 1640 medium containing 10% (v/v) FBS, streptomycin (100 µg/mL) and penicillin (100 U/mL) at 37 • C in 5% CO 2 . Cells were seeded at 5000 cells/well in 96-well plates (Corning Inc., New York, NY, USA) and allowed to grow into 70%-80% confluency. Then, cells were treated with GNS and GNS composite carriers in RPMI 1640 medium (200 µL) at concentrations ranging from 0.01 to 1000 µmol/L for 48 h. Cells were incubated in MTT reagent for 4 h and 150 µL DMSO was added to dissolve the intracellular formazan crystals. Optical densitie (OD) at 490 nm was detected using an Infinite F200 (TecanInc, Zurich, Switzerland). Cell viability (%) was calculated as the OD treated /OD control × 100%, where OD treated represents the OD in the presence of GNS and GNS composite carrier while OD control is the absorbance of cells not treated with GNS and the GNS composite carrier.

Cell Apoptosis Assay
PTX-resistant HepG2 cells were seeded in a 24-well plate at a density of 5 × 10 4 cells per well.

Statistical Analysis
A statistical analysis was performed using GraphPad Prism software (Version 5.01) (GraphPad Software, San Diego, CA, USA). A student's t-test was used to analyze the differences between two independent groups. A P value at 0.05 or less was considered statistically significant.

Characterization of DG-PEG-Cys-9R, 9R/DG-GNS and TSL-9R/DG-GNS
The chemical structure of PTX-TSL-siCOX-2(9R/DG-GNS) and the preparation process are illustrated in Scheme 1. The 1 H NMR spectra of DG-PEG-Cys demonstrated that 3.67-3.69 ppm and 4.02 ppm belonged to DG; 3.51 ppm and 3.34 ppm belonged to CH 2 of PE; 1.49-1.74 ppm and 5.57-5.59 ppm belonged to Cys and 2.50 ppm corresponded to DMSO peak. The 1 H NMR spectra from H3CO-PEG-Cys demonstrated that 3.34 ppm and 3.50 ppm belonged to CH 2 of PEG compared; 1.23-1.70 ppm and 5.56-5.58 ppm belonged to Cys and 2.50 ppm corresponded to DMSO peak. These data confirm the successful linkage of DG-PEG-Cys and H3CO-PEG-Cys. was 7 nM. The heating groups were incubated at 43 °C in 5% CO2 for 15 min). After being treated for 4 h, the cells were collected by centrifugation and washed once with PBS. After cell suspension in 200 μL binding buffer, staining was performed with propidium iodide (PI) and annexin V-FITC for 20 min at room temperature. The fluorescence intensities of cells were recorded by flow cytometry.

Statistical Analysis
A statistical analysis was performed using GraphPad Prism software (Version 5.01) (GraphPad Software, San Diego, CA, USA). A student's t-test was used to analyze the differences between two independent groups. A P value at 0.05 or less was considered statistically significant.

Characterization of DG-PEG-Cys-9R, 9R/DG-GNS and TSL-9R/DG-GNS
The chemical structure of PTX-TSL-siCOX-2(9R/DG-GNS) and the preparation process are illustrated in Scheme 1. The 1 H NMR spectra of DG-PEG-Cys demonstrated that 3.67-3.69 ppm and 4.02 ppm belonged to DG; 3.51 ppm and 3.34 ppm belonged to CH2 of PE; 1.49-1.74 ppm and 5.57-5.59 ppm belonged to Cys and 2.50 ppm corresponded to DMSO peak. The 1 H NMR spectra from H3CO-PEG-Cys demonstrated that 3.34 ppm and 3.50 ppm belonged to CH2 of PEG compared; 1.23-1.70 ppm and 5.56-5.58 ppm belonged to Cys and 2.50 ppm corresponded to DMSO peak. These data confirm the successful linkage of DG-PEG-Cys and H3CO-PEG-Cys. The successful conjugation of DG-PEG-Cys and 9R, H3CO-PEG-Cys and 9R was confirmed by gel electrophoresis. Figure 1A shows that DG-PEG-Cys-9R (a) and H3CO-PEG-Cys-9R (c) had a higher molecular weight, confirming their successful linkage. The successful conjugation of DG-PEG-Cys and 9R, H 3 CO-PEG-Cys and 9R was confirmed by gel electrophoresis. Figure 1A shows that DG-PEG-Cys-9R (a) and H 3 CO-PEG-Cys-9R (c) had a higher molecular weight, confirming their successful linkage.
The FTIR spectra of DG displayed a 3291 cm −1 for O-H; C-O for 1034 cm −1 . Moreover, the FTIR spectrum of HOOC-PEG-NH 2 revealed that the emerging peak at 1111 cm −1 was the C-O-C in PEG, and the peak at 2885 cm −1 was attributed to C-H in PEG; the peak at 1734 cm −1 was due to the presence of C=O in PEG. After the reaction of DG with HOOC-PEG-NH 2 , the peaks at 3329 cm −1 and 1577 cm −1 could be attributed to N-H, and the peak at the 1627 cm −1 was assigned to C=O, indicating the formation of amide in DG-PEG. After the reaction of Cys with DG-PEG, the peak could be found at 1629 cm −1 , which could be attributed to C=O. Moreover the 1572 cm −1 and 3330 cm −1 were assigned to N-H, which indicated the successful formation of amide in DG-PEG-Cys. Compared to the FTIR spectrum ( Figure 1B spectrum of HOOC-PEG-NH2 revealed that the emerging peak at 1111 cm −1 was the C-O-C in PEG, and the peak at 2885 cm −1 was attributed to C-H in PEG; the peak at 1734 cm −1 was due to the presence of C=O in PEG. After the reaction of DG with HOOC-PEG-NH2, the peaks at 3329 cm −1 and 1577 cm −1 could be attributed to N-H, and the peak at the 1627 cm −1 was assigned to C=O, indicating the formation of amide in DG-PEG. After the reaction of Cys with DG-PEG, the peak could be found at 1629 cm −1 , which could be attributed to C=O. Moreover the 1572 cm −1 and 3330 cm −1 were assigned to N-H, which indicated the successful formation of amide in DG-PEG-Cys. Compared to the FTIR spectrum ( Figure 1B) Figure 1D, Table 1). The 9R provided a positive surface charge and could adsorb negative siCOX-2. PEG modifications shielded the positive charge of GNS and 9R, which helped reduce the toxicity of the co-delivery system. The PDI of each sample group was ≤0.3, demonstrating an improved distribution of the sample particle sizes. GNS composite carriers were well dispersed as individual nanoparticles and star-shaped based on TEM images (Figure 2). The morphological features of 9R-GNS, 9R/DG-GNS and siCOX-2(9R/DG-GNS) did not differ from GNS. However, the thin film of PTX-TSL was clearly evident on the surface of GNS complexes after modification. The morphological features of PTX-TSL are shown in Figure 2F, in which a round and relatively uniform distribution was observed. It is worth mentioning that the TEM sizes of GNS composite carriers were measured in a dry state, which probably distorted the real sizes and sharpness. However, the hydrodynamic diameter is the real diameter under hydrated conditions that was larger than those observed by TEM. This phenomenon is commonly found in previous report [51,52].

Analysis of the siCOX-2 Encapsulation
The siCOX-2 binding abilities of carriers based on 9R-GNS or 9R/DG-GNS were analyzed by gel shift assay [53,54]. As shown in Figure 4A, the composite nanoparticle of GNS complexes (9R-GNS, 9R/DG-GNS) and siCOX-2 was prevented from moving towards the positive electrode in 4% agarose gels. When siCOX-2 was combined with the nanocarriers at the correct ratio, the positively charged ligand neutralized the negative charge of siCOX-2, and its movement towards the positive electrode was inhibited [55]. Then, the GNS complex was coated with PTX-TSL by mercapto coordination, which protects siCOX-2 and increases its stability. When the GNS core was coated by PTX-TSL, we found that the construction of PTX-TSL-siCOX-2(9R/DG-GNS) can be formed and optimized at the molar ratio of (molar ratio of siCOX-2/PTX-TSL-(9R/DG-GNS) was 1:40 or 1:60).

Analysis of the siCOX-2 Encapsulation
The siCOX-2 binding abilities of carriers based on 9R-GNS or 9R/DG-GNS were analyzed by gel shift assay [53,54]. As shown in Figure 4A, the composite nanoparticle of GNS complexes (9R-GNS, 9R/DG-GNS) and siCOX-2 was prevented from moving towards the positive electrode in 4% agarose gels. When siCOX-2 was combined with the nanocarriers at the correct ratio, the positively charged ligand neutralized the negative charge of siCOX-2, and its movement towards the positive electrode was inhibited [55]. Then, the GNS complex was coated with PTX-TSL by mercapto coordination, which protects siCOX-2 and increases its stability. When the GNS core was coated by PTX-TSL, we found that the construction of PTX-TSL-siCOX-2(9R/DG-GNS) can be formed and optimized at the molar ratio of (molar ratio of siCOX-2/PTX-TSL-(9R/DG-GNS) was 1:40 or 1:60)

Detection of Drug Loading and Release Capacity of PTX-TSL
The PTX peak areas of the concentration gradients were determined by HPLC. The PTX concentration was used as an abscissa and the peak area was used as the ordinate for standard curve construction: y = 26111 x ± 26707 (R 2 = 0.9986). The drug loading and encapsulation efficiency of PTX-TSL-(9R/DG-GNS) was calculated as 7.2% ± 1.19 and 92.98% ± 1.07, respectively.
The phase changes of the temperature sensitive liposomes by DSC were 42.6 • C. Both 37 • C and 43 • C were selected as the drug release conditions. As shown in Figure 4B, the liposomes were stable at 37 • C and the released PTX-TSL and PTX-TSL-siCOX-2(9R/DG-GNS) were ≤10% after 60 min. This was primarily due to the temperature being below the phase transition temperature (Tm). The temperature-sensitive liposome membranes were in a densely packed colloidal state through which drug diffusion was low. Surprisingly, PTX-TSL and PTX-TSL-siCOX-2(9R/DG-GNS) had rapid release kinetics in the first 20 min which remained at~80% under heating conditions (43 • C) ( Figure 4B). This was because at 42 • C, the outer membranes underwent phase transition, became permeable, and the encapsulated drug was rapidly released. Temperatures of 43 • C were slightly higher than the phase transition temperatures (42.6 • C) of temperature-sensitive liposomes, meaning that the release was faster [56,57].

In Vitro Formulation Compatibility
MTT assay was used to evaluate the cytotoxicity of free GNS and GNS complexes on HUVECs. As shown in Figure 4C, the viability of the HUVECs after treatment with GNS, 9R-GNS, 9R/DG-GNS and TSL-(9R/DG-GNS) was approximately 72%, 76%, 80% and 82% at the highest concentration (1000 µmol/L), respectively. Notably, the viability of cells treated by TSL-(9R/DG-GNS) at any other doses (0.01, 0.1, 1, 10, 100 µmol/L) were all ≥90%. Notably, the GNS complex displayed improved biocompatibility after modification with TSL. TSL could shield the positive charge of 9R and the toxic components of GNS, which enhanced biocompatibility.

In Vitro Cellular Uptake
We evaluated the cellular uptake of siCOX-2 and GNS complexes in paclitaxel-resistant HepG2 cells (HepG2/PTX cells) using confocal laser-scanning microscopy (CLSM) ( Figure 5). Fluorescence imaging ( Figure 5A) indicated that siCOX-2(GNS) was unable to enter HepG2/PTX cells. siCOX-2(9R-GNS) could partially penetrate after 4 h incubation, while the levels of siCOX-2(9R/DG-GNS) entry were high, particularly under heating conditions (43 • C, 15 min). Only small amounts of PTX-TSL-siCOX-2(9R/DG-GNS) entered the cells in the absence of laser irradiation. However, PTX-TSL-siCOX-2(9R/DG-GNS) under heating conditions displayed high levels of cell entry, evidenced by the increased CLSM fluorescence intensity. This was further confirmed by the quantitative analysis of the averaged fluorescent intensities of each sample at a 4 h incubation time ( Figure 5B). A significantly lower intracellular fluorescence intensity was found in the cells treated with the siCOX-2(GNS), compared to those of other nanoparticles. siCOX-2(9R-GNS) partially entered cells, which was related to the transmembrane effects of 9R. GNS modified with 9R and DG could be taken up by tumor cells through the targeting effects of DG and the transmembrane effects of 9R. At 43 • C, membrane permeability was higher and the levels of siCOX-2(9R/DG-GNS) entering the cells increased. The levels of endocytosed PTX-TSL-siCOX-2(9R/DG-GNS) were low. This was because the non-laser irradiation group of siCOX-2(9R/DG-GNS) was coated with TSL, which had shielding action on DG and 9R. However, when the temperature reached the phase transition temperature of TSL, PTX and internal siCOX-2(9R/DG-GNS) could be released. Hence, PTX-TSL-siCOX-2(9R/DG-GNS) entered cells to significantly higher levels under heating conditions (43 • C, 15 min).

Cell Apoptosis
Annexin V-FITC/PI double staining was performed to determine the apoptosis of HepG2/PTX cells treated by different nanoparticles. As shown in Figure 7B, compared to siNC(9R/DG-GNS) group, the cells treated with siCOX-2(9R/DG-GNS) under 37 • C displayed a significantly higher rate of apoptosis, 20.8% (sum of late apoptosis percentage (Q2) and early apoptosis percentage (Q3)). These results can be attributed to the combination of 9R/DG-GNS and siCOX-2. Compared to siCOX-2(9R/DG-GNS) group, the cells treated with PTX-TSL-siCOX-2(9R/DG-GNS) also displayed notable apoptosis, with apoptosis rates of~34.8%. These results can be mostly attributed to the chemotherapeutic effects of PTX and the existing of TSL, which can protect the stability of the encapsulated siCOX-2. In addition, the cells treated with PTX-TSL-siCOX-2(9R/DG-GNS) followed by 43 • C heating for 15 min had apoptosis rate increased to~47%. Upon heating, both the increased temperature and the enhanced drug release contributed to the induction of cell apoptosis. These results confirm that the hyperthermia combined with chemotherapy and gene therapy could significantly increase the cell growth inhibition rate.

Conclusions
In summary, we successfully prepared siCOX-2 loaded GNS composite nanoparticles siCOX-2(9R/DG-GNS). The PTX-TSL-siCOX-2(9R/DG-GNS) co-delivery system was further constructed. The delivery system has many advantages, including rapid release and rapid uptake by tumor cells under elevated temperatures. The results of the anti-tumor drug resistant assays showed that siCOX-2 could effectively overcome resistance to PTX. Additionally, drug-resistant cancer cells could be targeted by the photothermal conversion effects of GNS. Therefore, the PTX-TSL-siCOX-2(9R/DG-GNS) system holds promise for the treatment of anti-tumor drug resistance.