DTX-loaded star-shaped TAPP-PLA-b-TPGS nanoparticles for cancer chemical and photodynamic combination therapy

Abstract

A novel multifunctional material consisting of a four-armed star-shaped porphyrin-cored poly(lactide)-b-D-α-tocopheryl polyethylene glycol 1000 succinate amphiphilic copolymer (TAPP-PLA-b-TPGS) was synthesized through an arm-first approach and was characterized by ¹H NMR and gel permeation chromatography (GPC). This porphyrin-functionalized material has potential applications in both drug-delivery systems and photodynamic therapy (PDT). Docetaxel (DTX)-loaded or coumarin 6-loaded nanoparticles (NPs) were prepared by a modified nanoprecipitation technique. The resulting NPs were characterized through determination of their size and size distribution data, surface charge and surface morphology, drug loading content, drug encapsulation efficiency, as well as differential scanning calorimetry (DSC) experiments. In drug release assays, the DTX-loaded NPs showed excellent pH-dependent drug-release behavior. The NPs were also found to generate singlet oxygen (¹O₂) species and exhibit significant phototoxicity in HeLa cervical cancer cells after irradiation with light of 660 nm wavelength. The drug-loaded NPs demonstrated superior performance compared to the commercial drug, Taxotere®, which is likely due to synergistic effects between phototherapy and chemotherapy in the destruction of HeLa cells. This novel functionalized drug-delivery system shows considerable potential in providing a new multi-modality treatment approach for cancer.

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1. Introduction

Cervical cancer is not only the fourth most common type of cancer but also the fourth most common cause of cancer death among women worldwide. Studies show that nearly 530 000 women were diagnosed with cervical cancer in 2012, with 85% of them in developing countries.^1,2 The current clinical approaches for cervical cancer treatment are still limited to surgical resection, radiation and consolidation chemotherapy, which can be nonspecific and can have several side effects.³ Over the past two decades, the semi-synthetic taxane analog, docetaxel, has been widely used in the treatment of cervical cancer. Docetaxel is an anti-mitotic chemotherapy medication which works by inducing tubulin polymerization.⁴ However, because of its extremely low water solubility, the commercial drug form of docetaxel is dissolved in high concentrations of Tween 80 (polysorbate 80), which could lead to various side effects such as hypersensitivity reactions, gastrointestinal problems, cardiotoxicity and neurotoxicity.⁵

Recently, a new treatment option for cancer has been developed based on photodynamic therapy (PDT). PDT is a form of phototherapy in which irradiation with light of a specific wavelength light activates photosensitizers (PS), which then generate the powerful reactive oxygen species (ROS). ROS are capable of destroying the cancer cells by damaging the biomacromolecules and organelles within the cells.^6–8 Unlike other therapy, PDT is a precisely targeted treatment which is controlled by selecting appropriate wavelength light.^6,9,10 Moreover, PDT can be used to treat the same site repeatedly, and is non-invasive. However, due to the hydrophobicity of most photosensitizers, they show unsatisfactory biodistribution properties and skin toxicity.¹¹

Nanomedicines, especially the biodegradable polymeric nanoparticles (NPs), are commonly used as drug delivery systems,^12–14 and may overcome the above disadvantages. The amphiphilic biodegradable block copolymers have the ability to encapsulate various poorly soluble agents and form micelles through self-assembly due to hydrophobic–lipophilic interactions.^3,15–17 Polylactic acid (PLA), a biodegradable thermoplastic aliphatic polyester, is being increasingly explored in medical applications.^18,19 As the decomposition product of PLA is the harmless lactic acid, PLA can be safely used as medical implants.²⁰ However, as PLA is extremely hydrophobic, the drug carriers based on PLA are rapidly captured by the reticuloendothelial system (RES) when injected into the blood stream.^21,22 This can be solved by conjugating D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or simply TPGS) to PLA to lower its hydrophobicity. TPGS, a water-soluble derivative of natural vitamin E, is prepared by the esterification of vitamin E succinate with PEG 1000.²³ It has been reported that TPGS can serve as an emulsifier in NPs. TPGS also improves the solubility, permeability and stability of the formulated drug, and increases its cellular uptake.^23–26 Moreover, TPGS has been shown to reduce multi-drug resistance (MDR) of tumor cells through P-glycoprotein inhibition,^27–29 and it has been approved by the U.S. Food and Drug Administration (FDA) in drug delivery.²⁵ The preparation techniques of polymeric nanoparticles include nanoprecipitation, dialysis, solvent evaporation and multiple emulsions.^3,30,31

Star-shaped block copolymers have recently attracted considerable attention owing to their unique properties and applicability in nanomedicine development.³² The drug loaded star-shaped NPs have a lower solution viscosity, smaller hydrodynamic radius, higher drug loading content (LC) and higher drug encapsulation efficiency (EE) than the drug loaded linear NPs with the same molecular weight and composition.^33,34 One type of classic photosensitizer is porphyrin derivative, which has the excellent biocompatible porphyrin moiety and is widely used in PDT, such as Photofrin had been approved for the prophylactic treatment of bladder cancer in 1993 in Canada.^6,9 5,10,15,20-Tetrakis(4-aminophenyl)-21H,23H-porphine (TAPP) is a porphyrin-based photosensitizer. And it is a desirable core for synthesizing star-shaped amphiphilic copolymers, because it has multiple functional groups at its periphery.^35,36 For example, poly(3-caprolactone)–poly(ethyleneglycol) diblock copolymer with TAPP as core was synthesized for use in PDT-functionalized drug delivery system.¹¹ Therefore, TAPP-functionalized star-shaped copolymers would have great potential in nanomedicine and PDT for cancer therapy.

In the present study, we have attempted to create efficient docetaxel nanocarriers for PDT based cervical cancer treatment in the form of a novel amphiphilic star-shaped block copolymer of TAPP-centered, 4-armed star PLA-b-TPGS. The drug loading content, drug encapsulation efficiency, and singlet oxygen production ability of the drug-loaded star-shaped TAPP-PLA-b-TPGS NPs were evaluated, and their cytotoxicity and phototoxicity were also investigated in vitro.

2. Materials and methods

2.1 Materials

D-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS, C₃₃O₅H₅₄(CH₂CH₂O)₂₃) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 5,10,15,20-Tetrakis(4-aminophenyl)porphyrin (TAPP, C₄₄H₃₄N₈) was obtained from Tokyo Chemical Industry (TCI, Tokyo, Japan). D,L-Lactide (LA), acetonitrile and methanol (HPLC grade), stannous octoate (Sn(Oct)₂), coumarin-6, 1,3-diisopropylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), 4-(dimethylamino) pyridine (DMAP), 1,3-diphenylisobenzofuran (DPBF) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St Louis, MO, USA). Docetaxel (DTX) was provided by Shanghai Jinhe Bio-tech Co., Ltd. (Shanghai, PR China). All the commercial chemicals that were used in this study were of the highest grade. Anti-α-tubulin was purchased from Abcam (Cambridge, UK) and A FITC conjugated goat anti-rabbit IgG antibody was purchased from Santa Cruz (Texas, USA). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Sangon Biotech (Shanghai, China). Human cervix carcinoma cell line HeLa cells were grown in our lab.

2.2 Synthesis of star-shaped copolymer TAPP-PLA-b-TPGS

The star-shaped block copolymer TAPP-PLA-b-TPGS was synthesized as described in previous reports.^32,37 The synthetic route of the TAPP-PLA-b-TPGS star-shaped polymer is summarized in Scheme 1.

Scheme 1 Synthesis of star-shaped copolymer TAPP-PLA-b-TPGS.

2.2.1 Synthesis of linear copolymer PLA-b-TPGS. Initiator TPGS (M_n = 1500, 1.50 g, 1.0 mmol), D,L-lactide (M_n = 144, 8.6 g, 60 mmol) and catalyst Sn(Oct)₂ (0.024 g, 0.1 mol% of monomer) were added to a glass tube which was connected to a vacuum system. In order to remove air and moisture, the system was evacuated and refilled with inert argon three times. The evacuated tube was then sealed, and the ring opening polymerization reaction was allowed to proceed for 10 h at 150 °C. After the reaction, the tube was cooled to room temperature. The product was dissolved in dichloromethane. Then the polymer was precipitated in excess cold anhydrous ether, washed with a solvent mixture of ether and methanol. The final product, PLA-b-TPGS, was placed in a vacuum drying oven and dried at 40 °C for 24 h.

2.2.2 Synthesis of carboxyl-terminated TPGS-b-PLA-COOH. PLA-b-TPGS (M_n ≈ 10 100, 5.00 g, 0.5 mmol), succinic anhydride (200 mg, 2.0 mmol), DMAP (122 mg, 5.0 mmol) and TEA (101 mg, 1.0 mmol) were dissolved in anhydrous dioxane (50 mL) and stirred under inert argon at room temperature for 24 h. The solvent was completely removed by vacuum distillation in a rotary evaporator. The residue was re-dissolved in dichloromethane and washed with 10% HCl (3 × 30 mL) and brine (3 × 30 mL). The organic layer was then dried with anhydrous MgSO₄ overnight, filtered, and precipitated in cold diethyl ether. The precipitated product, carboxyl-terminated TPGS-PLA-b-COOH, was dried under vacuum at 40 °C for 24 h.

2.2.3 Synthesis of star-shaped copolymer TAPP-PLA-b-TPGS. TPGS-b-PLA-COOH (2.0 g, 0.2 mmol), NHS (34.5 mg, 0.3 mmol), DCC (41.3 mg, 0.2 mmol), and DMAP (36.6 mg, 0.3 mmol) were dissolved in anhydrous dichloromethane (25 mL) and stirred under inert argon at room temperature for 24 h. Then TAPP (45 mg, 0.0625 mmol) was added to the mixture, and stirring was continued under inert argon at room temperature for another 24 h. The reaction mixture was then filtered and precipitated in cold anhydrous ether. The precipitate was dried under vacuum at 40 °C for 24 h and re-dissolved in acetone. As the final purification step, the acetone solution was placed in a dialysis membrane bag (MW cut-off: 10 000 Da) and immersed in 500 mL acetone for 48 h to remove unreacted TPGS-b-PLA-COOH and other impurities. The solution was then evaporated in a rotary evaporator, and the resulting solid pure product was dried at 40 °C in a vacuum drying oven.

2.3 Characterization of polymers

The structures of copolymers PLA-b-TPGS and TAPP-PLA-b-TPGS were confirmed by ¹H NMR (Bruker AMX 500) with CDCl₃ as solvent. Molecular weights of the copolymers were measured by GPC (Waters GPC analysis system with RI-G1362A refractive index detector, Waters Corp., Milford, MA, USA) with THF as the eluent at the flow rate of 1 mL min⁻¹. Molecular weight and polydispersity index (PDI) were evaluated using standard polystyrene samples.

2.4 Preparation of NPS with and without DTX loading, and with coumarin-6 loading

DTX-loaded TAPP-PLA-b-TPGS and PLA-b-TPGS NPs were prepared by a nanoprecipitation method using an acetone/water system, as reported previously.^29,38 Briefly, 100 mg copolymer and 10 mg DTX were fully dissolved in 8 mL acetone. The mixture was then slowly added using a syringe into 100 mL aqueous solution (0.03% w/v TPGS solution) while stirring (800 rpm). The acetone was removed completely by stirring overnight at room temperature. Finally, the NPs were obtained through centrifugal separation at 20 000 rpm for 15 min at 4 °C, and washed three times with deionized water to remove the excess TPGS emulsifier and free DTX. The precipitate was re-suspended and placed in a −80 °C refrigerator overnight. The frozen suspension was lyophilized to get the NPs powder. The fluorescent coumarin-6-loaded NPS was prepared through the same procedure except that 1 mg of coumarin-6 was used in the reaction instead of 10 mg DTX.

2.5 Characterization of drug-loaded NPs

2.5.1 Drug loading and encapsulation efficiency. The drug loading content (LC) and drug encapsulation efficiency (EE) of the drug-loaded NPs were evaluated by high performance liquid chromatography (HPLC) according to previously reported methods.³⁹ Briefly, DTX-loaded nanoparticles (5 mg) were dissolved in 1 mL dichloromethane while stirring. Then the solution was poured into 5 mL of solvent mixture (chromatography grade acetonitrile and deionized water 50 : 50, v/v). The dichloromethane was evaporated by passing a nitrogen stream through the solution for about 15 min. Subsequently, the solution was filtered and the DTX content was determined at absorbance 227 nm by HPLC (LC 1200, Agilent Technologies, Santa Clara, CA, USA) equipped with a reverse-phase C-18 column (150 mm × 46 mm, GL Sciences Inc., Tokyo, Japan). The mobile phase consisted of deionized water and acetonitrile (50 : 50, v/v) at the constant flow rate of 1.0 mL min⁻¹. The value of LC and EE were determined by the following formulae, respectively.

2.5.2 Morphological analysis of NPs. The surface morphology of DTX-loaded NPs was investigated using field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). The NPs power was suspended by deionized water, and then the solution was dropped onto a piece of monocrystalline silicon and was dried in a vacuum drying oven at room temperature overnight. The prepared samples were coated with platinum layer by JFC-1300 automatic fine platinum coater (JEOL, Tokyo, Japan). After sample preparation, the NPs were analyzed by field emission scanning electron microscopy (FESEM, JEOL JSM-6301F, Tokyo, Japan). To further observe the morphology of NPs, transmission electron microscopy (TEM, TecnaiG2 20, FEI Company, Hillsboro, Oregon, USA) was used to scan the NPs. The solution of NPs power was dropped onto a copper grid coated with a carbon membrane and dried at room temperature before characterization.

2.5.3 Size and zeta potential. The particle size and zeta potential of the NPs were examined by Malvern Mastersizer 2000 (Zetasizer Nano ZS90, Malvern Instruments Ltd., UK). The freshly prepared nanoparticles were appropriately diluted before measurement. All measurements were performed in triplicate.

2.5.4 Differential scanning calorimetry (DSC) analysis. DSC analysis was used to characterize the physical status of DTX in amphiphilic copolymer NPs, using a differential scanning calorimetry (STA449F3, Netzsch, Germany). The NPs were purged with dry nitrogen at a flow rate of 20 mL min⁻¹ and were heated from room temperature to 200 °C at a rate of 10 °C min⁻¹.

2.5.5 In vitro drug release. In vitro DTX release from drug-loaded NPs was performed as described previously.³ In brief, 5 mg of DTX-loaded NPs powder was suspended in 1 mL release buffer (PBS, containing 0.1% Tween 80, pH = 7.4 or pH = 5.0) and transferred into a dialysis membrane bag (Spectra Por®, MWCO = 3500, Spectrum, Houston, TX, USA). Then the bags were immersed into 15 mL release buffer inside a centrifuge tube and incubated in a shaking bath at 37 °C, at 200 rpm. The release buffer was replaced with fresh release buffer every day. The released DTX was extracted from the collected release buffer by DCM, and the DCM solution of DTX was then placed into an evacuated container. After evaporation of DCM, the solid DTX residue was dissolved in methanol (HPLC grade) and analyzed by HPLC as described above. In this manner, the cumulative DTX amount released over 14 days was determined.

2.6 Reactive oxygen species (ROS) assay

In order to measure the amount of singlet oxygen (¹O₂) generated by TAPP-PLA-b-TPGS, 1,3-diphenylisobenzofuran (DPBF) was used as the singlet oxygen scavenger.¹¹ The TAPP-PLA-b-TPGS or PLA-b-TPGS was first dissolved in a small amount of DMSO. Then the solution was added into methanol to form a polymer-methanol solution. The DPBF scavenger solution in methanol was also prepared by the same procedure. The two solutions were then mixed to get a polymer concentration of 100 μg mL⁻¹. The mixtures were illuminated by light of 660 ± 10 nm wavelength using LEDs from Yabo Scientific and Technical Corporation (Shenzhen, China). The mean fluence rate of the device was 18.49 mW cm⁻². The decreased absorption rate of DPBF was measured by the microplate reader (Epoch, BioTek Instruments Inc., Highland Park, USA), using the absorption band at 410 nm.

2.7 Cellular uptake of coumarin 6-loaded NPs

In this study, coumarin-6 can be used as the fluorescent standard as it shows strong green fluorescence (Ex/Em = 430/485 nm). For qualitative analysis, cells were seeded in 12-well plates containing cover glasses (diameter 18 mm) overnight. Then the cells were cultured in the DMEM containing 100 μg mL⁻¹ coumarin-6 loaded TAPP-PLA-b-TPGS NPs. After 30 min, the cover glass was washed with PBS. The cells were fixed on the cover glass with 4% paraformaldehyde for 20 min and the nuclei were stained with DAPI. The samples were observed by laser scanning confocal microscope (CLSM, Olympus Fluoview FV-1000, Tokyo, Japan).

2.8 Immuno cytochemical staining

Cells were cultured in 12-well plates with cover glass (diameter 18 mm) and fixed in 95% air humidified atmosphere containing 5% CO₂ at 37 °C for 24 h to allow attachment. DTX/DTX-loaded NPs/drug-free NPs were added into the wells at 100 ng mL⁻¹ drug concentration for 30 min to allow cellular uptake. Then the cells were allowed to grow in DMEM with 10% FBS for 6 h. Cells were fixed and stained with a primary antibody against α-tubulin at a dilution of 1 : 100 at 4 °C overnight. A FITC-conjugated goat anti-rabbit IgG was used to combine the α-tubulin antibody. Cell nucleus were stained with DAPI and observed by laser scanning confocal microscope as described above.

2.9 Cytotoxicity of DTX-loaded NPs

Cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and incubated overnight. Then DTX-loaded or drug-free TAPP-PLA-b-TPGS nanoparticle suspension and commercial Taxotere® at equivalent docetaxel concentrations 0.25, 2.5, 12.5 and 25 μg mL⁻¹ were used to culture the cells for 24, 48 and 72 h. At a specific time, the medium was replaced with DMEM containing MTT (5 mg mL⁻¹) and cells were then incubated for an additional 4 h. After that, MTT was removed and DMSO was added to dissolve the formazan crystals. Absorbance was measured at 570 nm using the microplate reader as above. Untreated cells were taken as control with 100% viability. The IC₅₀ value, the drug concentration at which there is 50% inhibition of cell growth compared to the control sample, was calculated by curve fitting of the cell viability data.

2.10 Phototoxicity of drug-free and DTX-loaded NPs mediated PDT

In order to estimate the copolymer mediated PDT effect, cells were seeded into 96-well plates at a density of 3 × 10⁴ cells per well and incubated overnight. Then, the normal medium was replaced with the medium containing various concentrations of drug-free or drug-loaded NPs, and the cells were incubated at 37 °C for an additional 24 h. The cells were then washed with PBS three times. Thereafter, the cells were exposed to light emitted by the LED, for three different irradiation times of 5 min, 10 min and 15 min. Then the MTT assay was performed as described earlier. When the photosensitizing NPs were added into the medium, all the procedures were conducted in subdued light. IC₅₀ was calculated in the same manner as for the cytotoxicity determination of DTX-loaded NPs described above.

2.11 Statistical analysis

All experiments were repeated at least three times unless otherwise stated. Comparisons were performed using a two-tailed paired Student's t test, and probability (p) less than 0.05 was considered statistically significant.

3. Results and discussion

3.1 Synthesis and characterization of star-shaped copolymers

The synthesis procedure of star-shaped TAPP-PLA-b-TPGS is shown in Scheme 1. The first step is the preparation of the linear amphiphilic copolymer of PLA-b-TPGS by the ring-opening polymerization of D,L-lactide (LA) with TPGS as the initiator and Sn(Oct)₂ as the bulk catalyst, at 150 °C. Then, the carboxylated linear polymer of TPGS-b-PLA-COOH was conjugated to TAPP through the reaction between amino groups of TAPP and carboxyl groups of TPGS-b-PLA-COOH, to form the 4-armed amphiphilic copolymer TAPP-PLA-b-TPGS.

In order to characterize the formation of the copolymers, ¹H NMR was used and the spectrum is shown in Fig. 1. The characteristic resonances of both TPGS (δH^a = 3.65 ppm, TPGS repeating unit: –CH₂CH₂O–) and PLA (δH^b = 1.62 ppm, LA repeating unit: –CHCH₃; δH^c = 5.21 ppm, LA repeating unit: –CHCH₃) were observed, as seen in Fig. 1A, in accordance with those reported by Zeng and co-workers,²¹ indicating the successful synthesis of the linear polymer PLA-b-TPGS. The structure of star-shaped copolymer of TAPP-LA-b-TPGS was confirmed by characteristic peak signals of TAPP moieties (δH^d = 8.90 ppm, δH^e = 7.98 ppm, δH^f = 8.23 ppm) in Fig. 1B, which could not be observed in the ¹H NMR spectrum of the linear PLA-b-TPGS, and these results are similar to the study reported by Zhang et al.³⁵ These spectra indicate that the linear polymer PLA-b-TPGS and the star-shaped polymer TAPP-PLA-b-TPGS are successfully synthesized. The molecular weights (M_n) of the copolymer TAPP-PLA-b-TPGS were measured by ¹H NMR and GPC, respectively. The M_n estimated from ¹H NMR spectrum (M_n = 41 360) is larger than that from GPC (M_n = 35 780). This could be attributed to the fact that molecular weight estimation by GPC analysis uses the linear polymer as calibration, however the star-shaped copolymer TAPP-PLA-b-TPGS has a smaller hydrodynamic volume than the linear polymer with similar molecular weight and does not expand much in solution. Similar results were reported in our previous study.^21,39 Furthermore, the polydispersity index (PDI, M_w/M_n) of the copolymer is 1.24, which is rather narrow.

Fig. 1 Typical ¹H NMR spectra of copolymers (A) PLA-b-TPGS and (B) TAPP-PLA-b-TPGS.

3.2 Preparation and characterization of NPs

The commonly used chemotherapeutic medication DTX was encapsulated into the polymeric NPs by a modified nanoprecipitation technique using acetone as the solvent of choice. This method was used for the encapsulation as it is reported to perform well as a mild, facile, and low energy process for drug encapsulation of polymeric NPs.⁴⁰ As shown in Fig. 2A, the DTX was encapsulated in the core of TAPP-PLA-b-TPGS NPs without any chemical modification through the nanoprecipitation elaboration approach. Acetone was used to dissolve the polymers and DTX to form a homogenous and clear solution. Then, the acetone solution was slowly injected into aqueous TPGS solution while stirring. In the water, stable nanoparticles were spontaneously formed, with the hydrophobic PLA at the core surrounding water-insoluble DTX, and hydrophilic TPGS segment as the outer shell.⁴¹ Finally, in order to obtain stable DTX-loaded NPs, the reaction mixture was stirred overnight to evaporate the organic solvent acetone, followed by centrifugation and lyophilization to yield the NPs as loose powder.

Fig. 2 (A) Schematic diagram of the preparation of the DTX-loaded TAPP-PLA-b-TPGS NPs. (B) DLS size distribution of the DTX-loaded TAPP-PLA-b-TPGS NPs; (C) FESEM image of the NPs; (D) TEM image of the NPs.

3.2.1 Size, zeta potential, morphology and drug encapsulation efficiency. As particle size and surface properties of the NPs play an important role in cellular uptake, drug release, in vivo pharmacokinetics and biodistribution, the size and size distribution of the DTX-loaded NPs were estimated by dynamic light scattering (DLS) experiments and the results are displayed in Table 1. It has been previously reported that 100–200 nm sized NPs have higher cellular uptake efficiency.⁴² The mean hydrodynamic sizes of DTX-loaded PLA-b-TPGS and DTX-loaded TAPP-PLA-b-TPGS NPs were found to be about 100–130 nm in diameter, which falls in the excellent size range for increasing the cellular uptake, enhanced permeability and retention (EPR)^12,43 effect of the drug. The TAPP-PLA-b-TPGS NPs had an average size of around 127 nm, and also displayed a relatively narrow size distribution (PDI = 0.159), which makes them attractive choices in drug delivery systems. The size distribution data of the DTX-loaded TAPP-PLA-b-TPGS NPs is shown in Fig. 2B.

Table 1 Characterization of DTX-loaded nanoparticles^a

Polymer	Size (nm)	ZP (mV)	PDI	LC (%)	EE (%)
a ZP = zeta potential, PDI = polydispersity index, LC = loading content, EE = encapsulation efficiency, error bars represent standard deviation (SD) for n = 3.
PLA-b-TPGS	108.5 ± 6.83	−6.06 ± 2.15	0.173	7.40	81.41
TAPP-PLA-b-TPGS	127.2 ± 4.31	−9.09 ± 3.46	0.159	8.65	95.14

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The zeta potentials of DTX-loaded TAPP-PLA-b-TPGS NPs and DTX-loaded PLA-b-TPGS were −9.09 mV and −6.06 mV, respectively, which was appropriate for NPs dispersibility and cell accessibility.¹³ Compared to the DTX-loaded PLA-b-TPGS NPs, the DTX-loaded TAPP-PLA-b-TPGS showed higher drug loading content (8.65%) and encapsulation efficiency (95.14%) (Table 1). There are two reasons that may account for this phenomenon. At first, the star-shaped core region PLA would have strong binding affinity with hydrophobic DTX molecule. Then, many arms of star-shaped TAPP-PLA-b-TPGS could interact with DTX molecule from various directions, which show higher efficiency than the linear PLA-b-TPGS.

FESEM and TEM experiments were used to characterize the morphology of the TAPP-PLA-b-TPGS NPs. Fig. 2C and D present the FESEM and TEM data of the NPs, respectively. From the images, the nearly-spherical shaped NPs can be clearly recognized, with a mean size of nearly 100 nm. The diameter of NPs decreases somewhat when they are in a dry state, thus the size obtained from TEM and FESEM measurements is smaller than that from DLS experiments.

3.2.2 Differential scanning calorimetry (DSC). In order to investigate the physical state of docetaxel in DTX-loaded TAPP-PLA-b-TPGS and PLA-b-TPGS NPs, differential scanning calorimetry (DSC) was conducted. DSC is also helpful in determining whether the drug release occurs from the inner or outer region of the NPs. As shown in Fig. 3, the melting endothermic peak of pure docetaxel occurred at 171 °C. However, there is no such peak observed in either DTX-loaded TAPP-PLA-b-TPGS NPs or DTX-loaded PLA-b-TPGS NPs. This result allows us to conclude that the physical state of docetaxel in the NPs could be a disordered crystalline phase or a solid solution state. Similar results were reported in our previous study as well.³

Fig. 3 DSC analysis thermograms of the pure DTX, the DTX-loaded TAPP-PLA-b-TPGS NPs, and the DTX-loaded PLA-b-TPGS NPs.

3.2.3 In vitro drug release. To examine the drug release behavior in vitro, the DTX-loaded NPs were suspended in two separate PBS solutions with different pH (7.4 and 5.0), both containing 0.1% w/v Tween 80 which serves the purpose of increasing the solubility of DTX in the buffer solution and preventing the drug from adhering to the tube wall. The results of the drug release from the DTX-loaded TAPP-PLA-b-TPGS NPs in the first 14 days are shown in Fig. 4. In the first 7 days, the total drug released from the TAPP-PLA-b-TPGS, in acidic condition (pH 5.0), reached to 60.09% of the encapsulated drugs, and the value reached to 77.42% of the encapsulated drugs after 14 days. These drug release values are higher than the results in simulated physiological condition with pH 7.4 (45.67% and 60.15% in 7 and 14 days, respectively). The results indicated that the drug release from star-shaped polymer TAPP-PLA-b-TPGS NPs in acidic condition is faster than that in normal neutral pH condition. The accelerated hydrolysis of the ester bonds linking the arms and core of the structure, and the ester bonds in the PLA under acidic conditions, may contribute to the enhanced drug release at lower pH.⁴¹ The intracellular pH of tumors is reported to be lower than in healthy tissues and endosomal pH may range from 4.5 to 6.5.¹³ Therefore, this pH dependent release behavior of DTX-loaded TAPP-PLA-b-TPGS NPs is suitable for pH-triggered drug delivery in cancer therapy.

Fig. 4 In vitro drug release profile of DTX-loaded TAPP-PLA-b-TPGS NPs at pH 5.0 and pH 7.4. Error bars represent standard deviation (SD) for n = 3. *p < 0.05 (t-test).

3.3 Cellular uptake of coumarin 6-loaded NPs

It has been shown that internalization and sustained retention of the NPs by cancer cells can have a significant impact on the therapeutic effects of the drug-loaded NPs.⁴⁴ Coumarin 6-loaded NPs were used to investigate the cellular uptake efficiency of the NPs. The HeLa cells were cultured in 100 μg mL⁻¹ Coumarin 6-loaded TAPP-PLA-b-TPGS NPs suspension for 30 minutes, and then the cells were observed by CLSM after sample preparation. The images shown in Fig. 5 were obtained from DAPI channel (blue), FITC channel (green) and the overlay of the two channels. As shown in Fig. 5, the HeLa cells' cytoplasm emitted considerable green fluorescence, which indicated that the entire amount of coumarin 6-loaded TAPP-PLA-b-TPGS NPs had been internalized into the HeLa cells as quickly as in 30 minutes. This could be attributed to the small size of the TAPP-PLA-b-TPGS NPs, as the particle size plays a major role in the cellular uptake of biodegradable polymeric NPs. Thus, the star-shaped copolymer of TAPP-PLA-b-TPGS may have an excellent performance in the drug-delivery system.

Fig. 5 Confocal laser scanning microscopy (CLSM) images of HeLa cells after 30 min treated with the coumarin 6-loaded TAPP-PLA-b-TPGS NPs. Coumarin-6 is green and cell nucleus is stained with DAPI (blue).

3.4 Effects of paclitaxel on microtubules in HeLa cells

The mechanism of DTX is that the drug stabilizes microtubules and inhibits the depolymerization of microtubules during mitosis, thus resulting in cell death.⁴⁵ In this assay, anti-α-tubulin primary antibody and FITC-conjugated secondary antibody were used to determine whether the DTX released from the NPs still maintained its bioactivity. In order to ensure that the effects of DTX on microtubules came from the NPs internalized by the cells rather than released from NPs in the medium, the HeLa cells were incubated with the DTX loaded NPs at 100 ng mL⁻¹ equivalent DTX concentration for just 30 min. Then the cells were washed and incubated in normal DMEM medium for 6 h to allow the drug inside the cells to work, and the immunofluorescence stained microtubules were observed by CLSM. The images are shown in the Fig. 6. The microtubules of the drug-free TAPP-PLA-b-TPGS NPs incubated cancer cells show a clear and organized distribution which is the same as the control. Thus, it is evident that the blank NPs had no effect on the distribution of microtubules. However, the cells incubated with DTX-loaded TAPP-PLA-b-TPGS NPs showed bundled microtubules, which is similar to the results obtained with Taxotere®. On the one hand, these results are similar to cellular uptake of coumarin 6-loaded NPs experiment, that DTX-loaded TAPP-PLA-b-TPGS NPs were entrapped in the cancer cells within a short period of time (30 min). On the other hand, these results show that DTX can be released form the NPs and presented similar pharmaceutical effects as the Taxotere® at relatively low concentration. Thus, the star-shaped copolymer of TAPP-PLA-b-TPGS has the potential to provide excellent therapeutic effects of the drug-loaded NPs.

Fig. 6 Confocal laser scanning microscopy (CLSM) images of microtubule in HeLa cells which were incubated with DTX, drug-free TAPP-PLA-b-TPGS NPs, and DTX-loaded TAPP-PLA-b-TPGS NPs for 30 min, and were cultured with drug-free medium for another 6 h. Cells were stained with anti-a-tubulin antibody to determine the effect of the NPs on cell microtubule organization. Green: a-tubulin; blue: nuclear (DAPI).

3.5 Reactive oxygen species (ROS) assay

In PDT, the cells are killed mostly due to the singlet oxygen (¹O₂) species generated from the photosensitizer when irradiated by light of specific wavelengths.^6,10 To investigate the singlet oxygen (¹O₂) generation of TAPP-PLA-b-TPG, we used 1,3-diphenylisobenzofuran (DPBF) as the scavenger to react with singlet oxygen (¹O₂) and trap it. The decreased absorption value of DPBF is positively correlated to the ¹O₂ quantum yields. The singlet oxygen (¹O₂) generation profile is shown in Fig. 7. The absorbance of DPBF containing PLA-b-TPGS was almost unchanged under several different irradiation times. However, with increasing irradiation time, the absorbance of DPBF containing TAPP-PLA-b-TPGS decreased significantly. Therefore, TAPP-PLA-b-TPGS was able to exhibit efficient singlet oxygen (¹O₂) generation, which was the most important factor for potential photosensitizer. This property of TAPP-PLA-b-TPGS is likely attributed to the porphyrin core of star-shaped polymer, which helps to promote the singlet oxygen (¹O₂) generation.

Fig. 7 Singlet oxygen generation profile of TAPP-PLA-b-TPGS and PLA-b-TPGS. Error bars represent standard deviation (SD) for n = 3. ***p < 0.001 (t-test).

3.6 Cytotoxicity and phototoxicity of TAPP-PLA-b-TPGS

In order to evaluate the cytotoxicity of drug-free TAPP-PLA-b-TPGS NPs, DTX-loaded TAPP-PLA-b-TPGS NPs and commercial Taxotere®, the HeLa cells were used in the MTT assay in the absence of light. Fig. 8A–C show the cells viability after culturing with NPs at equivalent DTX concentrations of 0.25, 2.5, 12.5 and 25 μg mL⁻¹ for 12 h, 24 h, and 48 h. In the earlier microtubule polymerization assay, we have demonstrated that the star-shaped amphiphilic copolymer TAPP-PLA-b-TAPP had no effect on the microtubule protein. Furthermore, the polymer also did not display any cytotoxicity at various concentrations and during different time periods in this MTT assay. These results further indicate that the star-shaped copolymer of TAPP-PLA-b-TPGS would make a good biocompatible and non-toxic drug delivery system. On the other hand, the drug-loaded TAPP-PLA-b-TPGS NPs had an advantage in decreasing the HeLa cell viability, as the cell viability reached as low as 19.5% at equivalent DTX concentration of 25 μg mL⁻¹ after 72 h. Also, the cytotoxicity of the DTX-loaded TAPP-PLA-b-TPGS NPs is likely to increase by prolonging the culture time and increasing the NPs concentration, thus exhibiting a dose-dependent and time-dependent effect. Although the DTX-loaded NPs and the Taxotere® showed similar effects on the HeLa cells after 24 h (Fig. 8A), the DTX-loaded TAPP-PLA-b-TPGS NPs showed significantly higher cytotoxicity than the commercial Taxotere® after 48 h and 72 h cell incubation (Fig. 8B and C). This trend was also observed in the IC₅₀ values. The IC₅₀ values of HeLa cells after 48 and 72 h incubation with the DTX-loaded TAPP-PLA-b-TPGS NPs were much smaller than the values obtained with Taxotere® (Table 2). This may be due to the fact that the TAPP-PLA-b-TPGS NPs are able to increase the cellular uptake and the drug-loaded NPs within the cells can then release the drug to the cytoplasm.

Fig. 8 Viability of HeLa cells cultured with the DTX-loaded TAPP-PLA-b-TPGS NPs compared with that of Taxotere® at the same DTX dose and that of the drug-free TAPP-PLA-b-TPGS NPs with the same NPs concentration, in dark: (A) 24 h; (B) 48 h; (C) 72 h. Viability of HeLa cells after 24 hour, cultured with the DTX-loaded TAPP-PLA-b-TPGS NPs compared with that of Taxotere® at the same DTX dose and that of the drug-free TAPP-PLA-b-TPGS NPs with the same NPs concentration exposed to different irradiation times: (D) 5 min; (E) 10 min; (F) 15 min. Error bars represent standard deviation (SD) for n = 3. *p < 0.05; **p < 0.01; ***p < 0.001 (t-test).

Table 2 IC₅₀ values of Taxotere® and DTX-loaded TAPP-PCL-b-TPGS on HeLa cells after 24, 48, 72 h incubation and exposed to 5, 10, 20 min irradiation after 24 h incubation

Incubation time (h)	IC50 (μg mL^−1)
	Taxotere®	DTX-loaded TAPP-PLA-b-TPGS NPs
		Dark	5 min	10 min	15 min
24	24.77 ± 1.63	23.06 ± 1.71	19.25 ± 1.37	5.04 ± 0.52	1.93 ± 0.15
48	12.75 ± 1.52	4.32 ± 0.41	—	—	—
72	10.25 ± 0.98	0.088 ± 0.013	—	—	—

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To investigate phototoxicity of the drug-free TAPP-PLA-b-TPGS NPs and DTX-loaded TAPP–PLA–TPGS NPs, the viability of the HeLa cells were assessed after exposing them to light for 5, 10 and 15 min followed by culturing for 24 h. As shown in the Fig. 8D–F, the drug-free TAPP-PLA-b-TGPS NPs destroyed the HeLa cells at equivalent DTX concentration more than 12.5 μg mL⁻¹ after irradiating for 10 min or 15 min, while they did not show any cytotoxicity towards the HeLa cells in dark, based on the previous experiment. The DTX-loaded TAPP-PLA-b-TPGS NPs killed only 48.8% of HeLa cells at the highest concentration 25 μg mL⁻¹ in dark after culturing 24 h, while the drug-loaded NPs killed 71.7% and 78.8% of HeLa cells at the same concentration and same culturing time after 10 min and 15 min irradiation, respectively (Fig. 7E and F). Also, the DTX-loaded TAPP-PLA-b-TPGS exerted higher cytotoxicity on HeLa cells than the commercial Taxotere®, although there were no significant differences between drug-loaded NPs and Taxotere® after culturing for 24 h in dark (Fig. 7A). This trend could be quantitatively demonstrated in terms of IC₅₀ values. The values of IC₅₀ were 5.04 μg mL⁻¹ and 1.93 μg mL⁻¹ for irradiation time 10 min and 15 min, respectively, after culturing 24 h. These values are smaller than the IC₅₀ value of 23.06 μg mL⁻¹ in dark, at the same culturing time. These results indicate that PDT of TAPP-PLA-b-TPGS may promote synergistic effects with DTX. There may be three reasons that account for the enhancement of cytotoxicity. First, the excellent size of the NPs and the zeta potential likely contributed to the increased cellular uptake. Also, the TPGS is possibly able to prevent the P-glycoprotein on cell membrane from pumping DTX out of the cytoplasm.^46,47 Second, the photochemical internalization (PCI) effect is an important factor.⁴⁸ The DTX-loaded NPs are endocytosed and some of them are entrapped into the endosome and lysosome. In combination with exposure to light, reactive oxygen species generating from photosensitizers could breakdown the endosomal and lysosomal membranes to release the drug into the cytoplasm. Third, the ROS are generated by TAPP-PLA-b-TPGS after irradiation, which then kills the cells by destroying the organelle.⁹

4. Conclusions

In this work, a new type of four-armed star-shaped porphyrin-cored PLA-b-TPGS copolymer (TAPP-PLA-b-TPGS) has been successfully synthesized for use in drug delivery and PDT applications. The drug-loaded nanoparticles formed through TAPP-PLA-b-TPGS encapsulation of DTX showed significantly higher drug loading concentration and drug encapsulation efficiency, and achieved faster drug release in slightly acidic medium. The DTX-loaded TAPP-PLA-b-TPGS also showed superior in vitro anticancer performance with HeLa cells than commercial Taxotere® and exhibited excellent cytotoxicity towards cervical carcinoma after irradiation. We believe that a synergistic effect exists between PDT and DTX which is responsible for enhanced destruction of HeLa cells, when the multifunctional TAPP-PLA-b-TPGS material serves as the drug nanocarriers. Therefore, the star-shaped copolymer TAPP-PLA-b-TPGS has potential applications in the development of a new multi-modality treatment approach for cancer.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 31270019, 51203085), Program for New Century Excellent Talents in University (NCET-11-0275), Guangdong Natural Science Funds for Distinguished Young Scholar (no. 2014A030306036), Scientific and Technological Innovation Bureau of Nanshan District (no. KC2014JSCX0023A), Science, Technology & Innovation Commission of Shenzhen Municipality (no. KQC201105310021A, CYZZ 20130320110255352) and Tianjin Municipal Natural Science Foundation (no. 15JCZDJC38300).

References

1. G. Suneja, M. Bacon, W. J. Small, S. Y. Ryu, H. C. Kitchener and D. K. Gaffney, Front. Oncol., 2015, 5, 14.
2. J. Ferlay, H. Shin, F. Bray, D. Forman, C. Mathers and D. M. Parkin, Int. J. Cancer, 2010, 127, 2893–2917.
3. X. Zeng, W. Tao, L. Mei, L. Huang, C. Tan and S.-S. Feng, Biomaterials, 2013, 34, 6058–6067.
4. J. Baker, J. Ajani, F. Scotté, D. Winther, M. Martin, M. S. Aapro and G. von Minckwitz, European Journal of Oncology Nursing, 2009, 13, 49–59.
5. Z. Wang, X. Zeng, Y. Ma, J. Liu, X. Tang, Y. Gao, K. Liu, J. Zhang, P. Ming, L. Huang and L. Mei, J. Biomed. Nanotechnol., 2014, 10, 1509–1519.
6. D. E. Dolmans, D. Fukumura and R. K. Jain, Nat. Rev. Cancer, 2003, 3, 380–387.
7. B. W. Henderson and T. J. Dougherty, Photochem. Photobiol., 1992, 55, 145–157.
8. T. J. Dougherty, C. J. Gomer, B. W. Henderson, G. Jori, D. Kessel, M. Korbelik, J. Moan and Q. Peng, J. Natl. Cancer Inst., 1998, 90, 889–905.
9. S. B. Brown, E. A. Brown and I. Walker, Lancet Oncol., 2004, 5, 497–508.
10. C. A. Robertson, D. H. Evans and H. Abrahamse, J. Photochem. Photobiol., B, 2009, 96, 1–8.
11. C.-L. Peng, M.-J. Shieh, M.-H. Tsai, C.-C. Chang and P.-S. Lai, Biomaterials, 2008, 29, 3599–3608.
12. J. Nicolas, S. Mura, D. Brambilla, N. Mackiewicz and P. Couvreur, Chem. Soc. Rev., 2013, 42, 1147–1235.
13. B. Daglar, E. Ozgur, M. E. Corman, L. Uzun and G. B. Demirel, RSC Adv., 2014, 4, 48639–48659.
14. M.-C. Jones and J.-C. Leroux, Eur. J. Pharm. Biopharm., 1999, 48, 101–111.
15. K. S. Soppimath, T. M. Aminabhavi, A. R. Kulkarni and W. E. Rudzinski, J. Controlled Release, 2001, 70, 1–20.
16. N. Kamaly, Z. Xiao, P. M. Valencia, A. F. Radovic-Moreno and O. C. Farokhzad, Chem. Soc. Rev., 2012, 41, 2971–3010.
17. H.-B. Shang, F. Chen, J. Wu, C. Qi, B.-Q. Lu, X. Chen and Y.-J. Zhu, RSC Adv., 2014, 4, 53122–53129.
18. V. Lassalle and M. L. Ferreira, Macromol. Biosci., 2007, 7, 767–783.
19. A. Kumari, S. K. Yadav and S. C. Yadav, Colloids Surf., B, 2010, 75, 1–18.
20. Y. Cheng, S. Deng, P. Chen and R. Ruan, Front. Chem. China, 2009, 4, 259–264.
21. X. Zeng, W. Tao, Z. Wang, X. Zhang, H. Zhu, Y. Wu, Y. Gao, K. Liu, Y. Jiang, L. Huang, L. Mei and S.-S. Feng, Part. Part. Syst. Charact., 2015, 32, 112–122.
22. M. E. Fox, F. C. Szoka and J. M. J. Fréchet, Acc. Chem. Res., 2009, 42, 1141–1151.
23. Z. Zhang, S. Tan and S.-S. Feng, Biomaterials, 2012, 33, 4889–4906.
24. L. Mu and S. S. Feng, J. Controlled Release, 2002, 80, 129–144.
25. L. Mu and S. S. Feng, J. Controlled Release, 2003, 86, 33–48.
26. Z. Zhang, L. Mei and S.-S. Feng, Nanomedicine, 2012, 7, 1645–1647.
27. L. Z. Benet, T. Izumi, Y. Zhang, J. A. Silverman and V. J. Wacher, J. Controlled Release, 1999, 62, 25–31.
28. Y. Liu, L. Huang and F. Liu, Mol. Pharm., 2010, 7, 863–869.
29. H. Zhu, H. Chen, X. Zeng, Z. Wang, X. Zhang, Y. Wu, Y. Gao, J. Zhang, K. Liu, R. Liu, L. Cai, L. Mei and S.-S. Feng, Biomaterials, 2014, 35, 2391–2400.
30. A. A. Barba, A. Dalmoro, M. d'Amore, C. Vascello and G. Lamberti, J. Mater. Sci., 2014, 49, 5160–5170.
31. C. Vauthier and K. Bouchemal, Pharm. Res., 2009, 26, 1025–1058.
32. F. Wang, T. K. Bronich, A. V. Kabanov, R. D. Rauh and J. Roovers, Bioconjugate Chem., 2008, 19, 1423–1429.
33. O. G. Schramm, G. M. Pavlov, H. P. van Erp, M. A. R. Meier, R. Hoogenboom and U. S. Schubert, Macromolecules, 2009, 42, 1808–1816.
34. P. Dong, X. Wang, Y. Gu, Y. Wang, Y. Wang, C. Gong, F. Luo, G. Guo, X. Zhao, Y. Wei and Z. Qian, Colloids Surf., A, 2010, 358, 128–134.
35. L. Zhang, Y. Lin, Y. Zhang, R. Chen, Z. Zhu, W. Wu and X. Jiang, Macromol. Biosci., 2012, 12, 83–92.
36. E. D. Sternberg, D. Dolphin and C. Brückner, Tetrahedron, 1998, 54, 4151–4202.
37. T.-B. Ren, Y. Feng, Z.-H. Zhang, L. Li and Y.-Y. Li, Soft Matter, 2011, 7, 2329.
38. T. Govender, S. Stolnik, M. C. Garnett, L. Illum and S. S. Davis, J. Controlled Release, 1999, 57, 171–185.
39. W. Tao, X. Zeng, T. Liu, Z. Wang, Q. Xiong, C. Ouyang, L. Huang and L. Mei, Acta Biomater., 2013, 9, 8910–8920.
40. L. Mei, H. Sun, X. Jin, D. Zhu, R. Sun, M. Zhang and C. Song, Pharm. Res., 2007, 24, 955–962.
41. J. Zhu, Z. Zhou, C. Yang, D. Kong, Y. Wan and Z. Wang, J. Biomed. Mater. Res., Part A, 2011, 97, 498–508.
42. S. A. Kulkarni and S.-S. Feng, Pharm. Res., 2013, 30, 2512–2522.
43. L. Dai, C.-X. Li, K.-F. Liu, H.-J. Su, B.-Q. Chen, G.-F. Zhang, J. He and J.-D. Lei, RSC Adv., 2015, 5, 15612–15620.
44. Y. Gong and F. M. Winnik, Nanoscale, 2012, 4, 360.
45. H.-Y. Hwang, I.-S. Kim, I. C. Kwon and Y.-H. Kim, J. Controlled Release, 2008, 128, 23–31.
46. E.-M. Collnot, C. Baldes, U. F. Schaefer, K. J. Edgar, M. F. Wempe and C.-M. Lehr, Mol. Pharm., 2010, 7, 642–651.
47. J. Dintaman and J. Silverman, Pharm. Res., 1999, 16, 1550–1556.
48. L. Prasmickaite, A. Høgset, P. K. Selbo, B. Ø. Engesæter, M. Hellum and K. Berg, Br. J. Cancer, 2002, 86, 652–657.

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Footnote

† These authors contributed equally to this work.

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