PLGA nanocomposites loaded with verteporfin and gold nanoparticles for enhanced photodynamic therapy of cancer cells

Abstract

In this paper, PLGA nanocomposites were developed by incorporating a photosensitizer, verteporfin and gold nanoparticles into the polymeric matrix and utilised for enhanced photodynamic therapy of cancer cells. Both enhanced fluorescence and ¹O₂ generation from verteporfin were observed in this new formulation under both 425 nm LED and 405 nm laser illumination. A maximum enhancement factor of 2.5 for fluorescence and 1.84 for ¹O₂ generation was obtained when the molar ratio of gold : VP was 5 : 1 and excited at 425 nm, compared with PLGA doped with verteporfin alone. The experiment results could be explained by the local electric field enhancement of gold nanoparticles. Furthermore, improved therapeutic efficacy in human pancreatic cancer cells, PANC-1, was also demonstrated by using this new formulation following light exposure, indicating the utility of these nanocomposites for enhanced photodynamic therapy.

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1. Introduction

Photodynamic therapy (PDT) is a clinically approved cancer therapy that uses visible light in combination with FDA-approved photosensitizers (Photofrin®, Levulan®, Visudyne® and others). Due to its advantages such as less invasiveness and precise target, PDT was mainly employed in treatment of early stage of cancer, infections, and age-related macular degeneration.¹ Photosensitizers are drugs that can be activated by light of appropriate wavelength. Upon excitation their absorbed energy is transferred to neighbouring oxygen molecules, producing toxic compounds such as singlet oxygen and other reactive oxygen species (ROS). These species are highly active and can cause cell death.^2–5 Therefore, generation of sufficient amounts of singlet oxygen or other ROS in PDT is a key factor for an effective treatment.

Main drawbacks of photosensitizers are their poor solubility in a physiological environment and non-specific biodistribution. To overcome these limitations, different carrier systems have been developed including the use of polymeric nanoparticles as photosensitizer delivery devices. As a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA) has been widely employed as a drug/gene delivery platform due to its unique properties such as great biocompatibility, non-toxicity and biodegradability.^6,7 Moreover, the surface of PLGA nanoparticles can be modified with various molecules such as polyethylene glycol (PEG), chitosan, poloxamer or poloxamines.^8–10 These polymers can be further functionalised and conjugated with targeting ligands for targeted drug or gene delivery.^11,12 It's worth mentioning that PLGA has been approved by the US FDA and European Medicine Agency as a drug delivery system for different medical applications in humans,⁶ for example, imaging and targeting of tumour cells and carrying anti-tumour agents including photosensitizers to tumour cells.^13,14

Currently, there is increased interest in exploring application of various nanomaterials for diagnostics and therapy.^15,16 Among these nanomaterials, gold nanostructures are being explored in a wide range of biomedical applications due to their strongly enhanced and tunable optical properties, excellent biocompatibility, minimum cytotoxicity and photothermal properties in laser-mediated photothermal cancer therapy.^17–19 Recently, enhanced generation of singlet oxygen and other ROS by combining gold nanostructures (nanospheres and nanorods) and photosensitizers has been reported. Various gold–photosensitizer conjugates have been investigated, including Rose Bengal covalently conjugated with gold nanoparticles,²⁰ 5-aminolevulinic acid (5-ALA) and gold nanoparticle conjugation through electrostatic binding,²¹ silica-coated gold nanorods containing indocyanine green (ICG),²² porphyrin-doped silica/gold nanocomposites²³ and gold nanoparticle/ZnO nanorod hybrids.²⁴

In the present study, we encapsulated verteporfin (VP), an efficient photosensitizer clinically approved for PDT of neovascular macular degeneration,^25,26 and 3–5 nm gold nanoparticles at varying ratios of gold : VP inside the PLGA polymeric matrix. It should be mentioned that the small size nanoparticles (<5.5 nm) are more advantageous for clinical use than the larger nanostructures, because such small sizes are generally compatible with renal clearance.^27,28 Singlet oxygen (¹O₂) was generated from VP upon light illumination at excitation wavelengths of both 405 and 425 nm. The amount of ¹O₂ was assessed by using Singlet Oxygen Green Sensor (SOSG) that is highly specific to ¹O₂ generation.²⁹ Following these studies, we further investigated toxicity of PLGA nanoparticles and light illumination on PANC-1 cells and HEK-293 cells. Finally, the cellular PDT effect of these nanoparticles on PANC-1 cells under 405 nm laser irradiation was evaluated by means of MTS assays. Our study shows that gold nanoparticles and VP co-encapsulated PLGA nanocomposites generate more singlet oxygen than VP alone and thus they offer promise as an improved PDT agent.

2. Experimental details

2.1 Materials

Dichloromethane (DCM), VP, dodecanethiol-stabilized gold nanospheres, PLGA (lactide : glycolide (50 : 50), MW: 30 000–60 000), poly(vinyl alcohol) (PVA, MW: 31 000–50 000) and Triton X-100 were purchased from Sigma-Aldrich. Singlet Oxygen Sensor Green (SOSG) and TrypLE were obtained from Thermo Fisher Scientific Inc. CellTiter 96® AQueous One Solution Cell Proliferation Assay was purchased from Promega.

2.2 Synthesis and characterization of PLGA nanocomposites

PLGA nanocomposites with varying amounts of gold nanoparticles and a fixed amount of VP have been synthesized via a simple solvent evaporation single-emulsion method.^30,31 In short, 300 μL of DCM solution containing VP (2.3 mg mL⁻¹) was added to 2.5 mL of DCM solution containing 15 mg PLGA, followed by mixture with various amounts of gold nanoparticles (3–5 nm, 2% (w/v) in toluene). The mixture was added dropping to an aqueous PVA (1%, w/w) solution and sonicated for 1.5 min at 200 W output using a microtip probe sonicator (Branson Digital Sonifier, S-250D, Emerson Industrial Automation, USA). The solvent (DCM and toluene) was evaporated overnight at room temperature, under moderate magnetic stirring. PLGA nanocomposites were purified by centrifugation (10 min, 7500 rpm) and washed with distilled water for three times. The final product was freeze-dried at −51 °C and 0.09 mbar in a freeze dryer (Alpha 1–4 LDplus, John Morris Scientific Pty Ltd), yielding powdered nanoparticles. Scheme 1 briefly illustrates preparation of the nanocomposites.

Scheme 1 Schematic synthesis of PLGA nanocomposites loaded with gold nanoparticles and verteporfin.

2.3 Characterization of PLGA nanocomposites

The extinction spectra of PLGA nanocomposites doped with gold and VP, pure VP and pure PLGA were measured using a spectrophotometer (Cary 5000 UV-Vis-NIR, Varian Inc.). The luminescence spectrum of VP encapsulated inside of PLGA nanocomposites was obtained using a Fluorolog-Tau-3 system from HORIBA Scientific with 450 W Xe lamp excitation. Size distribution and zeta potentials of the PLGA nanocomposites were measured with a Zetasizer Nano Series from Malvern Instruments. The morphology of PLGA nanocomposites was determined using Transmission Electron microscopy (TEM). For TEM imaging, samples were prepared by placing a drop of nanoparticle suspension onto a copper grid and air-dried, followed by negative staining with one drop of 2% aqueous uranyl acetate for contrast enhancement. The air-dried samples were then imaged using a PHILIPS CM 10 system at an accelerating voltage of 100 kV. Images were captured with an Olympus Megaview G10 camera and iTEM software.

The encapsulation efficiency and drug loading were determined as follows:

The total amount of VP entrapped in nanocomposites was obtained by measuring the fluorescence intensity of VP after complete dissolution of nanocomposites in DCM and calculating its concentration from the standard curve of free VP solution. VP fluorescence (Ex/Em: 425 nm/690 nm) was recorded on a Fluorolog-Tau-3 system.

2.4 Differential scanning calorimetry (DSC)

Differential scanning calorimetry (DSC2010, TA Instruments, Delaware, US) was used to characterize the physical state of VP in PLGA nanocomposites. Briefly, about 1 mg of the each sample was placed on an aluminium pan and covered with an aluminium lid. The sample pans were heated in a linear gradient (10 °C min⁻¹, ranging from 25 °C to 150 °C), alongside with a reference pan in a nitrogen environment. DSC data was acquired from the Universal Analysis software.

2.5 In vitro singlet oxygen generation test

To quantify the amount of singlet oxygen generated from PLGA samples containing gold nanoparticles and VP with different molar ratios, 16 μL of SOSG (0.5 mM) was mixed with 3 mL PLGA suspension solution. The mixture was then placed in a cuvette and solution was irradiated with two different light sources for different time points. The light sources used were a 425 nm LED (61 mW cm⁻², irradiation for 8 min) and a 405 nm continuous wavelength laser (346 mW cm⁻², irradiation for 5 min). After irradiation, the SOSG fluorescence at 525 nm upon 488 nm excitation was recorded using a Fluorolog-Tau-3 system.

The percentage of SOSG fluorescence enhancement after irradiation at various time points was calculated as follows:

where F_t and F₀ are respectively the fluorescence intensities at various irradiation times and without irradiation.

2.6 In vitro VP release from PLGA nanocomposites

Freeze-dried PLGA nanocomposites loaded with VP and gold nanoparticles were dispersed in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Catalogue No. 16000044) containing 10% fetal bovine serum (FBS). The samples were incubated at 37 °C for 1–24 h and then centrifuged for 15 min at 7500 rpm. After centrifugation, the supernatants were assessed for presence of VP released from nanocomposites into the solution by VP fluorescence. The percentage of VP left in PLGA samples at various time points was calculated as follows:
where [VP]₀ and [VP]_s respectively indicate the concentration of VP entrapped in PLGA nanocomposites before incubation and in the supernatant after incubation at different time points.

2.7 Cell preparation

A PANC-1 cell line, an epithelioid carcinoma cell line derived from human pancreas, and a HEK-293 cell line, originally derived from human embryonic kidney, were purchased from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin, and maintained in a 37 °C incubator with 5% CO₂ humidified air. Passaging of cells was performed once the confluency reached around 80%, cells were washed with phosphate-buffered saline (PBS) and trypsinised with TrypLE. Following incubation for 5 min at 37 °C, a fresh medium was added to trypsinised cells. Cell suspension was centrifuged at 1500 rpm for 5 minutes. After removing the supernatant, cells pellet was resuspended in a fresh medium.

2.8 Imaging and quantitative analysis of cellular uptake of PLGA nanocomposites loaded with VP and gold nanoparticles

The PACN-1 cells (3 × 10⁴ per mL) were attached to glass-bottom Petri dishes and incubated at 37 °C for 24 h. After removing the culture medium, the cells were incubated with PLGA nanocomposite suspension (25 μM) in culture medium supplemented with 10% FBS for 5 h. The cells were then washed with PBS (1×, PH 7.4) three times to remove free PLGA nanocomposites. To assess the cellular uptake of PLGA, the cells were fixed with 2.5% paraformaldehyde for 10 min at room temperature, washed twice with PBS (1×, PH 7.4) and stained with Hoechst 33342 (5 μg mL⁻¹) for 10 min at room temperature before imaging. The cells were imaged using a Leica SP2 confocal laser scanning microscopy system. A violet laser at 405 nm was used for the excitation of VP loaded inside PLGA nanocomposites. The fluorescence emission was measured at 700 ± 25 nm for VP. To quantify the number of gold nanoparticles per cell, the cells need to be counted and lysed after cellular internalization of PLGA nanocomposites. This was done via the following procedure. After incubation with nanocomposites and wash with PBS (1×, PH 7.4) three times, the cells were detached with trypsin from the Petri dishes and counted using a cell counter (Countess II FL automated cell counter from Thermo Scientific). 100 μL NaOH (1 M) and 100 μL Triton X-100 (1% v/v) were subsequently added to 800 μL of cell suspension. The cells were lysed at R.T. for 2 h with constant shaking, followed by centrifugation (10 min, 7500 rpm). After removing supernatant, nitric acid (100 μL) and hydrochloric acid (300 μL) was added to the residue for 72 h to dissolve gold nanoparticles. Solution were then diluted to 2 mL with 2% nitric acid and analyzed via ICP-MS against standard gold ion solution.

2.9 Toxicity of PLGA nanocomposites and light illumination

Before treatment, PANC-1 cells and HEK-293 cells (2 × 10⁵ per well) were grown on 96-well plates for 24 hours in culture medium with 10% FBS. For PLGA nanocomposite treatment experiments, the cells were incubated with PLGA samples with a molar ratio of gold to VP of 5 : 1 for 5 hours, followed by incubation in fresh medium for further 24 hours. For light illumination experiments, cells were illuminated with 425 nm LED for 8 min and 405 nm laser for 5 min, respectively, followed by incubation in a fresh medium for further 24 hours. The toxicity of PLGA nanocomposites and light illumination on two cell lines was determined by the MTS test (Promega Co., WI, USA) as per manufacturer's instructions and compared with that of control cells without any treatment. Cell viability was then calculated as a percentage of the absorption intensity of the control, which was set to 100%.

2.10 In vitro PDT studies

The in vitro PDT of PLGA nanocomposites under light illumination were evaluated using adherent PANC-1 cells. Cells were seeded in 96-well plates at a density of 2 × 10⁵ cells per well and incubated with PLGA samples (molar ratio of gold to VP of 5 : 1) containing different concentrations of VP molecules for 5 h, followed by 405 nm laser irradiation over the cells for 5 min. Further, the cells were incubated for another 24 h prior to cell viability assessment by the MTS assay.

3. Results and discussion

3.1 Characterization of PLGA nanocomposites

Fig. 1b illustrates a typical TEM image of two PLGA samples. Gold nanoparticle clusters loaded inside PLGA were easily observed due to the higher electron density of gold. Fig. 1c shows the absorption spectrum of the PLGA nanocomposites containing gold and VP, where characteristic absorption peaks from both gold and VP were observed. The physiochemical parameters of two PLGA formulations such as size, zeta potential, drug loading and encapsulation efficiency were also determined (Table 1). Both PLGA formulations showed negative surface charges higher than −30 mV, which indicates relatively low stability and results in aggregation of nanocomposites in the aqueous suspension.³² Therefore, to keep samples stable in the long term after synthesis, the suspension need to be freeze-dried and nanocomposites are redispersed in the suitable medium prior to the experiments.

Fig. 1 Physical and optical properties of PLGA samples. (a) Photograph of PLGA loaded with gold nanoparticles and VP. (b) TEM images of two PLGA samples. (c) Absorption spectra of PLGA nanoparticles, gold nanoparticles and VP molecules.

Table 1 Mean size, zeta potential, VP loading and encapsulation efficiency of two PLGA nanocomposite samples used in this study

Formulation (molar ratio of gold to VP)	Size (nm)	ζ-Potential (mV)	VP loading efficiency (%)	VP encapsulation efficiency (%)
PLGA (0 : 1)	105 ± 23	−21.5 ± 2.4	3.27	4.13 ± 1.1
PLGA (5 : 1)	109 ± 19	−24.0 ± 1.8	1.34	4.21 ± 1.2

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Fig. 2 shows DSC curves of different PLGA samples, which provided information about the physical state of pure VP and VP loaded inside PLGA nanocomposites. There was no obvious melting peak observed from the DSC curve of pure VP, indicating VP appears amorphous in nature. The pure PLGA sample exhibits an endotherm peak at 46 °C. The DSC curves of PLGA nanocomposites (PLGA loaded with VP alone and PLGA loaded with VP and gold nanoparticles) also show an endothermic peak at 53 °C, which was close to the temperature peak characterising pure PLGA (46 °C). Apart from this peak, there were no other obvious endothermic peaks observed in the DSC curve of PLGA nanocomposites, indicating that VP loaded inside PLGA nanocomposites still remains an amorphous state.

Fig. 2 DSC thermograms of pure VP, pure PLGA, PLGA nanocomposites loaded with VP alone and PLGA nanocomposites loaded with VP and gold nanoparticles.

3.2 VP fluorescence measurement in different PLGA nanocomposites

We first checked the fluorescence intensities of VP in PLGA samples with different molar ratios of gold and VP. The presence of gold nanoparticles did not change the spectral shape of the emission. Indeed, in the samples with and without gold nanoparticles as shown in Fig. 3, we were able to observe a very similar spectrum, with the emission maximum typical of VP centred around 694 nm. Gold nanoparticles clearly enhanced fluorescence of VP, with the enhancement factor of 2.5 for the molar ratio (gold : VP) of 5 : 1, compared with the sample without gold nanoparticles present (molar ratio of 0 : 1). We attributed the observed enhancement to the interactions of the excited fluorophores with surface plasmon resonance (SPR) in metal nanostructure.^33–36 When the absorption band of a fluorophore overlapped with the SPR band of metal nanostructures (especially gold and silver), a remarkable fluorescence enhancement was observed. In our study, gold nanoparticles showed the SPR band maximum around 525 nm, which partially overlapped with the absorption band of VP. This coupling effect between VP and gold nanoparticles led to the plasmonic enhancement of fluorescence.^33,34 However, the size of our gold nanoparticles was smaller than 10 nm, which significantly weakened the SPR intensity.^37,38 In addition, in our study, VP molecules were randomly loaded inside the polymeric matrix and they tend to be located in varying positions, with some molecules less than optimally placed in term of the distance for maximum fluorescence enhancement. These factors may partially contribute to limited fluorescence enhancement observed in this study.

Fig. 3 Fluorescence spectra of VP in PLGA samples with two molar ratios (gold : VP) of 5 : 1 and 0 : 1, under 425 nm excitation. Bar graph (inset) highlights the fluorescence enhancement for different molar ratios of gold and VP (5 : 1, 10 : 1, 20 : 1) compared to PLGA sample without gold nanoparticles (0 : 1).

With an increase of the molar ratio of gold and VP, the fluorescence enhancement of VP was decreased (in the case of 10 : 1) and quenching effect became dominant (in the case of 20 : 1). As it is well known, when in direct contact with metal substrates the fluorescence enhancement effect is reduced by non-radiative energy transfer to a point of complete quenching.^39–41 This phenomenon has been widely reported.^42–44 In our study, with an increase of the amount of gold nanoparticles encapsulated in PLGA matrix, VP molecules tend to aggregate on the surface of gold nanoparticles due to physical adsorption, and this direct contact to gold surface resulted in a change from fluorescence enhancement to quenching.

3.3 Detection of singlet oxygen generation from PLGA nanocomposites in solution

The generation of singlet oxygen was monitored using the same concentration of SOSG in each sample, while varying the molar ratio of gold nanoparticles and VP molecules. The fluorescence enhancement of SOSG as a function of light irradiation time is plotted in Fig. 4. Fig. 4a shows that PLGA samples with molar ratio (gold : VP) of 5 : 1 and 10 : 1 generated more ¹O₂ than the control one (gold : VP = 0 : 1), with an increase percentage of 92% and 56% after 8 min LED illumination, respectively. However, with a further increase in the amount of gold nanoparticles (a molar ratio of 20 : 1), less ¹O₂ generation was observed compared with the control, with an increase percentage of only 23.5% achieved after 8 min illumination. Under 405 nm laser irradiation, among these samples, one with the molar ratio (gold : VP) of 5 : 1 still exhibited the highest efficiency of ¹O₂ generation compared with other samples, with an increase percentage of 89% being obtained after 5 min illumination (Fig. 4b). Similarly, the sample with the highest concentration of gold nanoparticles (a molar ratio of 20 : 1) produced less ¹O₂ compared with the control one, with an increase percentage of only 17.0% after 5 min exposure (Fig. 4b). Similar to the plasmonic enhancement of VP fluorescence induced by gold nanoparticles described above, the highest efficiency of ¹O₂ generation was also observed in the sample with the molar ratio (gold : VP) of 5 : 1.

Fig. 4 The increase percentage of SOSG fluorescence intensities in PLGA samples with various molar ratios of gold and VP molecules (0 : 1, 5 : 1, 10 : 1, 20 : 1) exposed to a 425 nm LED (a) and a 405 nm laser at different illumination time points (b).

To understand the phenomenon of enhanced ¹O₂ generation, we need to first explain the mechanism of ¹O₂ generation from VP under light illumination. This photophysical mechanism involves absorption of light by the ground-state VP molecule that subsequently undergoes excitation from the ground state to the first excited state. The first excited state is short-lived, and VP quickly undergoes intersystem crossing from the first excited state to the triplet state which can transfer energy directly to ground-state molecular oxygen, ³O₂, to generate ¹O₂.⁴⁵ It has been previously reported that the local field around metal nanoparticles affects the singlet oxygen generation from photosensitizer.⁴⁶ They increase the local intensity of the excited light when metal nanoparticles are excited with electromagnetic radiation.^47,48 This local field enhancement helps to increase the net system absorption which increases triplet yield of the photosensitizer, subsequently increasing the ¹O₂ generation.⁴⁹ In the sample containing gold and VP, the increased triplet-state VP in proximity to gold nanoparticles, due to the local field enhancement around gold nanoparticles, led to the enhancement of ¹O₂ generation.

3.4 In vitro VP release from PLGA nanocomposites

As shown in Fig. 5, a fast release of VP within 5 h incubation was observed for PLGA samples, with 22% of VP and 20% of VP released from PLGA loaded with VP alone and PLGA loaded with VP and gold nanoparticles, respectively. This was followed by a slower release between 5 and 24 h, with 74% and 78% of VP still present in two nanocomposites, respectively, after 24 h incubation. This could be attributed to rapid release of VP loosely attached to the PLGA surface in the beginning.

Fig. 5 Release study of VP from PLGA nanocomposites after incubation in DMEM with 10% FBS over a period of 1 h, 2 h, 3 h, 4 h, 5 h, 7 h and 24 h.

3.5 Cellular uptake of PLGA nanocomposites

As shown in Fig. 6, red fluorescence signal from VP loaded inside PLGA nanocomposites was clearly observed after 5 h incubation with nanocomposites, indicating the cellular internalization of PLGA. The mechanism of such internalization was probably attributed to liquid phase pinocytosis and clathrin-mediated endocytosis.⁵⁰ Based on ICP-MS data of gold solution, we estimated that 1.04 × 10⁵ gold nanoparticles were internalised by each PANC-1 cell.

Fig. 6 Confocal images of PANC-1 cells incubated with PLGA nanocomposites loaded with VP for 5 h. Scale bar was 20 μm.

3.6 Toxicity of PLGA nanocomposites and light source illumination

Gold nanoparticles and VP loaded PLGA nanocomposites (gold : VP = 5 : 1) did not affect the viability of PANC-1 cells and HEK-293 cells treated with sample concentrations up to 5 mg mL⁻¹, higher than those used for PDT treatment in our study. This was evidenced by the MTS assay which shows cell's viability was not clearly reduced at 24 h after treatment with PLGA, compared with the control (Fig. 7). Light illumination is another potential factor affecting cell's activity in this study. Therefore, a set of light illumination experiments were also performed by respectively irradiating the cell samples under 425 nm LED for 8 min and 405 nm laser for 5 min. The MTS test showed no clear decrease in survival of PANC-1 cells and HEK-293 cells at 24 h after light source illumination (Fig. 7).

Fig. 7 Cytotoxicity of PLGA loaded with gold nanoparticles and VP and two light sources (405 nm laser and 425 nm LED) on PANC-1 cells (a) and HEK-293 cells (b). The cells were either incubated with PLGA nanocomposites for 5 h or illuminated with light sources. They were then incubated in fresh medium for another 24 h. The cytotoxicity was determined by MTS tests and compared with control samples without any treatment.

3.7 In vitro PDT effect of PLGA nanocomposites on PANC-1 cells

As described above, enhanced ¹O₂ generation from VP was observed in the sample with the molecular ratio of gold : VP of 5 : 1 under both 425 nm LED and 405 nm laser illumination. Therefore, we further particularly evaluated 405 nm laser-mediated PDT efficacy of two PLGA samples (gold : VP = 5 : 1 and 0 : 1) on PANC-1 cells by varying the concentration of loaded VP molecules. As shown in Fig. 8, under 5 min illumination, approximately 57% and 66% of cell survival were respectively observed in PLGA samples loaded with (gold : VP = 5 : 1) and without gold nanoparticles (gold : VP = 0 : 1). With an increase of the VP dose loaded into nanocomposites, more significant reduction in cell survival was observed for PLGA containing gold nanoparticles compared with the sample containing VP only, while the in vitro PDT effect of both PLGA samples was observed. For example, cell survival as low as 31% was obtained after treatment with PLGA loaded with both gold and VP (VP concentration = 32 μM and gold : VP = 5 : 1), while 44% of cell viability was observed after treatment with PLGA having the same concentration of VP but without gold nanoparticles. These results demonstrate higher in vitro cytotoxic efficiency of such nanocomposites on PANC-1 tumour cells.

Fig. 8 Cytotoxicity of PLGA nanocomposites with different concentrations of VP in PANC-1 cells after 5 min 405 nm laser illumination. The molar ratios of gold and VP molecules in PLGA samples were 0 : 1 and 5 : 1, respectively.

4. Conclusion

In this study, PLGA nanocomposites doped with both gold nanoparticles and VP molecules were developed. We carried out the evaluation of enhanced ¹O₂ generation and their application in PDT treatment on PANC-1 tumour cells. For various molecular ratios of gold and VP, different enhancement factors of fluorescence and ¹O₂ generation from VP were observed under 425 nm LED and 405 nm laser illumination, with a maximum enhancement factor of 2.5 for fluorescence and 1.84 for ¹O₂ generation when the molar ratio of gold : VP was 5 : 1 and excited at 425 nm. Furthermore, dark toxicity of PLGA nanocomposites on both PANC-1 cells and HEK-293 cells was evaluated and compared with untreated cells; we found that cell viability was not significantly affected. Finally, we investigated the efficiency of PLGA nanocomposites loaded with VP and gold nanoparticles on PANC-1 cell killing under 405 nm laser irradiation, which was higher compared with PLGA containing VP only. PLGA nanocomposites are also able to be targeted to the tumour cells by surface conjugation with ligands that specifically recognize tumour-associated molecules. Therefore, we believe that this new PLGA formulation has potential to be used in improved PDT treatment regimens.

Acknowledgements

All TEM images in this work were performed in the Microscopy Unit, Faculty of Science and Engineering at Macquarie University. We thank Mr Peter Wieland from the Department of Earth and Planetary Science at Macquarie University for his assistance in ICP-MS measurement. We acknowledge Dr Ayad G. Anwer from Centre of Excellence for Nanoscale Biophotonics at Macquarie University for supplying us with the cell lines. This work is supported by Discovery Early Career Researcher Award scheme (DE130100894) and Centre of Excellence scheme (CE140100003) from Australian Research Council.

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