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Photodynamic therapy is currently used in the treatment of cancer with the drawback of toxic side effects due to the un-specificity of the photosensitising agent towards the tumour. Indocyanine green (ICG) has the benefits of lower toxicity and a higher wavelength region allowing deeper tissue penetration. Liposomes have been suggested as potential delivery vehicles of ICG. In this study we aimed to incorporate ICG into nanosomes and to determine if nanosomal ICG is a better photosensitising agent than the free ICG. This study revealed that nanosomal ICG produced more cytotoxic effects than free ICG when used in conjugation with PDT.
Photodynamic therapy (PDT) is a minimally invasive two stage procedure that requires administration of a photosensitising agent followed by illumination of the tumour with visible light usually generated by laser sources . The cytotoxic effect produced by PDT is achieved through generation of free radicals or through production of singlet oxygen through energy transfer from light to triplet oxygen . PDT has its fair share of toxic side effects due to unspecificity towards the tumour. PDT has been approved and is in use for the treatment of lung cancer, head and neck cancer, prostate and brain tumours . Currently the only approved photosensitising agent is Photofrin as it clears from normal skin and muscle tissues faster than from superficial tumour tissues . Previous studies of PDT and Photofrin have indicated that the depth of tumour within the body may be an issue as activation of photofrin requires a light of wavelength around 630nm. Light at this wavelength does not pass through tissues easily. The photosensitiser of choice for this study is Indocyanine green (ICG). ICG is very stable, does not cause photosensitivity, has very low toxicity and is inexpensive [8, 13]. It has been shown that it has a high absorbance in the wavelength region of 600 to 900nm thus offering the advantage of deeper tissue penetration [8, 14].
Another problem with Photofrin is that major late effects of skin photosensitisation occur for up to 6 weeks after PDT treatment . Previous studies have suggested that a more direct approach is the conjugation of the photosensitiser to a non-toxic tumour-selective molecule or particle [1, 3]. It has been stated that the last 2 decades has seen huge progress in the development of tumour-targeted therapeutic agents for cancer in general .
Liposome trials have shown that they increase the therapeutic index of many drugs and provide a barrier against opsoniztion therefore limiting photosensitisation of normal tissues . Liposomes are self-assembling colloid structures composed of lipid bilayers surrounding an aqueous compartment which is capable of encapsulating the photosensitising agent . Stealth liposomes, on the other hand, are liposomes coated with polyethylene glycol (PEG). This prevents the rapid uptake of the liposomes by the phagocytotic cells hence increasing the circulation time . The use of liposomes allow for increased local drug concentrations in the target region due to the enhanced permeability and retention (EPR) effect. This is caused by the liposomes prolonged circulation in blood, allowing their extravasation into solid tumours by virtue of the presence of capillary discontinuities . Previous studies have shown that in both animal and human patients, liposome delivery systems improved AUC (area under the concentration curve - a method of measurement of the bioavailability of a drug, it is directly proportional to the total amount of unaltered drug in the blood) resulting in significant improvements in drug targeting to tumours . Therefore it was suggested that stealth liposomes may be employed as a drug delivery vehicle with the aim of achieving specific targeting of active agents to pre-defined sites .
Liposomes are currently used as delivery vehicles of anti-tumour agents to targeted tumours . It has been observed that current liposomal anthracyclines provide improved pharmacokinetics, reduced toxicities to normal organ sites and also increased tumour uptake . From previous studies it was also noted that defects in the capillary endothelium of tumour vasculature are typically 200-600nm in size therefore liposomes of 100nm or less can effectively accumulate within the tumour interstitial space hence providing more specificity .
A problem with ICG is that it is found mostly bound to plasma proteins. By incorporating ICG into liposomes, ICG will not be able to bind to the plasma proteins hence increasing the circulation time and increasing the therapeutic index.
In this study we aim to incorporate ICG into nanosomes, to determine if nanosomes used as delivery vehicles of ICG are taken up more effectively than the free dye by the tumour cells and to use a cell viability assay to see if nanosomal ICG is a better photosensitising agent than free ICG.
2. Materials and methods
2.1 Cell culture
In this study the RIF-1 (radiation-induced fibrosarcoma) was cultured in RPMI 1640 medium and 10% foetal bovine serum. These cells were cultured at 37°C in 5% CO2 in a humidified atmosphere. Once a week, the cells were harvested by centrifugation after treatment with trypsin-EDTA solution.
2.2 Preparation of ICG-containing nanosomes
To prepare the ICG-containing nanosomes, distearyl phosphatidyl choline (DSPC) and cholesterol were dissolved in choloroform. The resulting solution was mixed with an ICG solution in methanol in a small spherical flask. The molar ratio of DSPC:cholesterol:ICG within the mixture was 2:1:1. The solvents were evaporated using a rotary evaporator, at 55°C. The resulting film was hydrated with water resulting in a multi-lamellar vesicle (MLV) suspension of 15mg/mL total lipid concentration. The MLV suspension was then passed through polycarbonate filters of 100nm pore size by using an extrusion device to adjust the liposome size.
2.3 Treatment with ICG/nanosomes
1x10^4 cells/ml were seeded in a 96-well tissue culture plate (initially 14 wells used) in 200µl of fresh medium and allowed to attach overnight. On the next day, 5µl of free ICG or nanosomal ICG were added to the relevant wells. Wells 1 and 2 contained cells but were to receive no treatment. Wells 3 and 4 contained cells and were to receive light treatment without the added presence of free ICG or nanosomal ICG. Well 5 contained cells with added free ICG but no light treatment was to be received and well 6 contained cells with nanosomal ICG but no light treatment was to be received. Wells 7 to 10 contained cells with added Nanosomal ICG and were to receive light treatment. Wells 11 to 14 contained cells with added free ICG and were to receive light treatment. After 15mins, the contents of wells 7 and 11 were removed and replaced with 200µl fresh medium. After 30mins, the contents of wells 8 and 12 were removed and replaced with 200µl fresh medium. After 60mins, the contents of wells 9 and 13 were removed and replaced with 200µl fresh medium. After 120mins, the contents of wells 10 and 14 were removed and replaced with 200µl fresh medium. Between all target times, the wells were re-incubated. Following complete removal of all free ICG or nanosomal ICG, the wells were treated for 3mins by an infrared light source (diode laser, 630nm and <1µW).
2.4 Cell viability assay (MTT)
Following treatment with free ICG or nanosomal ICG and light, the cells were left to incubate overnight. The next day the cytotoxic effects were measured using an MTT assay. The medium was removed from each well and replaced with 50µl of MTT solution (2mg/ml in serum free RPMJ). This was left to incubate for 90mins. The liquid was then removed very gently ensuring not to remove the precipitate. The precipitate was then dissolved in 200µl DMSO. The absorbance was measured on a plate reader at 490nm.
2.5 Fluorescence imaging.
As most photosensitisers are fluorescent, due to the excited photosensitiser returning back to the ground state, the absolute amount of free ICG or nanosomal ICG and the rate of uptake was determined using Fluorescence imaging to detect the presence of fluorescence using a xenogen IVIS_ Lumina imaging system supported by Living Image_ Software version 2.60.
As before, 6 wells were seeded in a 96 well plate with 1x10^4cells/ml and left overnight to attach. Two of these wells were controls with no treatment. Within two of the wells 5µl of free ICG were added and the other two received 5µl of nanosomal ICG. After 30mins, the first wells with the free ICG and the nanosomal ICG contents were removed, washed with medium and replaced with 200µl of fresh medium. The same was done with the other two wells at 60mins. The plate was then placed into the IVIS imaging instrument using ICG filters to determine the amount of uptake. The images were captured following 10-15second exposures.
Another 12 wells in a 96well plate was seeded with 1x10^4cells/ml and left overnight to attach. Wells 1-5 contained 5µl of free ICG, wells 7-11 contained 5µl of nanosomal ICG and wells 6 and 12 were controls with no treatment. After 1min, the contents within wells 1 and 7 were removed, washed twice with medium and replaced with 200µl fresh medium. The same was done for wells 2 and 8 after 5mins, wells 3 and 9 after 15mins, wells 4 and 10 after 30mins and wells 5 and 11 after 60mins. The plate was then placed into the IVIS imaging instrument using ICG filters to determine the rate of uptake. The images were captured following 10-15second exposures.
3.1 cell viability assay
In order to observe the cytotoxic effects occurring following treatment with nanosomal ICG and free ICG as a photosensitising agent, a MTT assay was performed.
From the cell viability assay, it was observed that no cell death occurred in wells 1 and 2 which contained cells but received no treatment. These were then used to calculate the % cell viability of the treated cells using the equation:
Figure 1- Data gained from a MTT assay performed at 490nm after treatment with free ICG or nanosomal ICG and light.
Results from the cell viability assay show that significantly more cytotoxic effects occurred when the cells were treated with nanosomal ICG and light than using free ICG with light or even using free ICG or nanosomal ICG on their own (fig 1). Fig 1 shows that the percentage cell viability decreases with time when treated with nanosomal ICG and light, with almost all cells killed at 120mins. An initial drop in percentage cell viability is observed with free ICG treated with light although percentage cell viability increases markedly before dropping again. The percentage cell viability is however still more than 100% at the longest time interval when treated with free ICG and light (fig.1).
A very slight almost non exsistent cell kill was observed in wells 3 and 4, treatment with light only. In well 5, treatment with nanosomal ICG only, 23.5% cell viability was observed. This indicated that nanosomal ICG without light treatment still produced sufficient cell kill but compared to the 9% cell viability observed in nanosomal ICG treated with light, light still produces better results. Treatment with free ICG only however didn't differ greatly at 100.96% cell viability from free ICG with light which resulted in 90% cell viability at its lowest.
3.2 Fluorescence imaging- absolute amount
In order to determine if the absolute amount of nanosomal ICG and free ICG taken up by the target cell was responsible for the cytotoxic effects observed, fluorescence imaging was performed. The higher the fluorescence, the more absolute amount of nanosomal ICG and free ICG was taken up by the target cells.
Figure 2 - Uptake of ICG and nanosomal ICG using the xenogen IVIS_ Lumina imaging system supported by Living Image_ Software version 2.60. Wells A and B contained 5µl of free ICG and Wells C and D contained 5µl of nanosomal ICG. The colour bar indicates the colours given off at various fluorescence units.
Figure 3- Bar chart to show the absolute amount of free ICG and nanosomal ICG taken up by the target cells. Data obtained from figure 2.
From figure 2 and 3, it is clear that more fluorescence occurred in wells A and B indicating that there was more absolute amount of free ICG taken up by the target cells than nanosomal ICG.
3.3 Fluorescence imaging - rate of uptake
In order to determine, if the rate of uptake of nanosomal ICG had an effect on the cytotoxic effects observed, fluorescence imaging was performed.
Figure 4 -The rate of uptake of nanosomal ICG and free ICG by cells determined by using xenogen IVIS_ Lumina imaging system supported by Living Image_ Software version 2.60. Wells A-F contained 5µl of free ICG. Wells H-M contained 5µl of nanosomal ICG. The colour bar indicates the colours observed at various fluorescence levels.
All wells in this experiment produced a signal too strong to enable differentiation between nanosomal ICG and free ICG (fig.4).
Figure 5- The same experiment as figure 4 although with a change to the time intervals and a 1 in 10 dilution of free ICG and nanosomal ICG was used. Wells A-E contained ICG. Wells G-K contained nanosomal ICG. Wells F and L were controls containing cells but no added free ICG or nanosomal ICG.
Figure 6 - graph to show the experimental data from figure 5, Fluorescence units against time. The higher the Fluorescence units, the more uptake observed.
The rate of uptake was greater with free ICG although it seemed to reach a plateau phase. As the absolute amount of nanosomal ICG taken up by the target cell was less than free ICG, it was decided to determine the rate of uptake. The rate of uptake of nanosomal ICG was a lot slower than free ICG resulting in less than half the total uptake of ICG (fig.6). Although the rate of uptake of nanosomal ICG was a lot slower, it appeared to be increasing at a steady rate.
Photodynamic therapy is a minimally invasive two stage procedure that requires administration of a photosensitizing agent followed by illumination of the tumour with visible light usually generated by laser sources . One of the problems with PDT however, is the lack of specificity to tumour cells resulting in cytotoxic effects also occurring in normal tissue resulting in adverse side effects. Previous studies have suggested that the use of a non-toxic nanoparticle may be used as a drug delivery system of the photosensitiser (ICG within this study) to the tumour hence minimising adverse side effects.
The use of Liposomes allow for increased local drug concentrations in the target region due to the enhanced permeability and retention (EPR) effect. Previous studies have indicated that liposomes are already in use as drug delivery systems of anti-cancer drugs and have proved to increase the therapeutic values and increased tumour uptake . Previous studies have also determined that particles less than 100µl in size provide more selectivity towards tumours due to defects in the capillary endothelium of tumour vasculature .
From the information gathered from previous studies, it was decided that for this experiment we would use nanosomes as drug delivery systems of the photosensitising agent ICG. Initial results indicated that a more cytotoxic effect occurred with cells treated with nanosomes (containing ICG) and treated with light than with ICG alone treated with light. It was also observed that nanosomes without treatment with light provided sufficient cytotoxic effects. Following these results, it was decided to measure the absolute amount of nanosomal ICG and free ICG taken up by the target cells. The results were not as expected. Less uptake of nanosomal ICG than free ICG was apparent. Following this, it was decided to measure the rate of uptake of nanosomal ICG compared to free ICG. If the rate of uptake of nanosomal ICG was sufficiently faster than free ICG, it may have resulted in more cell kill resulting in decreased nanosomal ICG present. As before the expected results were not observed. The rate of uptake of nanosomal ICG was actually significantly slower than free ICG.
From this experiment new windows of research may have been opened as to why using nanosomes as a delivery system of ICG to tumour cells produces more cytotoxic effects with and without treatment of light, even though the level and rate of uptake is lower. There could be multiple reasons why this occurs. It may be that the nanosomal ICG are presented to or enter the cells in a more toxic form. It may also be that being associated with the lipid in nanosomes, the level of fluorescence of ICG is reduced and a photodynamic, rather than a fluorescent event is occurring when treated with light.
Future studies could potentially involve the use of direct real time examination with a confocal laser scanning microscopy with the facilities for real-time observation to observe the events occurring during uptake of nanosomal ICG .