Spherical and rod shaped mesoporous nanosilicas for cancer-targeted and photosensitizers delivery in photodynamic therapy

Mesoporous silica nanoparticles (MSNPs) have attracted much attention in many biomedical applications. One of the fields in which smart functional nanosystems have found wide application is in cancer treatment. Here, we present new silica nanoparticle-based systems which have been explored as efficient vehicles to transport and deliver photosensitizers (PSs) into tumor tissues during photodynamic therapy (PDT). In this work, we report the preparation, characterization and, in vitro studies of distinct shaped MSNPs grafted with S -glycoside porphyrins (Pors). The ensuing nanomaterials were fully characterized, and their properties as third-generation PSs for PDT against two bladder cancer cell lines, HT-1376 and UM-UC-3, were examined. The best uptake results were obtained for MSNP-PS2 , while MSNP-PS1 showed the lowest cellular uptake among the nanocarriers tested, but revealed the best phototoxicity in both cancer cells . Overall, the phototoxicity was higher with MSNPs than with mesoporous silica nanorods (MSNRs) and higher uptake and phototoxicity were consistently observed in UM-UC-3 rather than in HT-1376 cancer cells.


Introduction
Among the new treatments developed for bladder cancer, photodynamic therapy (PDT) has shown promising survival rates, which increases the relevance of research on this therapy for this clinical condition. 1PDT combines three components: drug, light and oxygen, which by their own do not have any toxic effects on the biological systems.However, strong cytotoxic is observed in the target cells when reactive oxygen species (ROS) are produced while the drug (PS) is in contact with molecular oxygen and exposed to light with an appropriate wavelength. 2 As PS molecules do not show toxicity until activated by light irradiation, systemic side effects can be easily avoided in PDT compared to other cancer therapies. 3DT with Photofrin was first approved for bladder cancer treatment 27 years ago in Canada. 4Although many years past since then, PDT still has limitations as a general protocol for treating different types of cancer.Some of those limitations are related to the clinical use of PS, namely: 1) low generation of singlet oxygen production ( 1 O2) due to the hydrophobic character of the most used PS; 2) many PSs exhibit a nonspecific biodistribution and poor selectivity for tumor tissue over normal tissues, which can lead to prolonged skin sensitivity; 3) the lifetime of 1 O2 limits its diffusion to more or less 10 to 55 nm in cells making PDT a selective treatment but, unfortunately, its effectiveness also decreases with tissue thickness. 3,5 n this context, nanoparticles have emerged as promising vehicles for PS used in PDT, thus opening a new possibility to improve this therapy in bladder cancer treatment. 6he application of PSs nanoformulations in PDT can enhance the treatment by increasing the biocompatibility of hydrophobic PS and their blood circulation.Moreover, NPs can accumulate and remain selectively, in tumor tissues due to enhanced permeability and retention effect (EPR) -mediated passive tumor targeting. 7n the early 1990s, a new family of molecular sieves was discovered which are often called M41S. 8 In 2001, J. Perez-Pariente et al. 9 used for the first time MCM-41 as a host in a drug delivery system containing ibuprofen molecules as the guests.They reported that ibuprofen molecules occupied part of the MCM-41 mesopores and could diffuse out of the host by placing the loaded samples in a simulated body fluid.This led to Recent studies have demonstrated that grafting Pors inside or over the surfaces of MSNPs could limit the formation of Pors aggregates, increase their water compatibility and decrease the pre-mature release of Pors. 22 23 . Encouraged by these results and considering that the morphology of SiO2 NPs could affect both the biodistribution and cellular uptake 12,14,28 we decided to investigate the effect of particle shape using the same Pors attached to spherical and rod-like MSNPs.To the best of our knowledge, this is the first time that the behavior of spherical and rod-like MSNPs grafted with S-glycoside Pors is assessed by in vitro experiments performed with bladder cancer cells.For this purpose, different MSNPs bearing S-glycoside Pors were prepared.As a model PS 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (TPPF20, PS0, Figure 1) was chosen.For cancer cells targeting, 5,10,15,20tetrakis(4-1'-thio-glucosyl-2,3,5,6-tetrafluorophenyl)porphyrin (SGlc-Por, PS1, Figure 1) and 5,10,15,20-tetrakis(4-1'-thiogalactosyl-2,3,5,6-tetrafluorophenyl)porphyrin (SGal-Por, PS2, Figure 1) were investigated.These two PSs bear, respectively, glucose and galactose moieties which are crucial for tumor targeting.Since galectin 29 and glucose 29b, 30 binding proteins are expressed in high levels in cancer cells it is important to design PSs with glycol-molecules to obtain successful cancer treatment by precise tumor targeting.Thus, PS1 and PS2 were grafted on the surface of MSNPs using the synthetic strategy presented in Scheme 1. HT-1376 and UM-UC-3 bladder cancer cell lines, derived from transitional cell carcinoma, were chosen as models because both have different affinities for galactose and glucose derivatives.Both cells express glycobinding proteins but in different levels: glucose transporter (GLUT1) and galactosebinding protein (galectin-1).29b Moreover, in order to compare the silica particles' shape-dependent behavior in HT-1376 and UM-UC-3 cancer cell lines, sphere-shaped MSNPs and rodshaped mesoporous silica nanorods (MSNRs) were prepared in this research.Finally, the in vitro photodynamic efficacy of the new nanomaterials was undertaken on HT-1376 and UM-UC-3 bladder cancer cells.

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General methods
Absorption spectra were recorded using a JASCO V-660 UV-Visible Spectrophotometer.FT-IR spectra were recorded in KBr pellets using GRASEBY SPECAC or with Cary 630 FT-IR Spectrometer.The irradiation system used to determine the production of 1 O2 was the Newport irradiation system, consisting of a 300 W halogen lamp, FSQ-OG530 color glass filter for wavelengths <530 nm, and magnetic stirrer.The irradiation system used to determine the phototoxicity during biological experiments was a Lumacare system, model LC-122, consisting of a 250 W halogen lamp coupled to an optical fiber (with a cutoff filter for wavelengths <540 nm).The radiation power was measured with a potentiometer bright Spectra Physics, model 407A, and the sensor of the same brand, model 407A-2.Transmission electron microscopy (TEM) images were obtained using a JEOL JEM1010 transmission electron microscope operating at an acceleration voltage of 100 kV.All reagents were obtained from commercial sources and were used without further purification steps.Reverse phase column chromatography was carried out on Waters Sep-Pak C18 35 cm 3 cartridges.Analytical thin layer chromatography (TLC) was carried out on pre-coated silica gel sheets (Merck, 60, 0.2 mm).

Synthesis of NPs Synthesis of MSNPs and MSNRs
MSNPs and MSNRs were prepared after slight modification of the method presented in the literature. 31In a first step, CTAB (100 mg, 2.74 mM for MSNP-100, 200 mg, 5.49 mM, for MSNP-200, 400 mg, 10.98 mM for MSNP-400, 500 mg, 13.72 mM for MSNP-500,) was dissolved in water (100 mL) in Erlenmeyer flask equipped with magnetic bar.Next, to the above solution ethyl acetate (0.88 mL) and NH4OH (30%, 2.7 mL) were subsequently added.In the end, TEOS (500 µL, 2.24 mmol) was added and the solution was stirred for 5 min at RT.After that, 100 mL of water was added to slower the hydrolysis of TEOS and accelerates the silica condensation.After 24 h of stirring at RT, NPs were filtered and washed with EtOH.To remove the surfactant templates, nanoparticles were redispersed in 100 mL of ethanol/acetic acid (glacial) mixture (95/5, v/v) and the mixture was stirred for 30 minutes.In the end, nanoparticles were washed with EtOH and air-dried.

MSNPs' and MSNRs' functionalization with APTS
To the suspension of 30 mg of MSNPs or MSNRs in 2.5 mL H2O, 158 µL of APTS in 750 µL of EtOH was added.After adjusting the pH to 7 by adding 0.2 M HCl (2.25 mL) the reaction was stirred at RT for 24 h.MSNPs-NH2 and MSNRs-NH2 were washed with EtOH and air-dried.

Grafting of PS on the surface of MSNPs-NH2 and MSNRs-NH2
The grafting of porphyrins PS0, PS1, and PS2 on the nanomaterials MSNPs-NH2 and MSNRs-NH2 were carried out in DMSO at 160 °C according to the literature, with minor modifications. 3235 mg of MSNPs-NH2 or MSNPs-NH2 were resuspended in DMSO (1 mL).A solution of PS (2.38 µmol) in DMSO (4 mL) was added to that suspension and the resulting mixture was magnetically stirred for 96 h at 160 °C.After that time, red hybrid materials were obtained.The resulting hybrid materials were washed with DMSO and EtOH until no Soret and Q bands were observed in the rinse solvent and air-dried.The amount of unreacted porphyrin was calculated by UV-Vis spectrophotometry.

Cellular uptake of NPs
After incubation with NPs in the dark, UM-UC-3 and HT-1376 cells were washed with PBS buffer and mechanically scrapped in 1% (m/v) sodium dodecyl sulfate (SDS; Sigma) in PBS buffer at pH 7.0.NPs intracellular concentration was determined by spectrofluorimetry using a microplate reader (Synergy HT, Biotek, Winooski, VT, USA) with the excitation filter (set at This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins 360±40 nm) and emission filter (645±40 nm).Results were normalized for protein concentration (determined by bicinchoninic acid reagent; Pierce, Rockford, IL, USA).

PDT treatments on cells
Photodynamic irradiation was carried out in a fresh culture medium, in the absence of NPs, covering UM-UC-3 and HT-1376 cell monolayers with RPMI medium and exposing them to white light delivered by the illumination system LC-122 LumaCare at 8.4 mW/cm -2 for 40 min.As a control, sham-irradiated cells were used.These cells were kept in the dark for the same durations and under the same conditions as the irradiated cells.In all trials, triplicate wells were settled under each experimental condition, and each experiment was repeated at least three times.

MTT assay
MTT assay was used to determine cell metabolic activity after NPs incubation in the dark, irradiation, or both after 24 h.This colorimetric assay is measuring the ability of bladder cancer cells to reduce yellow 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma), to a purple formazan on a microplate reader (Synergy HT).The results are expressed in percentage of control (i.e.optical density of formazan from cells not exposed to NPs).

Statistical analysis
Statistical data analysis was carried out using Excel's Analysis ToolPak (Student's t test).The values are represented as mean ± s.e.m.Level of significance was set at values *P<0.05,**P<0.01and ***P<0.001.

Preparation and characterization of MSNPs and MSNRs
MSNPs and MSNRs have been prepared under alkaline conditions using tetraethoxysilane (TEOS) as the silica precursor and cetyltrimethylammonium bromide (CTAB) as the structure directing agent. 31In a first approach, the sample MSNP-PS0 was prepared (Scheme 1) and then evaluated for 1 O2 generation in in vitro studies using two human bladder cancer cell lines, HT-1376 and UM-UC-3.These experiments using MSNP-PS0 allowed a preliminary assessment of this type of MSNPs and, based on the results obtained, other samples with better cancer cells recognition have been prepared.Thus, nanomaterials bearing PS with sugar moieties (MSNP-PS1, MSNP-PS2, MSNR-PS1, MSNR-PS2) were prepared (Scheme 1) and fully characterized.For that purpose, PS1 and PS2 were synthesized according to literature procedures described elsewhere. 33esoporous silicas with distinct aspect ratios were obtained by varying the amount of CTAB (2.74 mM for MSNP-100, 5.49 mM for MSNP-200, 10.98 mM for MSNP-400, 13.72 mM for MSNP-500, Table 1) dissolved in the reacting mixture.In this reaction, CTAB serves as an organic template for the formation of mesoporous silica frameworks.The average size of the NPs was estimated by transmission electron microscopy (TEM) after dispersing the sample in water and drying it at ambient temperature.(Figure 2, Table 1).Table 1 indicates that the shape of the particles depends on the CTAB concentration used in the synthesis of mesoporous silicas.Hence, the higher the concentration of CTAB in the reaction mixture, the higher the aspect ratio (length:width) of the particles, which is in agreement with previous reports. 18,34 o compare shape-dependent behavior in HT-1376 and UM-UC-3 cancer cell lines, sphere-shaped MSNP-100 (117 ± 16 nm) and rod-shaped MSNR-500 (175 ± 39 nm x 36 ± 7 nm) were chosen and named, respectively as MSNP and MSNR.The PSs molecules were covalently bound onto the surfaces of MSNPs and MSNRs using (3-aminopropyl)triethoxysilane (APTS) as the molecular linker.Firstly, MSNPs and MSNRs were functionalized with APTS by dispersing the NPs in water and then adding a solution of APTS in ethanol to produce mesoporous silicas with amine terminal groups at the surfaces (MSNPs-NH2 and MSNRs-NH2).In the last step of the preparation, the grafting of Por: PS0, PS1, and PS2 on the surface of MSNPs-NH2 and MSNRs-NH2 was carried out in DMSO at 160 °C. 32,35 he final hybrid materials were washed with DMSO and EtOH until the typical Soret and Q bands of Pors were not observed in the rinsed solvent.The amount of Por covalently attached to NPs was calculated using UV-Vis spectrophotometry by subtracting the unreacted Por (from the rinse solvent) from Por taken into the reaction mixture.The highest amount of PS in the mesoporous silica samples was observed for MSNP-PS0 (26.4 nmol/mg, Table 2), which could be explained by the fact that PS0 is not bearing any sugar molecules.Thus, PS0 has the higher reactive para-fluorine atoms, which allows easier conjugation with nanomaterials compare to PS1 and PS2.The UV-VIS absorption spectra of all PSs and their nanoformulations were collected after dissolving and dispersing the samples in DMSO, respectively (Figures SI 3- ).The aminated NPs exhibited the presence of the N−H bending band at ~1600 cm -1 and broad bands in 2800 to 3800 cm -1 region, corresponding to stretching vibrations of primary amines, which indicated that the amino groups were bound onto the NPs surface. 36After covalent functionalization with Pors, a new band appeared at ~1590 cm - 1 corresponding to the C=C vibrational modes of Pors.The band at ~1700 cm -1 could be attributed to the bending vibration of the C=N of the Por ring.

Singlet oxygen generation study
The ability of the functionalized nanomaterials to generate 1 O2 was determined by an indirect chemical method in which 1,3diphenylisobenzofuran (DPBF) acts as a 1 O2 quencher.DPBF has an absorption maximum at 415 nm and forms a colorless endoperoxide product when it reacts with singlet oxygen (Figure SI 1).In this method, the ability of PSs or NPs to generate 1 O2 is measured by following the DPBF absorption decay at its maximum absorption (415 nm).
In this study, all solutions or suspensions for analysis were prepared in DMSO and stirred under irradiation for defined time intervals, at room temperature.Probes were exposed to the light of a 300 W halogen lamp.Incident light was filtered through an orange filter to take out light below 530 nm.Tetraphenylporphyrin (TPP) was used as a reference compound.Under these conditions, all MSNPs and MSNRs tested were photostable (Table SI 1).The 1 O2 generation was determined for all nanoformulations (MSNP-PS0, MSNP-PS1, MSNR-PS1, MSNP-PS2, MSNR-PS2) and the corresponding PSs (PS0, PS1, and PS2) (Figure 3).The free PS0, PS1, and PS2 were tested at concentrations of 0.5 µM and new nanomaterials were tested at concentrations of PS: 0.5 µM and 2.5 µM.Free PS 1 and PS 2 oxidized DPBF in the same This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins way.PS0 oxidized DPBF slightly less than PSs bearing sugar moieties.From the 1 O2 generation study, we could observe that free PS0, PS1, and PS2 oxidized DPBF almost in the same way (Figure 4).DPBF kinetic decay was similar in all sphere-shaped particles (MSNP-PS0, MSNP-PS1, MSNP-PS2).After 30 min of irradiation, these nanoformulations were able to reduce about 50% of DPBF with 2.5 µM of PS concentration.All sphere-shaped particles produced more 1 O2 than rod-shaped particles (Figure 4), suggesting that MSNPs could be more efficient than MSNRs in terms of cancer treatment in PDT.This could be due to the fact that in smaller NPs (NPs with smaller size aspect ratio, Table 1), there is a higher surface area.Thus, our MSNPs are more effective in 1 O2 production than MSNRs, under equal experimental conditions.

In vitro studies
In vitro studies were carried out into two human bladder cancer cell lines, HT-1376 and UM-UC-3.These cell lines express the glyco-binding proteins (glucose transporter, GLUT1 and the galactose-binding protein, galectin-1) in different levels, 29b which have a key role in the uptake and phototoxicity of galactodendritic porphyrinoids.29b GLUT1 is expressed at higher levels in HT-1376 cancer cells than in UM-UC-3 cells, whereas galectin-1 is expressed at higher levels in UM-UC-3 cells than in HT-1376 cells.29b

Cellular uptake of PSs and their nanoformulations
Preliminary uptake studies were performed using free PS (PS0, PS1, PS2).Bladder cancer cells were incubated in dark conditions with increasing concentrations of PS (2.5, 5, 10 µM prepared in PBS, maximum 0.5% DMSO v/v) for 4 h.Fluorescence spectroscopy studies demonstrated that PS1 accumulation was higher in HT-1376 than in UM-UC-3 cancer cells (Figure 5).On the other hand, the uptake of PS0 and PS2 was higher in UM-UC-3 than in HT-1376 cancer cells.The use of porphyrins with sugar moieties demonstrated higher uptake when compared with PS0, suggesting the sugar-tumor binding in cells expressing GLUT-1 and galectin-1.In the case of the bioconjugates PS1 and PS2, the uptake was dependent on the concentration of the PS and cell line.Among the studied PSs, the highest uptake was observed for PS1.For 10 µM of gluco-PS1, the intracellular accumulation was almost twice in both cancer cell lines compared to galacto-PS2.This could be caused by the fact that both HT-1376 and UM-UC-3 cancer cells have higher levels of glucose receptors when compared with galactose receptors.33a Thus, the presence of sugar moieties is important during the uptake of PSs by both cancer cells.Uptake studies were performed by incubating cancer cells with PS nanoformulations overnight.When the cells were incubated overnight with medium containing NPs solutions there was uptake dependent on the concentration of PS and shape of the NPs but not on the cell line (which was observed with free PSs).Interestingly, the uptake of MSNP-PS1 was almost three times lower when compared with the uptake of MSNR-PS1.On the other hand, although the uptake of MSNP-PS2 and MSNR-PS2 by HT-13-76 cells was similar, UM-UC-3 cells displayed a tendency to increase the uptake of MSNP-PS2.The presented results suggested that the aspect ratio of MSNPs could not be considered in these studies as a sole parameter to explain cellular uptake results.These studies pointed out that besides the shape of NPs, their chemistry (e.g.concentration of PS on the surface of NPs) and the type of cancer cells are crucial factors for predicting the uptake of NPs in cells (Table 3).

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The comparison between the amount of PS on the surface of NPs and the corresponding cell uptake value shows that there is an inverse trend relating to this data, i.e. as the amount of PS per mg of NP increases, there is a decrease in the cell uptake (Table 3, Scheme 2).Scheme 2. Schematic representation of suggested uptake process in cancer cells which is in inverse proportion to the concentration of PS on the surface of NP per mg of NP.The same mechanism is proposed for MSNPs and MSNRs.
In the present uptake studies, although the same amount of PS was used, the nanoformulations had different amounts of PS on their surfaces, thus different amounts of NPs were used in the experiments.When the concentration of PS on the NPs' surface was higher, the smaller was the amount of NPs (in mg) taken into the experiment resulting in a smaller uptake.In conclusion, the uptake depends not only on the concentration of PS on the surface of NPs but also on the amount of NPs (in mg or numbers of NPs).Furthermore, it was observed that the uptake of all new nanoformulations was higher in UM-UC-3 cancer cells (with a high level of galectin-1) when compared with HT-1376 cells (with a high level of GLUT1 protein).This could be a result of a better delivery process of MSNPs and MSNRs in UM-UC-3 than in HT-1376 cells.Interestingly, MSNP-PS0 was the only sample that accumulated better in both cancer cell lines than the corresponding non-immobilized Por (Figures 7, 8).The uptake of these sphere-shaped particles was ten times better in cancer cells when compared to free PS0.MSNP-PS0 (117 ± 16 nm) had the tendency to accumulate in cancer cells much more than smaller molecules of PS0.
From uptake results, it could be concluded that the cell uptake depends not only on the shape of nanocarriers but also on the type of PS, the concentration of PS on the surface of NPs, and the amount of NPs employed (in mg or numbers of NPs).

Dark toxicity and phototoxicity
The dark toxicity of PS0, PS1, PS2 and its corresponding nanoformulations (MSNP-PS0, MSNP-PS1, MSNP-PS2 and MSNR-PS1, MSNR-PS2) was evaluated using the well-known MTT assay (Figures SI 10, 11) in UM-UC-3 and HT-1376 bladder cancer cells.This colorimetric assay uses the ability of viable cells to reduce yellow 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), to purple formazan.Thus, it is a straightforward tool to determine cell metabolic activity.After overnight incubation of cancer cells (in dark) with NPs (0-20 µM of PS in RPMI medium) or 4 h incubation with PSs (0-10 µM in PBS buffer), none of the PSs or new NPs induced dark toxicity in cancer cells.This outcome is crucial in the PDT concept since the ideal therapeutic drug should not show cytotoxicity until photoactivation.
Following the confirmation of the uptake and non-dark toxicity of PSs and their new nanoformulations in UM-UC-3 and HT-1376 bladder cancer cells, their toxicity after light irradiation was evaluated using the MTT assay (Figures 7, 8).
In this study, UM-UC-3 and HT-1376 bladder cancer cells were incubated with PSs (0-10 µM in PBS buffer) for 4 h or with NPs (0-20 µM of PS in RPMI medium) overnight and then irradiated with an optical fiber emitting white light for 40 min (8.4 mW/cm 2 ).No cytotoxicity was observed in the sham irradiated, or left untreated (cells incubated in the absence of NPs).When the cells were incubated for 4 h in PBS containing PSs (0-10 µM in PBS buffer) there was phototoxicity dependent on the concentration of the PSs (Figure 7).PS1 and PS2 led to higher phototoxicity in both cancer cells than PS0 sample.This could be explained by the fact that these two PSs with sugar units presented much better uptake properties in HT-1376 and UM-UC-3 cancer cells compared to PS0.PS1 and PS2 presented slightly higher phototoxicity in UM-UC-3 cancer cells than in HT-1376 cancer cells.
After UM-UC-3 and HT-1376 bladder cancer cells were incubated overnight in medium with NPs (0-20 µM of PS) there was phototoxicity dependent on the concentration of the PSs(Figure 8).Although all new NPs induced phototoxicity in UM-UC-3 and HT-1376 bladder cancer cells in a concentrationdependent manner, the overall phototoxicity was higher with PSs than with NP formulations.In general, the phototoxicity was higher with MSNPs than with MSNRs (Table 3).All NPs presented higher phototoxicity in UM-UC-3 cancer cells than in HT-1376 cancer cells.Which could be the result of better uptake observed in these cell lines (Figure 8).Although the best cellular uptake was observed for MSNP-PS2 in both UM-UC-3 and HT-1376 bladder cancer cells, MSNP-PS1 with the lowest uptake results presented the highest phototoxicity in the same cancer cells lines (Table 3).
There is a clear relationship between the uptake behavior and phototoxicity results in terms of cell line.For all nanovehicles, a higher uptake was observed in UM-UC-3 than in HT-1376 cancer cells.The same behavior was spotted during phototoxicity studies.The phototoxicity was higher in UM-UC-3 than in HT-1376 cancer cells.Moreover, MSNPs showed higher phototoxicity in both cancer cells compared to MSNRs.

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Please do not adjust margins  Although MSNP-PS1 showed the lowest cellular uptake among other nanocarriers tested in both cancer cell lines, these NPs produced the highest phototoxic effect in both cancer cells.These results suggested that other factors are also relevant in the phototoxic effect of these NPs.For example, MSNP-PS1 has a higher concentration of PS on the surface of NPs [25 nmol of PS/mg of NP] comparing to the other tested nanomaterials, which eventually could result in better production of ROS.Other factors, such as subcellular localization of NPs or PSs also play a role in the outcome of PDT. 37The subcellular localization of NPs depends, for example, on the chemical nature of the NPs, particle size and shape, surface charge, targeting ligands, exposure duration as well as cell type. 38Thus, further studies are required to investigate the parameters that guide the PDT effect of these new NPs.Please do not adjust margins Please do not adjust margins Although all new NPs induced phototoxicity in UM-UC-3 and HT-1376 bladder cancer cells in a concentration-dependent manner, the overall phototoxicity was higher with free PSs than with NPs.Concerning the phototoxicity of all new nanovehicles, a second light irradiation treatment could increase in vitro photodynamic efficacy, as was already demonstrated by us. 39In the period between single and repeated irradiation, NPs could accumulate in different cellular organelles, which could enhance phototoxicity in both cancer cell lines.

Conclusions
The resulting new nanocarriers were fully described and their properties as new third-generation PSs for PDT against two bladder cancer cell lines, HT-1376 and UMUC-3 were proved.We revealed that MSNPs not only produced more 1 O2 during singlet oxygen study but also have higher phototoxicity in both cancer cell lines compared to MSNRs.The final results suggested that our new sphere-shaped nano-systems could be successfully used in PDT of bladder cancer which is the fourth most commonly diagnosed cancer 40 with a high rate of recurrence.Further advances towards improving the therapeutic efficacy of PDT treatment by these NPs and understanding the parameters that guide their PDT effect are under research.

Figure - 2
Scheme 1. Schematic preparation of the hybrid nanomaterials grafted with PSs showing the difference between the preparation of MSNP (A) and MSNR (B).

Figure 4 .
Figure 4. DPBF depletion in the presence of PS0, PS1, PS2 and its corresponding NPs (MSNP-PS0, MSNP-PS1, MSNP-PS2, and MSNR-PS1, MSNR-PS2) at different concentrations (0.5 and 2.5 µM) after 30 min of irradiation.Concentrations indicated for all nanomaterials refer to the equivalent concentration of non-immobilized Pors.Data are the mean value of at least three independent experiments and have a standard deviation lower than 3 %.

Table 1 .
Size of NPs estimated from TEM. Information on mean size and standard deviation was calculated from measuring more than 100 NPs in random fields of TEM grids.*NPs used as cores for further experiments.

Table 2 .
Concentration of PS on the surface of NP.

Table 3 .
Relation between the concentration of PS on the surface of NPs and its cellular uptake behavior with phototoxicity results (*the concentration is presented as the concentration of PS in the NP; **the uptake is presented for 20 µM of PS into each experiment; ***the phototoxicity is presented after 30 min of light irradiation as a MTT reduction (% from control)).