Zn phthalocyanines loaded into liposomes: characterization and enhanced performance of photodynamic activity on glioblastoma cells

Journal Pre-proofs Zn phthalocyanines loaded into liposomes: characterization and enhanced performance of photodynamic activity on glioblastoma cells Mariana Miretti, Tomas C. Tempesti, César G. Prucca, Maria T. Baumgartner PII: DOI: Reference:

S0968-0896(20)30146-2 https://doi.org/10.1016/j.bmc.2020.115355 BMC 115355

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

9 September 2019 16 January 2020 31 January 2020

Please cite this article as: M. Miretti, T.C. Tempesti, C.G. Prucca, M.T. Baumgartner, Zn phthalocyanines loaded into liposomes: characterization and enhanced performance of photodynamic activity on glioblastoma cells, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115355

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Elsevier Ltd. All rights reserved.

Graphical Abstract

To create your abstract, type over the instructions in the template box below. Fonts or abstract dimensions should not be changed or altered.

Zn phthalocyanines loaded into liposomes: characterization and enhanced performance of photodynamic activity on glioblastoma cells

Leave this area blank for abstract info.

Mariana Miretti a,*, Tomas C. Tempesti a, César G. Prucca b and Maria T. Baumgartnera,* a INFIQC (CONICET), Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina b CIQUIBIC (CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina

Light

DPPC Cholesterol Phthtalocyanine (DMSO)

Pc-liposomes

T98G Cells

Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com

Zn phthalocyanines loaded into liposomes: characterization and enhanced performance of photodynamic activity on glioblastoma cells Mariana Mirettia,*, Tomas C. Tempestia, César G. Pruccab and Maria T. Baumgartnera,* a b

INFIQC (CONICET), Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina CIQUIBIC (CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina

ARTICLE INFO

ABSTRACT

Article history: Received Received in revised form Accepted Available online

Photodynamic therapy (PDT) is considered a promising strategy for cancer treatment. PDT utilizes light in combination with a photosensitizer (PS) to induce several phototoxic reactions. Phthalocyanines (Pcs), a second generation of photosensitizers, have been studied in several cancer models. Among these, Pcs, have become of interest for the treatment of glioblastomas which are one of the most common and aggressive forms of tumors of the central nervous system. Due to the lipophilic nature of Pcs and their limited solubility in water, Pcs can be loaded in liposomes. In this work, we characterized liposomes of ZnPc and TAZnPc and their effectiveness to photoinactivate glioblastoma cells, was evaluated. Both Pcs show an increase in their photosensitizing activity when they were administrated in Dipalmitoylphosphatidylcholinecholesterol liposomes compared to Pcs administrated in dimethylformamide.

Keywords: Photodynamic Therapy Zn-phthalocyanines Liposomes Glioblastoma cells Photosensitizer

1. Introduction Photodynamic Therapy (PDT) is a novel therapeutic strategy for the treatment of several diseases, among them, tumors. Over the past few decades, PDT has gained increasing attention in clinical applications for the treatment of numerous kind of solid tumors, such as skin, lung esophagus, bladder, prostate, brain, head and neck, bone, cervix, and ovarian carcinomas.1 The main advantages of PDT compared to the other classical treatments against cancer, such as chemotherapy, radiotherapy, and surgery are that PDT induces cellular death that will reduce tumor mass, while minimizing damage to surrounding tissue and possible undesirables side effects.2 PDT is based on the combination of light and a photosensitizer (PS), both components innocuous if they act separately.1,2 When the PS is excited by light in the presence of molecular oxygen a series of photochemical reactions take place, Type I and Type II photochemical reactions, leading to the generation of reactive oxygen species (ROS) or singlet oxygen (1O2) respectively and resulting in cellular injury and in consequence inducing cellular death.3-7 Intracellular cytotoxic effects of PDT start at the site where PS is accumulated since the migration capacity and mean life of the singlet oxygen is its very brief.8,9 PSs present differences in their degree of hydrophobicity, their structural asymmetries and in the net ionic charge. These differences are crucial for the successful incorporation into cells as well as for the subcellular accumulation.3

2009 Elsevier Ltd. All rights reserved.

Several PSs have been used in different studies showing their potential in PDT.4,10,11 Phthalocyanines (Pcs) are second generation of PSs which are characterized to have high photo and chemical stability, long-wavelength absorption with high extinction coefficients, and high singlet oxygen quantum yields.12 Pcs had been studied in several cancer models12 and have received particular attention due to their strong absorption band in the therapeutic window of 600–800 nm3,13,14 and Pcs have become of interest for the treatment of glioblastomas since their effects are limited to the site of irradiation and adjacent tissue.15 Glioblastoma are extremely aggressive tumors that are resistant to conventional therapies used to treat other tumor types.16 Usually few patients survive longer than 5 years, with a median survival of approximately 14.6 months.17,18 The location of these tumors in the brain difficult the elimination by surgery, and in addition, the blood-brain barrier limits the access of drugs to reach their site of action resulting in a reduction in the efficacy of the treatment.19,20 Currently, the treatment most commonly applied in glioblastoma involves surgical resection followed by chemo and radiotherapy.18 The choice of drug therapy for glioblastoma is still limited to a handful of compounds.21 The efficiency of Pcs in aqueous media decrease as consequence of their lipophilic nature and limited hydrophilicity, which leads to the formation of aggregates.22 To avoid aggregation, Pcs can be encapsulated using delivery systems such as liposomes, micelles, gold nanoparticles, carbon nanotubes, etc.6,23 Liposomes are lipidic vesicles with an aqueous

 Corresponding author. Tel.: +54-51-5353867; fax: +54-51-5353867; e-mail: [email protected], [email protected]

compartment in their interior which can incorporate hydrophilic drugs and a hydrophobic center of phospholipid bilayers in where lipophilic compounds can be accommodated.24 Different studies have suggested that Pcs administered into liposomes can improve the efficacy of PDT.25-28 Moreover, liposomes could be a promise for drug delivery in glioblastoma therapy since their clinical use has been approved in different applications and stand among most used nanotechnology for brain delivery.29,30 The aim of this work was to characterize different liposomal formulation of ZnPc and one of its derivatives, Zn(II)tetraminephthalocyanine, (TAZnPc) loaded into DPPC-cholesterol liposomes and evaluate their use in the PDT on glioblastoma cells.

2. Experimental section All starting materials were purchased from Sigma-Aldrich and used without further purification. DPPC and cholesterol (chol) were purchased from Avanti Lipids. Microwave monomode CEM-Discovery reactor was used in the synthesis of both Pcs. 2.1 Phthalocyanines ZnPc was obtained from phthalonitrile in DMF (dimethylformamide).31 TAZnPc was synthesized from TNZnPc Zn(II)tetranitro-phthalocyanine as previously we reported.32 See HNMR in supplementary material.

immediately after preparation and also after seven and fifteen days of storage in dark at 25°C. 2.3.4 Long term stability Liposomes freshly prepared were freeze-dried. 10%v/v Dextrose solution was utilized as lyoprotectant.36 Liposomes with or without dextrose were frozen with liquid nitrogen. The frozen samples were placed into the drying chamber (Freezone-6, Labconco). Drying was performed at a pressure of 12 Pa during 12 hours. Lyophilized liposomes were stored at 25°C for nine months in the dark until further treatments. The lyophilized liposomes (powder) were rehydrated using ultrapure water to its original volume. 2.3.5 Encapsulation efficiency The percentage of encapsulation efficiency (%EE) of Pcs was determined by ultracentrifugation technique.37 Liposomes were centrifuged (Beckman Coulter Optimal LE-80K Ultracentrifuge) at 45000 RPM for 2h at 4°C. Liposomes sediment was collected with phosphate buffer pH 7.4 and dimethylformamide (DMF) was added. Measurements in UV-Vis spectroscopy were performed for Pcs quantification. The Pcs %EE was calculated as follows:

2.2 Preparation of liposomes Liposomes were prepared according to a modified protocol of the ethanol injection procedure developed by Kremer et al.33 Pcs were dissolved in dimethyl sulfoxide (DMSO). Pcs solutions ~ 0.2 and 1 mM, were stored at 4°C. Lipid solution was prepared at concentration 7.68 mM of dipalmitoylphosphatidylcholine (DPPC) and 1.19 mM of cholesterol (chol) in absolute ethanol. For preparation of Pc-liposomes, 0.265 ml of each Pc solution was added to 0.735 ml of the phospholipid-cholesterol solution; 0.375 ml of this mixture was injected into 5 mL of pH 7.4 phosphate-buffered aqueous solution. The injection was performed at speed of 1µL/s with magnetic stirring at 55°C,34 temperature above the main transition temperature of DPPC (41°C).35 2.3 Liposomes characterization For liposomes characterization, various parameters were analyzed. The determinations included average mean size and polydispersity index analysis, encapsulation efficiency and stability study. 2.3.1 Size measurements of liposomes Size and polydispersity index of liposomes were determined by dynamic light scattering (DLS), in a Delsa Nano C instrument (Beckman Coulter) equipped with a 658 nm laser diode, at a 165° scattering angle. 2.3.2 Spectroscopic characterization of liposomes Absorption and fluorescence spectra were recorded at 25.0 ± 0.5 °C using 1 cm path length quartz cells on a Shimadzu UV1800 Spectrophotometer and on Agilent Cary Eclipse Fluorescence Spectrophotometer respectively. 2.3.3 Short term Stability Stability parameters such as fluorescence, absorbance, size and polydispersity index of Pc-liposomes were measured

λabs ZnPc: 670 nm, λabs TAZnPc: 702 nm The encapsulation efficiency was determined in triplicate. 2.4

Light source

Irradiation was performed using a 150 W/21V quartz halogen lamp (see the spectrum in supplementary material). The light was filtered through a 2.5 cm glass cuvette filled with water to absorb heat at 5cm of the lamp. The sample was at 15.0 cm of the lamp. The light fluence rate at the treatment site was 12.5 mW/cm2 and the light doses of 10 and 27 J/cm2, of irradiation respectively. The light dose was determined using a SE-9087 Digital Light Meter (Extech Instruments). 2.5

Cell culture conditions

T98G human glioblastoma cell line (ATCC American Type Culture Collection) was cultured in DMEM (Dulbecco’s modified Eagle medium) supplemented with 10% of fetal bovine serum (FBS) and penicillin/streptomycin as antibiotic (Gibco) at 37 °C in a humidified incubator with 5% carbon dioxide atmosphere. 2.6

Cell viability determination

Cell viability was determined using alamarBlue Cell viability reagent (Invitrogen) following the protocol provided by the manufacturer. After the experiment, cells were incubated during 4 hours at 37 °C with alamarBlue reagent (10% v/v) dissolved in DMEM supplemented with 10% FBS and antibiotics. Fluorescence measurements were done using a Biotek microplate reader with an excitation wavelength of 540–570 nm and the fluorescence emission reads at 580–610 nm. 2.7

Dark cytotoxicity

T98G cells were seeded in 96 well plates (7000 cells/well) and incubated for 24 hours in DMEM supplemented with 10% of FBS plus antibiotics. After, medium was discarded, and cells

were incubated in presence of different concentration of the Pcs in DMF and into liposomes dissolved in DMEM supplemented with 4% of FBS plus antibiotics during 18 hours in absence of light. Then, medium was discarded, and viability was determined using alamarBlue (Invitrogen) as describe above. 2.8

Photocytotoxicity

Cells were seeded in 96 well plates at a density of 7000 cells/well and grown overnight at 37°C in DMEM supplemented with 10% of FBS plus antibiotics. Then, cells were incubated for 18 hours at 37 °C with Pcs at desired concentration dissolved in DMEM supplemented with 4% of FBS plus antibiotics. Later, the culture medium was replaced with 10% FBS supplemented DMEM medium and cells were irradiated at the desired light dose (10 or 27 J/cm2). Viability was examined 24 hours after illumination using alamarBlue (Invitrogen) as described previously and expressed referred to control cells (cells nonirradiated without Pc). Results are the mean ± SEM of two independent experiments performed in triplicate.

heterogeneity) of liposomes formulation are descripted in Table 1. No changes in size and polydispersity index were observed in ZnPc-liposomes formulations. Mean size of ZnPcA was little higher than ZnPcB, 133 and 121 nm respectively. Liposomes presented similar size to reported previously.29 However, in TAZnPc-liposomes formulations mean size was increased, 102 nm TAZnPcB and 190 nm TAZnPcA, when liposome/Pc ratio was higher. PI was less than 0.3 in all cases, which indicated particles size were homogenously distributed. Table 1. Characterization of liposomes. Size, polydispersity index and % encapsulation efficiencya Molar rate Liposome DPPC: chol: Pc 20:6:0

139 ± 20 0.26±0.02

--

ZnPc

20:6:1

133 ± 25 0.26±0.01

74 ±2

20.6

20:6:0.2

121 ± 30 0.27±0.01

75 ±3

4.1

20:6:1

190 ± 22 0.29±0.02

74 ±3

18.9

20:6:0.2

102 ± 18 0.27±0.02

75 ±3

3.7

ZnPc

B

The development of new PSs for treatment of cancer in which other therapies have not resulted (as in glioblastoma) is a current challenge. In terms of PDT effectivity, the light penetration depth is a parameter to be taken into account. When light enters into the tissue, can be either scattered or absorbed. The degree of both processes depends on tissue type and light wavelength.3 Some PSs which are excited at longer wavelengths have the potential to increase treatment depths in PDT of tumors.38 The Pcs used in this work ZnPc and TAZnPc (Figure 1) have λ max of absorption higher than 670 nm coinciding with the range of therapeutical window.3 They were synthetized as described under Materials and Methods. NH2

H 2N N

N

N

TAZnPc

B

PI: Polydispersity index. a The results are presented as mean ±SD of 3 independent experiments.

For spectroscopic characterization, UV-Vis spectra were measured in each liposome formulation (in PBS), as well as in DMF solution. Control liposomes no show fluorescence emission or absorbance. Absorption spectra of ZnPc-liposomes formulations non-shown relevant changes than in DMF (Figure 2). A reduction in the absorbance was observed in liposomes (PBS) respect to DMF solution, and this is related with opacity of formulation44 and aggregation45. ZnPcB shows less absorbance than ZnPcA due to the final concentration of Pc in liposomes; furthermore, aggregation was observed in the higher liposome/Pc ratio (ZnPcA respect ZnPcB). a)

3

N

N ZnPc

N

N

A

N

Zn

N

N

N

N

N

N Zn

TAZnPc

H 2N

TAZnPc

NH2

Absorbance / au

N N

Final Encapsulation concentration Efficiency % µM

PI

Control A

3. Results and discussion

Mean diameter (nm)

Figure 1. Structure of phthalocyanines ZnPc and TAZnPc

2

ZnPc DMF (20.6M)

1

ZnPc A (20.6M)

Enhanced drug delivery to glioblastoma using liposomes represents a promising therapeutic strategy.30 We loaded Pcs into liposome modifying the reported methodology in which Pc were encapsulated into liposomes by injection method using pyridine as co-solvent34 and instead, we use DMSO as co-solvent. Intravenous DMSO´s LD50 is fifteen times less toxic than intravenous LD50 of pyridine in rat, 5.36 g/kg39 and 0.36 g/kg40, respectively. DMSO is incorporated in the handbook of pharmaceutical excipients,41 and is used in several regulated products for healthcare and drug delivery applications, including stabilizing product formulations, sustained-release applications, and for the delivery of medical polymers.42,43 In the USA DMSO is available for irrigation in the treatment of interstitial cystitis as a 50% solution.41 This change in co-solvent could improve the formulation and reduce the toxicity associated with the use of pyridine.

Figure 2. Absorption spectra of ZnPc in DMF and ZnPc loaded into DPPC-chol liposomes (PBS). a) ZnPc in DMF 20.6µM and ZnPcA 20.6µM. b) ZnPc in DMF 4.1µM and ZnPcA 4.1µM

In this work, different liposomes formulations were obtained after changing the liposome/Pc ratio. The average size and polydispersity index (PI) (i.e., the measurement of size

ZnPc emission intensity into liposomes show a decrease, respect to DMF solution comparable to observed in absorbance (see figure 3). ZnPcB shows higher fluorescence intensity than

0 400

500

600

700

Wavelength nm

b)

0.8

Absorbance / au

0.6

ZnPc DMF (4.1M)

0.4

ZnPc B (4.1M)

0.2 0.0

400

500

600

700

Wavelength nm

ZnPcA related to aggregation tendency of ZnPcA. Similar results were observed in TAZnPc liposomes (see supplementary data).

TAZnPcB in 7 and 15 days had the same behavior compared to those observed in ZnPcA. In all formulation, after 15 days after preparation, particle size was less than 250 nm. PI measurements at 7 and 15 days shown no differences compared with initial particle size, being in all cases ≤ 0.3. Regarding to absorbance and emission fluorescence of Pcs into liposomes, they were measured 7 and 15 days after initial measure (after preparation, day 1). Emission fluorescence and absorbance of ZnPc-liposomes as well as all formulations of TAZnPc-liposomes did not show relevant changes. These values indicated the physicochemical stability of formulations for 15 days.

Figure 3. Emission spectrum of ZnPc in DMF 0.5µM and ZnPc loaded into DPPC-chol liposomes in PBS 0.5µM (formulation A and B). ZnPc λex:640 nm.

Encapsulation efficiency (EE) is associated with the solubility of the drug within the liposome compartments. EE of Pcs into liposomes was determined by UV-Vis as describe under material and methods section. Table 1 shown %EE of each formulation of ZnPc and TAZnPc. In all cases, the values of %EE were upper than 70%, in agreement with literature data reported previously for other lipid formulations used in glioblastoma cell lines.29 The stability of liposomes was evaluated by comparing the initial particle size, polydispersity index, emission and absorbance with those stored during seven and fifteen days after preparation. Table 2 shows the results of stability parameters measurements. The initial mean size of ZnPcA was 133 nm, after 7 and 15 days of storage the mean size was 141 nm and 176 nm respectively. Mean size changes in ZnPcB, TAZnPcA and

In order to evaluate the long-term stability, liposomes were lyophilized and stored for 9 months in the dark at 25°C. To preserve the membrane integrity during lyophilization, sugars were used as lyoprotectant agents,46 due to their ability to interact with the phospholipid’s polar head groups and to stabilize the membranes.36 In this work, dextrose was used as lyoprotectant. Size, fluorescence emission and absorbance parameters were measured in lyophilized liposomes with and without lyoprotectant (see Table 3). Mean size of ZnPc-liposomes was ~623 nm and ~296 nm for liposomes lyophilized without and with dextrose, respectively. The average size of lyophilized TAZnPcA was 958 nm and 354 nm without and with dextrose, respectively; while TAZnPcB was 789 nm and 263 nm (without and with dextrose, respectively). Particle size was increased after freeze-drying respect to initial values (see Table 2) in liposomes lyophilized with and without dextrose. PI shows an increment in all formulation with and without lyoprotectant, which indicated heterogeneity of particle size distribution. Size increment of liposomes with lyoprotectant was lower in all formulations.

Table 2. Stability of liposomes: mean diameter, polydispersity index, emission and absorbance. Measured after preparation (day 1), day 7 and day15. Liposome

Mean diameter (nm)

Polydispersity index (PI) Day 1

Day 7

Day 15

Fluorescence emission (a.u.) Day 1 Day 7 Day 15

Absorbance (a.u.)

Day 1

Day 7

Day 15

Day 1

Day 7

Control

139 ± 20

145 ± 25

168 ± 24

0.26± 0.02 0.28± 0.01

0.29± 0.01

__

__

__

__

__

Day 15 __

ZnPcA (20.6µM)

133 ± 25

141 ±28

176 ± 30

0.26± 0.02 0.30± 0.02

0.29± 0.01

432

398

385

1.63

1.31

1.25

ZnPcB (4.1µM)

121 ± 30

138 ± 35

163 ± 32

0.27± 0.01 0.28± 0.01

0.28± 0.02

650

642

631

0.48

0.39

0.36

TAZnPcA (18.9µM)

190 ± 22

192 ± 29

241 ± 33

0.25± 0.03 0.27± 0.02

0.30± 0.03

129

125

123

0.20

0.19

0.19

TAZnPcB (3.7µM) 102 ± 18 117 ± 22 152 ± 29 0.27± 0.01 0.29± 0.02 0.29± 0.02 314 301 298 0.11 0.11 0.10 Day 1 (immediately after preparation). Day 7 and Day 15 (7 and 15 days after preparation, respectively). The results are presented as mean ± SD of 3 independent experiments.

Table 3. Long term stability of liposomes after lyophilization and 9 months of storage at 25°C in the dark, with and without dextrose as lyoprotectant. Liposome ZnPcA (20.6µM) ZnPcB (4.1µM) TAZnPcA (18.9µM) TAZnPcB (3.7µM)

Lyophilizated, after 9 months of storage Mean Fluorescence Absorbance diameter PI emission (a.u.) (a.u.) (nm) ± SD 623 ± 103 1.53±0.13 385 1.01 625 ± 98 1.60±0.18 415 0.34 958± 130 1.62±0.20 110 0.11 789 ± 114 1.57±0.15 272 0.13

Lyophilizated, after 9 months of storage with dextrose (lyoprotectant) Mean diameter (nm) ± SD

PI

Fluorescence emission (a.u.)

Absorbance (a.u.)

296 ± 45 287 ± 52 354 ± 68 263 ± 56

1.51±0.10 1.25±0.15 1.79±0.19 0.82±0.12

347 405 95 258

1.00 0.32 0.11 0.09

PI: Polidispersity Index. The results are presented as mean ±SD of 3 independent experiments.

After to determine the characteristics of different liposomal formulations, we evaluated their use in PDT on glioblastoma cells line in culture. Photocytotoxicity on T98G glioblastoma cells using TAZnPc and ZnPc, both in DMF solution compared with the liposomal formulation of DPPC and chol was assessed at different concentrations in combination with two light doses: 10 or 27 J/cm2. In all experiments, the incubation time was 18 hours. It has been reported that incubation time affect the cellular death mechanisms. Incubation time upper to 2 hours lead to apoptosis, meanwhile less time lead to necrosis.25,47,48,49 Both Pcs were innocuous at concentrations tested in the absence of light (See supplementary data). The comparison of effect of ZnPc loaded into liposomes (ZnPcA) and in DMF solution on T98G is shown in Figure 4.

A ~90% reduction in cell survival was observed using ZnPcA or ZnPcB at 0.5, 0.1 and 0.05 μM (concentration of ZnPc) combined with 10 or 27 J/cm2. The viability of glioblastoma cells after irradiation was similar with both ZnPc liposome formulation which suggests that a difference in ratio liposome/Pc doesn´t affect the behaviour of de Pc for PDT on glioblastoma cells. Both ZnPcA as ZnPcB were more effective than ZnPc in DMF solution. Liposomes formulation allow diminishing ~10 times the ZnPc concentration, from 0.5µM to 0.05 µM, to obtain the same reduction of cellular viability. Effects of TAZnPc loaded into liposomes or in DMF solution on T98G cells were determined, and is shown in Figure 6. Cytotoxicity of TAZnPc in DMF and into liposomes show a strong reduction of cell viability at 0.5 μM and higher light dose (27 J/cm2). The survival of T98G cells after PDT using TAZnPcA shown differences compared to DMF solution at lower concentrations, suggesting that the liposome formulation enhance the photodynamic capacity of TAZnPc.

Figure 4. Photocytotoxicity of ZnPc in DMF solution (bars without lines) and ZnPcA liposomes (bars with horizontal lines) on T98G cells at different concentrations, white 0.05 μM and grey 0.5 μM.

Photocytotoxicity of ZnPcA liposomes is shown in bars with horizontal lines. As can be observed a ~90% reduction in cell survival is attained using ZnPcA at a final concentration of ZnPc 0.5 and 0.05 μM, the same effect was obtained only with a concentration of 0.5 µM for ZnPc in DMF, combined with both light doses. These results suggest that ZnPcA formulation is more effective at lower concentrations than ZnPc in DMF. To determine whether cell photoinactivation depend of liposome/Pc ratio two ZnPc liposomes formulations, ZnPcA and ZnPcB, were evaluated. In Figure 5, the results of photocytotoxicity of both ZnPc liposomes formulations are shown.

Figure 5. Effect of two formulations of ZnPc liposomes at different concentration on T98G cells, white 0.05 μM, light grey 0.1μM and grey 0.5μM. ZnPcA bars with horizontal lines and ZnPcB bars with oblique lines.

Figure 6. Cytotoxic effects of TAZnPc DMF solution (bars without lines) and TAZnPcA (bars with horizontal lines) at different concentrations on T98G cells, white 0.05 μM, light grey 0.1 μM and grey 0.5 μM.

The cytotoxicity effect of TAZnPcA and TAZnPcB was compared in Figure 7. At higher concentration and light dose (0.5 μM and 27 J/cm2), TAZnPcA present a slight difference in their photoinactivation capacity compared to that observed for TAZnPcB. Previously, has been reported that Pcs concentration affect aggregation degree.26,50,51 Hence, we hypothesized that this slight difference of both formulations in the citotoxcity, could be due to the aggregation tendency of TAZnPc in formulation “A” with higher liposome/Pc ratio.

Figure 7. Effect of two formulations of TAZnPc liposomes at 0.5 μM on T98G cells. TAZnPcA bars with horizontal lines and TAZnPcB bars with oblique lines.

15. 16.

4. Conclusion In this work, we report the characterization and the enhancing in photocytotoxicity of TAZnPc and ZnPc delivered using a liposomal formulation of DPPC and chol compared with DMF solutions on glioblastoma cells. Pcs loaded into liposomes were successfully prepared by injection method using DMSO as co-solvent. Both ZnPc as TAZnPc show a direct relation between cell photoinactivation and concentration and light dose delivered. Pcs loaded into liposomes improved the efficiency of PDT on glioblastoma cells respect the Pcs in DMF solution. Therefore, liposomes formulation allows diminishing the concentration of Pcs to be used in PDT. These results suggest that ZnPcs delivered into liposomes could be applied as adjuvant in treatment of glioblastoma.

17. 18.

19. 20.

21.

References 1.

2. 3.

4, 5.

6.

7.

8.

9. 10. 11. 12.

13. 14.

Agostinis, P.; Berg, K.; Cengel, K. A.; Foster, T. H.; Girotti, A. W.; Gollnick, S. O.; Hahn, S. M.; Hamblin, M. R.; Juzeniene, A.; Kessel, D., Photodynamic therapy of cancer: an update. CA: a cancer journal for clinicians 2011, 61 (4), 250-281. Josefsen, L. B.; Boyle, R. W., Photodynamic therapy and the development of metal-based photosensitisers. Metal-based drugs 2008, 2008, 276109. Castano, A. P.; Demidova, T. N.; Hamblin, M. R., Mechanisms in photodynamic therapy: part one—photosensitizers, photochemistry and cellular localization. Photodiagnosis and photodynamic therapy 2004, 1 (4), 279-293. Abrahamse, H.; Hamblin, M. R., New photosensitizers for photodynamic therapy. The Biochemical journal 2016, 473 (4), 347364. Bacellar, I. O.; Tsubone, T. M.; Pavani, C.; Baptista, M. S., Photodynamic Efficiency: From Molecular Photochemistry to Cell Death. International journal of molecular sciences 2015, 16 (9), 20523-20559. Calixto, G. M.; Bernegossi, J.; de Freitas, L. M.; Fontana, C. R.; Chorilli, M., Nanotechnology-Based Drug Delivery Systems for Photodynamic Therapy of Cancer: A Review. Molecules 2016, 21 (3), 342-360. Mfouo-Tynga, I.; Abrahamse, H., Cell death pathways and phthalocyanine as an efficient agent for photodynamic cancer therapy. International journal of molecular sciences 2015, 16 (5), 10228-10241. Dysart, J. S.; Patterson, M. S., Characterization of Photofrin photobleaching for singlet oxygen dose estimation during photodynamic therapy of MLL cells in vitro. Phys Med Biol 2005, 50 (11), 2597-2616. Skovsen, E.; Snyder, J. W.; Lambert, J. D.; Ogilby, P. R., Lifetime and diffusion of singlet oxygen in a cell. J Phys Chem B 2005, 109 (18), 8570-8573. Sekkat, N.; van den Bergh, H.; Nyokong, T.; Lange, N., Like a bolt from the blue: phthalocyanines in biomedical optics. Molecules 2011, 17 (1), 98-144. Szaciłowski, K.; Macyk, W.; Drzewiecka-Matuszek, A.; Brindell, M.; Stochel, G., Bioinorganic Photochemistry:  Frontiers and Mechanisms. Chemical reviews 2005, 105 (6), 2647-2694. Li, X.; Zheng, B.-D.; Peng, X.-H.; Li, S.-Z.; Ying, J.-W.; Zhao, Y.; Huang, J.-D.; Yoon, J., Phthalocyanines as medicinal photosensitizers: Developments in the last five years. Coordination Chemistry Reviews 2019, 379, 147-160. Doiron, D. R.; Svaasand, L. O.; Profio, A. E., Light dosimetry in tissue: application to photoradiation therapy. Advances in experimental medicine and biology 1983, 160, 63-76. Wilson, B. C.; Jeeves, W. P.; Lowe, D. M., In vivo and post mortem measurements of the attenuation spectra of light in mammalian tissues. Photochemistry and photobiology 1985, 42 (2), 153-162.

22.

23.

24. 25.

26.

27.

28.

29.

30.

31. 32.

33.

de Paula, L. B.; Primo, F. L.; Tedesco, A. C., Nanomedicine associated with photodynamic therapy for glioblastoma treatment. Biophysical reviews 2017, 9 (5), 761-773. Krakstad, C.; Chekenya, M., Survival signalling and apoptosis resistance in glioblastomas: opportunities for targeted therapeutics. Molecular cancer 2010, 9, 135-149. Glaser, T.; Han, I.; Wu, L.; Zeng, X., Targeted Nanotechnology in Glioblastoma Multiforme. Frontiers in pharmacology 2017, 8, 166180. Stupp, R.; Mason, W. P.; van den Bent, M. J.; Weller, M.; Fisher, B.; Taphoorn, M. J.; Belanger, K.; Brandes, A. A.; Marosi, C.; Bogdahn, U.; Curschmann, J.; Janzer, R. C.; Ludwin, S. K.; Gorlia, T.; Allgeier, A.; Lacombe, D.; Cairncross, J. G.; Eisenhauer, E.; Mirimanoff, R. O., Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. The New England journal of medicine 2005, 352 (10), 987-996. Laquintana, V.; Trapani, A.; Denora, N.; Wang, F.; Gallo, J. M.; Trapani, G., New strategies to deliver anticancer drugs to brain tumors. Expert opinion on drug delivery 2009, 6 (10), 1017-1032. van Tellingen, O.; Yetkin-Arik, B.; de Gooijer, M. C.; Wesseling, P.; Wurdinger, T.; de Vries, H. E., Overcoming the blood-brain tumor barrier for effective glioblastoma treatment. Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy 2015, 19, 1-12. Ozdemir-Kaynak, E.; Qutub, A. A.; Yesil-Celiktas, O., Advances in Glioblastoma Multiforme Treatment: New Models for Nanoparticle Therapy. Frontiers in Physiology 2018, 9 (170)1-14. van Leengoed, H. L.; Cuomo, V.; Versteeg, A. A.; van der Veen, N.; Jori, G.; Star, W. M., In vivo fluorescence and photodynamic activity of zinc phthalocyanine administered in liposomes. British journal of cancer 1994, 69 (5), 840-845. Yin, R.; Agrawal, T.; Khan, U.; Gupta, G. K.; Rai, V.; Huang, Y. Y.; Hamblin, M. R., Antimicrobial photodynamic inactivation in nanomedicine: small light strides against bad bugs. Nanomedicine (London, England) 2015, 10 (15), 2379-2404. Torchilin, V. P., Recent advances with liposomes as pharmaceutical carriers. Nature reviews. Drug discovery 2005, 4 (2), 145-160. García, A. M.; de Alwis Weerasekera, H.; Pitre, S. P.; McNeill, B.; Lissi, E.; Edwards, A. M.; Alarcon, E. I., Photodynamic performance of zinc phthalocyanine in HeLa cells: A comparison between DPCC liposomes and BSA as delivery systems. Journal of photochemistry and photobiology. B, Biology 2016, 163, 385-390. García, A. M.; Alarcon, E.; Munoz, M.; Scaiano, J. C.; Edwards, A. M.; Lissi, E., Photophysical behaviour and photodynamic activity of zinc phthalocyanines associated to liposomes. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology 2011, 10 (4), 507-514. Huang, L.; Wei, G.; Sun, X.; Jiang, Y.; Huang, Z.; Huang, Y.; Shen, Y.; Xu, X.; Liao, Y.; Zhao, C., A tumor-targeted Ganetespib-zinc phthalocyanine conjugate for synergistic chemo-photodynamic therapy. European Journal of Medicinal Chemistry 2018, 151, 294303. Weijer, R.; Broekgaarden, M.; Kos, M.; van Vught, R.; Rauws, E. A. J.; Breukink, E.; van Gulik, T. M.; Storm, G.; Heger, M., Enhancing photodynamic therapy of refractory solid cancers: Combining second-generation photosensitizers with multi-targeted liposomal delivery. Journal of Photochemistry and Photobiology C: Photochemistry Reviews 2015, 23, 103-131. Silva, E.; Franchi, L.; Tedesco, A., Chloro-aluminium phthalocyanine loaded in ultradeformable liposomes for photobiology studies on human glioblastoma. RSC Advances 2016, 6 (83), 79631-79640. Bellavance, M. A.; Poirier, M. B.; Fortin, D., Uptake and intracellular release kinetics of liposome formulations in glioma cells. International journal of pharmaceutics 2010, 395 (1-2), 251259. Gerdes, R.; Lapok, L.; Tsaryova, O.; Wohrle, D.; Gorun, S. M., Rational design of a reactive yet stable organic-based photocatalyst. Dalton transactions 2009, (7), 1098-1100. Velazquez, F. N.; Miretti, M.; Baumgartner, M. T.; Caputto, B. L.; Tempesti, T. C.; Prucca, C. G., Effectiveness of ZnPc and of an amine derivative to inactivate Glioblastoma cells by Photodynamic Therapy: an in vitro comparative study. Scientific reports 2019, 9 (1), 3010-3025. Kremer, J. M.; Esker, M. W.; Pathmamanoharan, C.; Wiersema, P. H., Vesicles of variable diameter prepared by a modified injection method. Biochemistry 1977, 16 (17), 3932-3935.

34.

35.

36.

37.

38.

39. 40. 41. 42. 43. 44.

45.

46.

47.

48.

49.

50. 51.

Ginevra, F.; Biffanti, S.; Pagnan, A.; Biolo, R.; Reddi, E.; Jori, G., Delivery of the tumour photosensitizer zinc(II)-phthalocyanine to serum proteins by different liposomes: studies in vitro and in vivo. Cancer letters 1990, 49 (1), 59-65. Mabrey, S.; Sturtevant, J. M., Investigation of phase transitions of lipids and lipid mixtures by sensitivity differential scanning calorimetry. Proceedings of the National Academy of Sciences 1976, 73 (11), 3862-3866. Glavas-Dodov, M.; Fredro-Kumbaradzi, E.; Goracinova, K.; Simonoska, M.; Calis, S.; Trajkovic-Jolevska, S.; Hincal, A. A., The effects of lyophilization on the stability of liposomes containing 5-FU. International journal of pharmaceutics 2005, 291 (1-2), 79-86. Laouini, A.; Jaafar-Maalej, C.; Sfar, S.; Charcosset, C.; Fessi, H., Liposome preparation using a hollow fiber membrane contactor-application to spironolactone encapsulation. International journal of pharmaceutics 2011, 415 (1-2), 53-61. Lee, L. K.; Whitehurst, C.; Pantelides, M. L.; Moore, J. V., In situ comparison of 665 nm and 633 nm wavelength light penetration in the human prostate gland. Photochemistry and photobiology 1995, 62 (5), 882-886. Willson, J. E.; Brown, D. E.; Timmens, E. K., A toxicologic study of dimethyl sulfoxide. Toxicology and Applied Pharmacology 1965, 7 (1), 104-112. Leslie, G. B.; Hanahoe, T. H. P.; Ireson, J. D.; Sturman, G., Some pharmacological properties of pyridine. Pharmacological Research Communications 1973, 5 (4), 341-365. Rowe, R. C.; Sheskey, P.; Quinn, M., Handbook of pharmaceutical excipients. Libros Digitales-Pharmaceutical Press: 2009. McKim, A.; Strub, R., Dimethyl sulfoxide USP, PhEur in approved pharmaceutical products and medical devices. Pharmaceutical Technology 2008, 32 (5), 74. Strickley, R. G., Solubilizing excipients in oral and injectable formulations. Pharmaceutical research 2004, 21 (2), 201-230. Oliveira, C. A.; Machado, A. E.; Pessine, F. B., Preparation of 100 nm diameter unilamellar vesicles containing zinc phthalocyanine and cholesterol for use in photodynamic therapy. Chemistry and physics of lipids 2005, 133 (1), 69-78. Diz, V. E.; Gauna, G. A.; Strassert, C. A.; Awruch, J.; Dicelio, L. E., Photophysical properties of microencapsulated phthalocyanines. Journal of Porphyrins and Phthalocyanines 2010, 14 (03), 278-283. Li, B.; Li, S.; Tan, Y.; Stolz, D. B.; Watkins, S. C.; Block, L. H.; Huang, L., Lyophilization of cationic lipid-protamine-DNA (LPD) complexes. Journal of pharmaceutical sciences 2000, 89 (3), 355-364. Fabris, C.; Valduga, G.; Miotto, G.; Borsetto, L.; Jori, G.; Garbisa, S.; Reddi, E., Photosensitization with zinc (II) phthalocyanine as a switch in the decision between apoptosis and necrosis. Cancer research 2001, 61 (20), 7495-500. Soriano, J.; Villanueva, A.; Stockert, J.; Cañete, M., Regulated necrosis in HeLa cells induced by ZnPc photodynamic treatment: A new nuclear morphology. International journal of molecular sciences 2014, 15 (12), 22772-22785. Soriano, J.; Villanueva, A.; Stockert, J. C.; Canete, M., Vehiculization determines the endocytic internalization mechanism of Zn(II)-phthalocyanine. Histochemistry and cell biology 2013, 139 (1), 149-160 Isago, H., Optical spectra of phthalocyanines and related compounds. Springer: 2015. Valduga, G.; Reddi, E.; Jori, G.; Cubeddu, R.; Taroni, P.; Valentini, G., Steady state and time-resolved spectroscopic studies on zinc(II) phthalocyanine in liposomes. Journal of Photochemistry and Photobiology B: Biology 1992, 16 (3), 331340.

Acknowledgments This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) of Argentina, SECYT UNC and FONCYT. MM. acknowledges CONICET for his doctoral fellowship.

Supplementary Material

Highlights 

DPPC-col liposomes loaded with ZnPc and TAZnPc were efficiently synthesized.



Liposomes were synthesized using co-solvent less toxic than conventional used.



ZnPc-liposomes

or

TAZnPc-liposomes

efficiently photoinactivate Glioblastoma cells in culture. 

Liposomes formulation allows diminishing the concentration of Pcs to be used in PDT.