**6. Study of the photodynamic effects of selected photosensitizers on human biological samples**

#### **6.1. Incorporation of nanoparticles in human blood**

Significant nowadays research efforts are focused on finding new photosensitizers with antineoplastic activity and an acceptable toxicological profile. Although consistent informa‐ tion exists regarding PDT in solid tumors, relatively few data are available for PDT of blood cancers. Therefore, we carried out a comparative study on lymphoblastic K562 cells and human normal peripheral blood mononuclear cells (PBMC) treated at a density of 2 x 105 cells/mL with 5,10,15,20-tetra-sulphophenyl-porphyrin (TPPS4) and then irradiated with He-Ne laser light (λ = 632.8 nm). The following cell functions were investigated: viability, multiplication, RNA synthesis, total RNA levels and apoptosis. Human normal PBMC subjected to TPPS4 loading and laser-irradiation develop a different cellular response, their viability and prolif‐ erative capacity not being altered by experimental PDT. Accordingly, it appears that TPPS4 is a non-aggressive compound for cellular physiology and becomes cytotoxic only by irradiation; moreover laser-activated TPPS4 affects only cells that have a tumoral pattern [112].

There are several differences between lymphocytes obtained from healthy donors undergoing artificial activation in vitro and genuinely leukemic cells. The cells membrane structure is different in healthy and malignant cells, resting and stimulated cells can be compared using fluorescence spectroscopy [27]. In the concentration range 10–100 μg/mL, TPPS4 loaded for 24h in normal PBMC does not significantly alter cell viability (Figure 10).

**Figure 10.** The viability of human normal PBMC loaded with various concentrations of TPPS4 for 24h

Irradiation of K562 cells, either loaded or not loaded with TPPS4, drops off cellular viability (assessed by the Trypan Blue exclusion test 4hrs after irradiation) for both tumor cells and normal PBMC (Figure 11). TPPS4-loaded K562 cells are almost similarly affected by irradiation as the corresponding control. TPPS4 alone has no significant effect on cell viability.

**Figure 11.** The viability of K562 cells and human normal PBMC at 4h post-irradiation (assessed by the Trypan Blue exclusion test). C/TPPS4 = non-irradiated cells; Ci = irradiated unloaded cells; TPPS4i = irradiated loaded cells.

Human normal PBMC react differently at the PDT procedure than tumor cells. Their viability and capacity to incorporate uridine are not altered by laser-activating of TPPS4 loaded into cells (Figure 12).

4-5 ml of human peripheral blood were fast collected. Blood mononuclear cells (PBMC) were separated by gradient dental equipment screened using Histopaque 1077 (Sigma), washed twice with RPMI 1640 culture medium (Sigma) and then normalized to 105 cells / ml RPMI 1640 culture medium. Samples containing cell lines loaded with various concentrations of TPPS4 24h were investigated in flow cytometry fluorescence recorded at wavelengths above 670nm. Increasing the concentration of TPPS4 fluorescence intensity increases cell suspension directly proportional both total fluorescence and fluorescence maximum. In flow cytometry, the regions of lymphocytes, granulocytes and monocytes are clearly distinguished. The fluorescence of sample can be measured by using properly chosen filter. Fluorescence is Photodynamic Nanomedicine Strategies in Cancer Therapy and Drug Delivery http://dx.doi.org/10.5772/59624 271

**Figure 12.** The viability and multiplication rate of normal PBMC at 24h post-irradiation. C = non-irradiated unloaded cells; TPPS4 = non-irradiated loaded cells; Ci = irradiated unloaded cells; TPPS4i = irradiated loaded cells.

**Figure 10.** The viability of human normal PBMC loaded with various concentrations of TPPS4 for 24h

as the corresponding control. TPPS4 alone has no significant effect on cell viability.

Irradiation of K562 cells, either loaded or not loaded with TPPS4, drops off cellular viability (assessed by the Trypan Blue exclusion test 4hrs after irradiation) for both tumor cells and normal PBMC (Figure 11). TPPS4-loaded K562 cells are almost similarly affected by irradiation

**Figure 11.** The viability of K562 cells and human normal PBMC at 4h post-irradiation (assessed by the Trypan Blue exclusion test). C/TPPS4 = non-irradiated cells; Ci = irradiated unloaded cells; TPPS4i = irradiated loaded cells.

Human normal PBMC react differently at the PDT procedure than tumor cells. Their viability and capacity to incorporate uridine are not altered by laser-activating of TPPS4 loaded into

4-5 ml of human peripheral blood were fast collected. Blood mononuclear cells (PBMC) were separated by gradient dental equipment screened using Histopaque 1077 (Sigma), washed twice with RPMI 1640 culture medium (Sigma) and then normalized to 105 cells / ml RPMI 1640 culture medium. Samples containing cell lines loaded with various concentrations of TPPS4 24h were investigated in flow cytometry fluorescence recorded at wavelengths above 670nm. Increasing the concentration of TPPS4 fluorescence intensity increases cell suspension directly proportional both total fluorescence and fluorescence maximum. In flow cytometry, the regions of lymphocytes, granulocytes and monocytes are clearly distinguished. The fluorescence of sample can be measured by using properly chosen filter. Fluorescence is

cells (Figure 12).

270 Advances in Bioengineering

analyzed separately for each type of cells. A flow cytogram represents the graphical repre‐ zentation of light scattering vs. right angle scattering and help to determine lymphocytes and granulocytes. The aspect of flow cytometry (mean fluorescence intensities) could offer data about dye aggregation or dye interaction with cellular membrane [41,113, 114]. The number of stained cells decrease from 91.02% (for control cells), to 89,27% for 10 μg/ml TPPS4, 87,80 % for 20 μg/ml TPPS4 and 86.93% for 40 μg/ml TPPS4 (Figure 13.)

**Figure 13.** The flow cytometry results for the PMBC cells incubated with TPPS4 at different concentrations

Ion [115] and Frackowiak [27] evaluated the incorporation of sulphonated porphyrin TPPS4, which is better incorporated in cells by comparison with the non-sulphonated ones, most probably due to the spatial forms of J-aggregated forms (helical forms) able to penetrate the membrane and recovering to the monomeric forms after penetration. It is shown that during irradiation cells are actively destroyed (Figures 14-17), [116].

**Figure 14.** The absorption and emission properties of TPPS4

**Figure 15.** The aggregation equilibrium of TPPS4

For a longer photodynamic activity it is important to correlate the photophysical activity (the lifetimes of the excited states) with photochemical activity (singlet oxygen efficiency and the photodegradation rate. The aggregated forms (J-aggregates) of porphyrins favor the penetra‐ tion of the membranes. The porphyrins incorporation in cells is well correlated with singlet oxygen generation capacity. In monomeric forms the non-sulfonated porphyrins are better incorporated than the sulfonated ones which are better incorporated in leukocytes than in granulocytes (Figures 18,19), [113].

The non-sulphonated porphyrins (TNP and TPP) in DMSO-water mixture (0.05% DMSO) exist as monomeric and J-aggregated (dicationic - aggregated forms), the last ones with comparable fluorescence properties with the monomers [117].

Photodynamic Nanomedicine Strategies in Cancer Therapy and Drug Delivery http://dx.doi.org/10.5772/59624 273

**Figure 16.** The flow cytometry of different forms of TPPS4

**Figure 14.** The absorption and emission properties of TPPS4

272 Advances in Bioengineering

**Figure 15.** The aggregation equilibrium of TPPS4

granulocytes (Figures 18,19), [113].

fluorescence properties with the monomers [117].

For a longer photodynamic activity it is important to correlate the photophysical activity (the lifetimes of the excited states) with photochemical activity (singlet oxygen efficiency and the photodegradation rate. The aggregated forms (J-aggregates) of porphyrins favor the penetra‐ tion of the membranes. The porphyrins incorporation in cells is well correlated with singlet oxygen generation capacity. In monomeric forms the non-sulfonated porphyrins are better incorporated than the sulfonated ones which are better incorporated in leukocytes than in

The non-sulphonated porphyrins (TNP and TPP) in DMSO-water mixture (0.05% DMSO) exist as monomeric and J-aggregated (dicationic - aggregated forms), the last ones with comparable

**Figure 17.** The flow cytometry of leukocytes and granulocytes incorporated with TPP and TNP

**Figure 18.** Emission properties of TPP and TNP in DMSO:water and in lymphocytes

**Figure 19.** The correlation graphic of absorption and emission for TPP and TNP

## **6.2. Application of B2 Vitamin in liposomes for ophthalmologic diseases**

After PDT treatment was possible to see by angrography a rapid and complete vasooclusion (Figure 20), because the vessels were filled with erythrocytes and due to platelet aggregates. Another observed effects are: vacuolization of mitochondria and endoplasmatic reticulum (ESR), clumping of nuclear chromatin and finally, a subconjunctival hemrrhage, chemosis and cyanotic color of the neovascular areas (Figures 21 and 22).

**Figure 20.** Fluorescein angiography after PDT treatment with Riboflavin on rabbit. Dark area delimitates the irradiated sites where hypofluorescence indicates vascular occlusion

**Figure 21.** Angiofluorography for a patient with malignant melanoma before PDT treatment with Rb (left), and 6 months (right) after PDT treatment with Riboflavin.

From Figure 21 could be observed the dissapearance of peritumoral neovascularization and complete dissapearance of tumoral neovascularization and tumoral atrofia (6 luni). Angiog‐ raphy showed an immediate and complete vasooclusion (Figure 22).

Before PDT: VPD=1/8 fc, VOS =1/6 fc; 2 months after PDT: VOD=1/6 fc; VOS=1/12 fc; 4 months after PDT: VOD=1/10 fc; VOS = 1/8 fc; 10 months after PD: VOD=1/15 fc; VOS=1/20 fc

**Figure 22.** Macular degenerescence for a patient with neovascular membrane for right eye and atrofic pseudotumoral form at left eye

#### **6.3. Dermatological applications**

**Figure 18.** Emission properties of TPP and TNP in DMSO:water and in lymphocytes

274 Advances in Bioengineering

**Figure 19.** The correlation graphic of absorption and emission for TPP and TNP

cyanotic color of the neovascular areas (Figures 21 and 22).

sites where hypofluorescence indicates vascular occlusion

**6.2. Application of B2 Vitamin in liposomes for ophthalmologic diseases**

After PDT treatment was possible to see by angrography a rapid and complete vasooclusion (Figure 20), because the vessels were filled with erythrocytes and due to platelet aggregates. Another observed effects are: vacuolization of mitochondria and endoplasmatic reticulum (ESR), clumping of nuclear chromatin and finally, a subconjunctival hemrrhage, chemosis and

**Figure 20.** Fluorescein angiography after PDT treatment with Riboflavin on rabbit. Dark area delimitates the irradiated

PDT produces cytotoxic effects through photodamage of cellular organelles and biomolecules. It is known that PDT mediates tumor destruction by three mechanisms: direct cell killing, tumor vasculature damage and immune response activation. The combination of the three mechanisms is required to obtain long-term tumor control [122].

Actinic keratosis (AK) is the most common skin lesion with malignant potential, with a prevalence ranging from 11% to 25% in the Northern Hemisphere and from 40% to 50% in Australia [123]. There are some factors responsible for skin lessions (squamous cell carconoma (SCC)) as follows: UV light, heat and pollutants resulted from carbon processing. The most sensitive persons are Fitzpatrick I and II phototype and men by comparison with women [124]. In time, these lesions could remain unchanged, could spontaneously regress or could progress to SCC and further developing on the support of pre-existing actinic keratosis.

In our experimental approach, we have obtained from untreated skin biopsies a mean of 2.8x106 keratinocytes/cm2 skin with a mean of 65% viability. After therapy from the same skin region and from the same surface isolated keratinocytes were less than half compared to the control skin, displaying as well a lower viability (Figure 23).

**Figure 23.** Primary keratinocytes isolated from normal human skin before (control) and after PDT with TPPS4 (viability and proliferative capacity)

Primary keratinocytes were further cultivated until the culture could not be maintained. The proliferation capacity of primary keratinocytes extracted from PDT skin biopsies was signifi‐ cantly lower compared to control skin (Figure 23). Annexin-V and propidium iodide labelling of isolated keratinocytes after in vivo PDT compared to control keratinocytes yielded to the following values: control = 100 %; An-PI- = 10 %; An+PI- = 18 %; An+PI+ = 65 %.

The tested TPPS4 showed an effective *in vivo* destructive effect on keratinocytes in the patient with actinic keratosis doubled by a good clinical response (Figure 24).

**Figure 24.** The aspect of the skin with AK before PDT (left) and after PDT (right) with TPPS4
