Photocytotoxicity studies of polyamidoamine (PAMAM) dendrimer and its glucoheptoamidated derivative as delivery drug systems in local PUVA photochemotherapy

Katarzyna Borowska1, Sebastian Radej2, Ryszard Galus3, Dariusz Śladowski4, Wiktor Paskal5, Dawid Mehlich5, Stanisław Wołowiec6

1Department of Histology and Embryology with Experimental Cytology Unit, Medical University of Lublin, 11 Radziwiłłowska Str., 20–080 Lublin, Poland, 2Department of Human Anatomy Medical University of Lublin 4 Jaczewskiego Str., 20-090 Lublin, Poland, 3Department of Histology and Embryology, Center for Biostructure, Medical University of Warsaw, 5 Chałubinskiego Str., 02-004 Warsaw, Poland, 4Departament of Transplantology and Central Tissue Bank Medical University of Warsaw 5 Chalubinskiego Str., 02-004 Warsaw, Poland, 5Department of Histology and Embryology, Laboratory of Centre for Preclinical Research, Medical University of Warsaw, Banacha 1b, 02-097 Warsaw, Poland, 6Centre for Innovative Research in Medical and Natural Sciences, Faculty of Medicine, University of Rzeszów, 1a Warzywna Str., 35-310 Rzeszów, Poland.

 

Corresponding author: Prof. Katarzyna Borowska, E-mail: k_borowska@wp.pl

 

Submission: 11.10.2017; Acceptance: 15.10.2017

DOI: 10.7241/ourd.2017e.12

How to cite this article: Borowska K, Radej S, Galus R, Śladowski D, Paskal W, Mehlich D, Wołowiec S. Photocytotoxicity studies of polyamidoamine (PAMAM) dendrimer and its glucoheptoamidated derivative as delivery drug systems in local PUVA photochemotherapy. Our Dermatol Online. 2017;8(4e):e1.

ABSTRACT

8-methoxypsoralen (8-MOP) given topically in local PUVA photochemotherapy poorly penetrates through the skin to its deeper layers which restricts its effectiveness.  Poly(amidoamine) (PAMAM) dendrimers enhanced 8-MOP permeation through the skin. The aim of this study was to investigate photocytotoxicity of the third generation PAMAM dendrimer (G3) and its glucoheptoamidated derivative (GD), a new delivery drug systems in local PUVA photochemotherapy. PAMAM dendrimer third generation (G3) as well as its glucoheptoamidated derivative (GD) were synthesized and used as macromolecular host to encapsulate 8-MOP. In our study, this is the first time we investigated the potential cytotoxicity of 8-MOP encapsulated in GD (4MOP@GD) to NIH-3T3 cells by a colorimetric method MTT compared to cytotoxicity of 4MOP@G3, 8-MOP and G3. The 4MOP@GD may be used as pharmaceutical substances applied systemically or topically which is why we tested the photoreactivity and estimated the phototoxic potential by the method based on the protocol of the validated NIH-3T3 NRU phototoxicity test. We confirmed our suspicions of lower toxicity 4MOP@GD compared to others testing substances. Increasing the concentration of the 4MOP@GD 200 times more than 4MOP@G3 maintains the cell viability at identical level. We are convinced that the use of the 4MOP@GD have a significant importance in the therapy by increasing the concentration of 8-MOP in the application site, together with increase safety for the patient. Using 8-MOP encapsulated in glycodendrimer at 4:1 stoichiometry (4MOP@GD) in local photochemotherapy with long-wavelength radiation makes possible to decrease dose UVA irradiation. The lower dose of local UVA results reducing risk of side effects like photocarcinogenesis. The 4MOP@GD is less cytotoxic than the encapsulate of 8-MOP in PAMAM G3 dendrimer (4MOP@G3). 4MOP@GD makes it possible to achieve concentrations five times higher than 4MOP@G3 in local PUVA photochemotherapy.

Key words: PUVA, 8-methoxypsoralen, 8-MOP, Poly(amidoamine) dendrimers, PAMAM.

 

INTRODUCTION

PUVA (Psoralen-UV-A) treatment is photochemotherapy that combines psoralen with long-wavelength radiation (UVA). The psoralen mainly used in PUVA is 8-methoxypsoralen (8-MOP). 8-MOP can be administered orally (systemic PUVA) or topically (local PUVA). Oral administration of 8-MOP may be associated with side effects. To reduce side effects of systemic PUVA, topical PUVA therapy has been developed. 8-MOP given topically poorly penetrates through the skin to its deeper layers which restricts its effectiveness. Consequently we developed the carrier which enables more efficient 8-MOP penetration through skin and helps to achieve appropriate concentration of 8-MOP in the deeper layer of the skin [1-3]. Poly(amidoamine) (PAMAM) dendrimers are good carriers for this purpose. PAMAM dendrimers were the first complete dendrimer family to be synthesized, characterized and commercialized [4,5]. Our previous study showed that PAMAM dendrimers enhanced 8-MOP permeation through the skin [1-3]. The aim of this study was to investigate photocytotoxicity of the third generation PAMAM dendrimer (G3) and its glucoheptoamidated derivative (GD), a new delivery drug systems in local PUVA photochemotherapy.

MATERIAL AND METHODS

Chemical syntheses
Dendritic hosts 
Polyamidoamine dendrimer PAMAM of third generation (G3) was synthesized according to protocol described in [5] and used as a host for 8-methoxypsoralen (8-MOP). Another host compound was obtained from G3 in reaction with excess of D-glucoheptono-1.4-lactone (GHL) in modified procedure described in [6,7] as follows: 242.2 mg (1160 μmoles) dissolved in 5 ml dimethylsulfoxide (DMSO) was added stepwise into 150 mg G3 (28.9 μmole) dissolved in 2 ml dimethylsulfoxide (DMSO). The mixture was stirred vigorously at 50 °C for 1 hour, transferred into cellulose dialyzing tube (MW cut off = 1.5 kDa) and triple dialyzed against water for three days. The solvents were removed under reduced pressure. The obtained yellow solid (360 mg, 26.5 μmoles) was identified by the 1H NMR spectroscopy as glycodendrimer (GD) of third generation with all 32 amine groups of G3 substituted with D-glucoheptoamide. Based on the formula the yield was 91.7 %. The 1H NMR spectrum of GD is presented at Figure 1.

Figure 1: The 1H NMR spectrum resonances of GD in DMSO-d6. The residual solvent resonance at 2.50 ppm was used as internal chemical shift reference. The glucoheptoamide (gh) substituent resonances (4.1 – 3.4 ppm region) are separated from broad resonances from PAMAM G3 core (3.3 – 2.1 ppm region). Integration of gh signals versus one of G3 multiplets of integral intensity 128H indicates the presence of 32 gh substituents. For detail analysis of the 1H NMR spectrum of this compound as well as low molecular analogues of PAMAM G0 substituted with one, two, and three gh substituents see (Uram et al., 2017).

Host-guest complexes of 8-MOP encapsulated in dendrimers; MOP@D
Host-guest complexes of 8-MOP were prepared at 10 umolar scale by addition of stock solution of 8-MOP in methanol (0.02 mol/dm3) into methanolic solution of G3 according to the stoichiometry of 4 equivalents of 8-MOP per one equivalent of the host. The methanol was removed from obtained solutions by rotary evaporation, the residue was dried overnight under high vacuum (0.2 mm Hg). The obtained homogenous oil of 4MOP@G3 was dissolved in 10 ml of water and used as stock solution for biological studies. The 8-MOP encapsulated in glycodendrimer, 4MOP@GD was obtained in similar way except the amount of the methanol to dissolve 86.2 mg of GD was about 50 ml. The obtained solid host-guest complexes were perfectly soluble in water. 
MTT cell viability assay
NIH-3T3 (European Collection of Cell Cultures ECACC, Salisbury, Wiltshire) cells were seeded in 96-well plates at a density 1 x 104 cells per well in 100 μl of DMEM (Biochrom GmbH, Berlin, Germany) supplemented with 10% FBS. After 24h, the medium was aspirated from each well and replaced by 100 μl of medium with nine different concentrations of G3 and 4MOP@G3 (200 μM – 0.78 μM reconstituted logarithmically from 1 mM stock). GD and 4MOP@GD concentrations were set to 750 μM – 7.8125 μM. The cells were incubated for 24h, then the medium was aspirated and cells were treated with 20 μL of MTT solution (5mg/ml dissolved in PBS; thiazolyl blue tetrazolium bromide; Sigma-Aldrich, Saint Louis, MO, USA), along with 80 μL of culture medium for additional 2h. Then, 100 μl of isopropanol was added to each well to dissolve the formazan crystals generated by living cells and the absorbance of each well was measured using a microplate reader at a wavelength of 540 nm (FLUOstar OMEGA, BMG Labtech, Ortenberg, Germany). All experiments were performed in triplicate, and the relative cell’s viability (%) was expressed as a percentage relative to the untreated control cells. The half maximal inhibitory concentration (IC50) was calculated with GraphPad Prism (GraphPad Software, La Jolla, CA USA). 
Phototoxicity tests
Tested substances were diluted in PBS with calcium (Sigma Aldrich). Phototoxicity testing was based on the protocol of the validated NIH-3T3 NRU phototoxicity test [8]. NIH-3T3 NRU phototoxicity test is based on a comparison of the cytotoxicity of a chemical when tested in the presence and in the absence of exposure to a non-cytotoxic dose of simulated solar light. Viability of the cells is measured using Neutral Red Uptake (NRU) test, which is based on the decreased uptake and binding of NR by damaged/dead cells. Dr Hoenle Sollar Simulator was used as a light source identical to that used in the validation trial. 
A permanent mouse fibroblast cell line, Balb/c NIH-3T3 (European Collection of Cell  Cultures ECACC, Salisbury, Wiltshire) DMEM (Dulbecco's Modified 2/15 OECD/OCDE  432 Eagle's Medium) supplemented with 10% new-born calf serum, 4 mM glutamine,  penicillin  (100 IU), and streptomycin (100 µg/mL). The NIH-3T3 cells were cultured in 96-well plates (104 per well) for twenty-four hours before treatment with tested substances. Serial dilutions of the tested substances were prepared on a 96 plates (eight dilutions) and transferred using multichannel pipette to the plates seeded with target cells. The plates were exposed to UVA (5 J/cm²). A parallel culture plate exposed to chemicals was not irradiated (kept in the dark). After irradiation chemicals were washed from the plates and NRU test was performed. The cells were incubated in the culture medium containing neutral red for three hours and then washed. The optical density of NRU (540nm) was measured with a microplate reader. For each chemical, measurements were performed on three independent occasions. From each run, ID50 values for irradiated (UV) and non irradiated samples were calculated using “Excell Curve fitting add on” (SRS1 software). 
Phototoxic Factor (PIF) was calculated for each substance according to formula PIF= ID50(Dark)/ID50 (UV).

RESULTS

The chemical syntheses
The chemical syntheses of host dendrimers were performed according to known procedures (Fig. 1). Two PAMAM dendrimer of third generation (G3; MW = 6909 Da, 3.6 nm diameter sized) with 32 amine groups was used as host to encapsulate 4 equivalents of 8-MOP according to protocol described earlier [1]. Another host molecule was a glycodendrimer (GD) obtained by the exhaustive amidation of G3 with D-glucoheptono-1.4-lactone [9]. The 4MOP@G3 and 4MOP@GD encapsulates, containing four equivalents of 8-MOP per macromolecular dendrimeric carrier were tested for cytotoxicity and phototoxicity (vide supra).
Cytotoxicity of the dendrimers and encapsulates of 8-MOP in dendrimers; MOP@D
MTT cell viability assay revealed heterogeneous results on cytotoxicity of the examined particles. G3 4MOP@G3 showed the highest cytotoxicity (Figs. 2B and 2D), IC50=11.43 µM. However, G3 – without active agent showed almost 4 times lower cytotoxicity. Following, the cytotoxic effect of the Gh30 and its methoxypsoralen-conjugated derivate was ambiguous. Cells viability decreased only in the highest range of concentrations of both particles (750 µM and 500-750 µM respectively, Figs. 2 C – D). IC50 of Gh30 was estimated to 2942 µM and Gh30 MOP to 2195 µM for NIH-3T3 murine fibroblast cell line.

Figure 2: (A-D) Graphs comprising results of relative MTT cell viability assay on NIH-3T3 cell line for the particles G3, 4MOP@G3, GD and 4MOP@GD, respectively.


Photocytotoxicity of the dendrimers and encapsulates
Results of the NRU phototoxicity test are presented in Table 1 and Figure 3. 
According to the prediction model of the test PIF values >5 indicate phototoxic potential of the tested substance. It has been clearly demonstrated in the case of 8-MOP (PIF>125).
G3–non phototoxic (PIF=1.3; PIF < 5)
4MOP@G3–phototoxic (PIF=8.9; PIF >5)
4MOP@GD-phototoxic (PIF>14.3)

Table 1: Results of the NRU phototoxicity test.
 
Figure 3: (A-D) Graphs of cell viability in phototoxicity response assay on NIH-3T3 cell line for 8-MOP, G3, 4MOP@G3, 4MOP@GD.

DISCUSSION

PUVA photochemotherapy is effective in the treatment of various skin diseases, e.g. psoriasis [10,11], vitiligo [11], atopic dermatitis [12], lichen planus [13], graf versus host disease (GVHD) [14], scleroderma [15] and cutaneous T-cell lymphoma [16]. The most commonly clinically used psoralen is the furanocoumarin, 8-methoxypsoralen (8-MOP), which is used in systemic and topical PUVA photochemotherapy. However, systemic use 8-MOP is sometimes limited by acute gastrointestinal side effects (nausea and vomiting) and neurological (giddiness and headache). Patients undergoing oral 8-MOP should be instructed to protect their eyes from UV light. Long term side effects include glaucoma and photocarcinogenesis (melanoma and non-melanoma skin cancer). The risk of photocarcinogenesis increases when PUVA requires a high dose of UVA radiation. To reduce dose of UVA and side effects of systemic PUVA therapy, local PUVA has been developed. Unfortunately 8-MOP given topically poorly penetrates through the skin to its deeper layers. This is particularly important during therapy diseases like scleroderma or cutaneous T-cell lymphoma. PAMAM dendrimers are effective enhancers of transdermal delivery of 8-MOP resulting in higher concentration of 8-MOP in epidermis and dermis in relation to standard 8-MOP solution [1-3]. Higher concentration 8-MOP in tissue results in the clinical use of lower dose of UVA radiation. The lower dose of local UVA results in reducing the risk of photocarcinogenesis in PUVA therapy. Therefore, the high level of 8-MOP guarantees successful UVA therapy, with the main role of the carrier which will guarantee safety of the therapy. The carriers must have local effects and be free of sides effects. The ideal carrier wouldn't interfere with morphology of the cells. Poly(amidoamine) (PAMAM) dendrimers are a good carrier for this purpose. 
Dendrimeric globular macromolecules like PAMAM G3 (G3) and its glucoheptoamidated derivative (GD) are able to encapsulate 9-MOP for up to 6 molecules of a guest at one molecule of the host [1]. The fluorescent labeled host molecules G3F and GDF were demonstrated to enter fibroblast cells, accumulated eventually in nuclei and induced apoptosis at 5 µM and 50 µM concentrations, respectively [7,9]. Lower cytotoxicity of GD in comparison with G3 prompted us to use GD as the host molecule for 8-MOP. We have replaced G3 with GD according to the concept of eradication of terminal cationic amine groups, which seems responsible for toxicity of PAMAM dendrimers [17]. We have loaded both hosts with 4 equivalents of 8-MOP. The 4MOP@GD and 4MOP@G3 encapsulates were water soluble up to 3 mM concentration. These stock solutions were used to prepare the media for cell cultures. The equilibrium between encapsulate and free 8-MOP, expressed by simple equilibrium reaction: 4MOP@D – 3MOP@D + MOP (where D – the G3 or GD dendrimer, MOP = 8-MOP) enables to release 8-MOP photosensitizer. No precipitation of 8-MOP was noticed upon dilution of stock solution, thus we have assumed that free 8-MOP concentration was kept below the value of its solubility, i.e. 1×10-2 mM. Although no concentration of free 8-MOP could be estimated, the availability of the photosensitizer for photochemical activity was studied for such solution in function of encapsulate concentration. Our investigations about the 4MOP@G3 and 4MOP@GD complexes started with indication of the cell viability using the MTT cytotoxicity assay on the model cell line NIH-3T3. The study confirmed our suspicions of lower toxicity GD compared to G3 (IC50-2195 µM and 11.43 µM, respectively). This gives the opportunity to increase the concentration of the 4MOP@GD more than 200 times while maintaining the cell viability at identical level as 4MOP@G3.
The aim of our study was to investigate photocytotoxicity of the third generation PAMAM dendrimer (G3) and its glucoheptoamidated derivative (GD), a new delivery drug system for local PUVA therapy. According to our knowledge this is the first observation indicating the cytotoxicity of PAMAM G3 dendrimer and its GD derivative and their host-guest complexes with the 8-MOP guest.  Phototoxicity testing was based on the protocol of the validated NIH-3T3 NRU phototoxicity test [8]. That will reflect the conditions of similar cells and in vivo. The most important question was regarding the optimum concentrations which could be used in the therapy. It is known that despite the treatment effects of PUVA, it has phototoxicity, mutagenic and carcinogenic effects on various physiological pathways and cell components [18,19]. In addition, PUVA induces chromosomal aberrations, sister chromatid exchanges, mutations, damage and cross-links in DNA of human cells in vitro [20]. Considering the above, we define the parameter IC50 and PIF for the NIH-3T3 cell line. The 4MOP@GD makes possible to achieve concentrations five times higher than 4MOP@G3, while maintaining the viability of the cells at the level greater than 90% in the dark. We believe that using the 4MOP@GD gives opportunity to increase concentration of the 8-MOP five times or decrease UVA irradiation for the patients. The effect is similar to 8-MOP reaction with NIH-3T3 cell line. In conclusion we convinced that the use of the 4MOP@GD have a significant importance in the therapy by increasing the concentration of 8-MOP in the application site, together with increase safety for the patient. The 8-MOP is a strong factor that triggers apoptosis of different types of cells in different levels of apoptosis. Franklin at al. [21] found that 8-MOP and UVA leads to an increase of arginase-1 release into the medium after 24h by neutrophils. Together with pro apoptotic ability of 8-MOP this may lead to central and peripheral tolerance by induction of chronic stimulation CD4+ T cells. The CD4 can activate macrophages, tissue fibroblasts and B cells. Macrophages can further enhance fibroblast activation. 8-MOP induces apoptosis under UVA irradiation in concentration of 3.9 µM- result of 68% nonviable cells; with no effect on NIH-3T3 cells in dark conditions (114% of control). The optimum concentration of 8-MOP should be over 65 µM as its results in degradation in cells viability below 100%. Roberts at al. found that G3 dendrimers affected cell growth only at high concentrations more than 1.0 mM [22]. In our study, the G3 critical concentration for NIH-3T3 (control cells) is 7.8 µM and 0.9 µM for cells irradiated UVA. Naha et al. [23] found that intracellular ROS generation by PAMAM dendrimers is clearly one of the toxic pathways and a clear generation dependence of intracellular increased ROS production. This response indicates that the cationic surface amino groups play a direct role in the production of ROS.  In addition, Lee et al. [24] demonstrated that the ROS, cytokine production, and cytotoxicity cascade could be initiated from the internalization of PAMAM dendrimers and their localization in the mitochondria. Therefore, although there may be external stress leading to some degree of indirect toxic response, it is proposed that the principal response is a direct result of internalization of the nanomaterials. In summary it is critical to discover the concentration of G3 which will eliminate its effects on apoptosis induction in target cells, otherwise modification of G3 structure in order to prevent mitochondrion damage and apoptosis induced by production of ROS. Our research shown that 4MOP@G3 has apoptosis activity in all investigated concentration for control cells and cells with UVA irradiation. The modification of G3 carrier to glycodendrimer GD will allow the concentration of 125 µM glucoheptoamidated 4MOP@GD to be used in the treatment with no effects on 3T3 cells  (result of 101% control group) and degradation of 71% UVA irradiated cells.

CONCLUSION

Using 8-MOP encapsulated in glycodendrimer (4MOP@GD) in local photochemotherapy with long-wavelength radiation makes possible to decrease the dose of UVA irradiation. The lower dose of local UVA results reducing risk of side effects like photocarcinogenesis. The 4MOP@GD is less cytotoxic than the encapsulate of 8-MOP in PAMAM G3 dendrimer (4MOP@G3). 4MOP@GD makes possible to achieve concentrations five times higher than 4MOP@G3 in local PUVA photochemotherapy.
 

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Notes

Source of Support: Nil,

Conflict of Interest: None declared.

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