Abstract
Background: Clinically-approved anticancer photodynamic therapy (PDT) is now extensively studied for various cancer diagnoses. We focused on the treatment efficacy of topical administration of hydroxy-aluminum phthalocyanine (AlOH-PC) entrapped in liposomes against in vivo models of prostate carcinomas. Materials and Methods: LNCaP and PC3 cells were subcutaneously injected into the right flank of athymic nude mice. Mice with grown tumours were used for in vivo efficacy studies. Firstly, we applied different doses of AlOH-PC to less aggressive LNCaP tumours to determine the effective dose. In later studies, we focused on more aggressive prostate tumours (PC3) using doses of liposomal-AlOH-PC gel formulation. Topical application of photosensitizers was followed by PDT irradiation (600-700 nm, 635 nm peak). Tumour growth was measured three times-a-week. Results: Comparison of PDT of aggressive PC3 and less aggressive LNCaP prostate carcinomas showed that both tumour types are sensitive and treatable by liposomal formulation of AlOH-PC. For LNCaP tumours the efficient dose (100% experimental animals cured, n=8/8) was 4.5 mg/ml of AlOH-PC in the gel. Whereas, in the case of PC3 carcinomas, a dose of 4 mg/ml significantly postponed tumour growth, but no animals were cured (n=0/8); a sufficient curative dose (100% mice cured, n=8/8) was 6 mg/ml of AlOH-PC in the gel. Conclusion: Liposomal AlOH-PC gel has potential for effective PDT of prostate carcinomas.
The fact that despite progress in cancer research leading to the design of new generations of targeted drugs, only a relatively low number of new clinically approved drugs is available; this places an emphasis on other under-utilized therapeutic approaches. Photodynamic therapy (PDT) has been a clinically-accepted approach for cancer treatment for a long time, but is still clinically rather under-appreciated (1). It consists of three key components: a light source, photosensitizer and tissue oxygen (Figure 1). When the photosensitizer is exposed to light of specific wavelength, it shifts oxygen into an excited state. As it returns into the former unexcited state, the released energy is utilized for production of singlet oxygen, which in turn directly mediates a cytotoxic effect (2). The mechanism of tumour destruction lies mainly in apoptotic or necrotic cell death pathways; an additional described mode of action is direct damage of tumour blood vasculature (1, 3). Moreover, it has been reported that PDT can induce a host immune reaction against the tumour cells (4, 5).
A member of the family of classical photosensitizers, porfimer sodium, received Food and Drugs Administration (FDA) approval as early as 1993 under the name Photofrin II (Axcan Pharma, Montreal, Canada) (6). Systemically administered Photofrin II as a treatment for non-dermatological malignancies has poor specificity for neoplastic tissues, moreover, it also requires a long interval between administration and consequent irradiation (48-72 h) (7). Additionaly, skin photosensitivity after systemic administration is the major drawback of Photofrin II application (6). Other intravenously-administered photosensitizers also face this problem (8, 9). With topical administration, this obstacle can be overcome; the clinically-approved photosensitizer methylester of δ-aminolevulic acid (Metvix; Galderma & PhotoCure ASA, Inc., Oslo, Norway) serves as a successful example (10).
The irradiated photosensitizer assumes its activated form. Returning to the unexcited form is accompanied by energy release, which can be transferred to tissue oxygen. This leads to generation of reactive oxygen species (ROS), such as free radicals and singlet oxygen, which then cause toxicity. Adapted from Dolmans et al. (2).
New photosensitizers aim to shorten the interval between administraton and PDT even more. This would bring better comfort to patients and hopefully enable PDT to be used more in clinical practice (7). Another approach within the family of new-generation photosensitizers is utilization of longer wavelengths for their activation. Due to better tissue penetration for light with longer wavelengths, this enables for deeper tumour treatment opportunities (11). Different phthalocyanines are being studied as candidates from this new generation of photosensitizers (12-14). In accordance with the demand for long wavelength utilization, they do absorb in the red spectrum (~670 nm) (15), and additionally, they have a high extinction coefficient (over 100,000/M/cm) together with a high coefficient of singlet oxygen production (16). Over time, metal-conjugated and metal-free phthalocyanines have been studied and the metal-conjugated ones showed better sensitizing properties (16). Among these phthalocyanines, due to its strong photodynamic activity (7) and simple synthesis, hydroxy-aluminum phthalocyanine (AlOH-PC) has emerged as a potential candidate for utilization in PDT (Figure 2).
Chemical structure of tested photosensitizer: hydroxy-aluminum phthalocyanine.
We recently showed that a liposomal formulation of AlOH-PC is suitable for efficient treatment of mammarian carcinoma (17). In this study, we tested the potential of PDT with AlOH-PC as a photosensitizer against prostate carcinomas.
Materials and Methods
Preparation of gel containing phthalocyanine liposomes. The detailed preparation of AlOH-PC-containing liposomes is a patented procedure (18). Briefly, sterile lecithin solution of concentration 1-40 mg/ml underwent microfluidization, using a semi-industrial microfluidizer (M-110L; Micrifluidics Inc., Newton, MA, USA), to produce particles smaller than 1 μm. Lyophilized AlOH-PC powder was added to the suspension, and this mixture underwent further microfluidization in a smaller chamber leading to the formation of liposomes of size under 500 nm. Such preparation leads to the production of organic solvent-free liposomes. Final liposomes containing AlOH-PC were then mixed with a translucent gel (Gel 2, clear pharmaceutical magistral gel based on 4% carboxymethylcellulose; Pharmgest, Ltd., Pribram, Czech Republic) at a 1:1 ratio by volume (ml/ml).
Experimental cell line. Human prostate carcinoma cell lines LNCaP and PC3 were purchased from the European Collection of Cell Cultures (Salisbury, UK; distributed by Sigma-Aldrich, Ltd., Prague, Czech Republic). Before application, cells had been cultivated in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2% penicillin/streptomycin, 1.25% L-glutamine, and 1% sodium pyruvate.
Antitumour efficacy of liposomal (AlOH-PC) gel against LNCaP xenografts: A: Untreated control group; B: control group treated with liposomal AlOH-PC gel without subsequent irradiation; C: irradiated control group without treatment with photosensitizer; D: group treated with 4.5 mg/ml liposomal AlOH-PC gel followed by irradiation; E: group treated with 9 mg/ml liposomal AlOH-PC gel followed by irradiation; F: group treated with 18 mg/ml liposomal AlOH-PC gel followed by irradiation. Number of experimental animals in each group was 8 (n=8). These results are representative of three independent experiments, p<0.05.
In vivo efficacy of liposomal phthalocyanine in gel on LNCaP tumours. All animal experiments depicted here after were performed in accordance with the Act on Experimental Work with Animals (Public Notice of the Ministry of Agriculture of the Czech Republic No. 246/1992, No. 311/1997, No. 207/2004; Decree of the Ministry of the Environment of the Czech Republic No. 117/1987; and Act of the Czech National Assembly No. 149/2004) of the Czech Republic, which is fully-compatible with the corresponding European Union directives.
Harvested LNCaP cells were administered subcutaneously, 1×106 cells as a mixture with BD Matrigel™ (I.T.A.-Intertact, Ltd., Prague, Czech Republic) into the abdominal right flank of athymic nu/nu mice (obtained from AnLab, Ltd. and Charles River Laboratories International, Inc., Prague, Czech Republic). When the tumours had reached a size of about 6 mm in diameter, mice were divided randomly into groups (n=8). Control groups were as follows: a) group without any compound and without irradiation; b) group with irradiation, but without any compound; and c) group with AlOH-PC gel (highest dose), but without irradiation. The experimental groups were treated with irradiaton gels at three different concentrations of AlOH-PC (4.5, 9, and 18 mg/ml of AlOH-PC). For treated groups, each gel was applied topically to the tumour (0.2 ml per tumour). Irradiation took place 10 min [according to (7)] after application of gel using a xenon lamp (ONL 051; Preciosa Crytur, Trutnov, Czech Republic) at 0.97 W, with a total energy of 100 J/cm2 from a distance of 1 cm behind the transmission filter. In all experimental cases, the duration of irradiation was precisely 8 min. Tumour growth was recorder twice-a-week and recorded data were statisticaly analysed using one-tailed t-test.
In vivo efficacy of liposomal phthalocyanine in gel on PC3 tumours. Growing PC3 tumours in athymic mice were assessed as described for LNCaP line above. Similarly, mice were randomly divided into groups (n=8) and gel application was followed by irradiation 10 min after application (irradiation time was precisely 8 min). We tested two different treatment schemes consisting of 4.5, 9, and 18 mg/ml (data not shown) and 2, 4 and 6 mg/ml AlOH-PC concentrations. Control groups were as follows: a) group without any compound and without irradiation; b) group with irradiation, but without any compound; and c) group with AlOH-PC gel (highest dose), but without irradiation. All recorded data were statisticaly analysed using one-tailed t-test.
Results
In vivo efficacy of liposomal phthalocyanine in gel on LNCaP tumours. Neither irradiation itself nor applied photosensitizer, had any significant effect on tumour growth (Figure 3A-C).
Three different AlOH-PC gel preparations were tested in order to determine the efficient concentration of photosensitizer for the treatment of a less aggressive prostate carcinoma model with low metastatic potential (LNCaP tumours). Treatment with gels containing liposomal AlOH-PC at concentrations of 4.5, 9 and 18 mg/ml led to shrinkage of tumours immediately after the irradiation (Figure 3D-F). Within a 15-day period, tumours decreased to an impalpable size, and as all treated experimental mice remained in this state up to the defined end dates, we considered those mice as cured.
Antitumour efficiency of liposomal (AlOH-PC) gel against PC3 xenografts: A: Untreated control group; B: control group treated with liposomal AlOH-PC gel without subsequent irradiation; C: irradiated control group without treatment with photosensitizer; D: group treated with 2 mg/ml liposomal AlOH-PC gel followed by irradiation; E: group treated with 4 mg/ml liposomal AlOH-PC gel followed by irradiation; F: group treated with 6 mg/ml liposomal AlOH-PC gel followed by irradiation. Number of experimental animals in each group was 8 (n=8). These results are representative of three independent experiments, p<0.05.
The lowest tested dose of 4.5 mg/ml only slightly reduced the tumour volume when compared with the higher doses, in the first days after treatment (Figure 3D). However, the curative effect was delayed and this was statistically insignificant, while final destruction of the tumour mass occurred within the same time, as that with the other doses (9 and 18 mg/ml).
In vivo efficacy of liposomal phthalocyanine in gel on PC3 tumours. More aggressive PC3 tumours were treated with the same gels as previously described for LNCaP tumours (i.e. 4.5, 9 and 18 mg/ml). Doses of 9 and 18 mg/ml were sufficient for complete tumour eradication, whereas 4.5 mg/ml gel postponed tumour growth significantly [size of treated tumours was over 20 times smaller than controls at the endpoint of the experiment (data not shown)]. However, the growth was only delayed.
We wanted to determine the curative dose, so we tested three different gels with narrower concentration intervals (2, 4 and 6 mg/ml). The possible effect of the photosensitizer itself (without irradiation) and irradiation-alone on the growth of the PC3 tumours was also assessed. As shown in Figure 4A-C, single usage of either irradiation or AlOH-PC liposomes had no effect on PC3 tumour growth.
In accordance with previous tests (data not shown), neither 2 nor 4 mg/ml gels were efficient for total eradication of the tumour mass (Figure 4D and E). However, both doses showed significant prolongation of tumour growth, with postponed growth outburst. Furthermore, we saw a total obliteration of tumour mass after treatment with 6 mg/ml AlOH-PC gel (Figure 4F).
Discussion
PDT has a potential utilization for many new cancer indications (19, 20). Modern fiber-optic systems together with improved endoscopy techniques can deliver light to almost any part of the body, therefore PDT is no longer limited to the field of superficial tumours (21). Additionally, new-generation photosensitizers have been developed to utilize long wavelength light activation (e.g. porphyrins ~630 nm; phthalocyanines ~670 nm; texafrins ~734 nm; bacteriochlorins ~740 nm). Easier tissue penetration of light with longer wavelengths compared to light with shorter wavelengths opens the door for usage of topically-administered PDT not restricted to superficial tumours, as the biological response can be documented at two-to three-times greater depths.
Moreover, the topical administration of photosensitizer brings site benefits compared to systemic administration, such as reducing systemic toxicity, by-passing first-pass elimination/metabolism in the liver, and also minimizing the induction of photosensitivity. A potential obstacle for the use of topical PDT may be the long interval between photosensitizer application and consequent irradiation, such as that for approved Metvix (4-6 h), which is inconvenient for patients (22). Our group described a liposomal system for phthalocyanine that overcomes this obstacle. We reported the unsurpassed drug-to-light time interval of 10 min sufficient for complete eradication of tumour in various models (7). Thus patients would not be forced to spend hours in hospitals waiting for irradiation of the treated area, which would be better for patients and less costly for both them and the health system.
The described “proof-of-principle” study shows the efficient treatment of two different prostate carcinomas using topically-administered liposomal AlOH-PC. It highlights the need for extensive further study of photosensitizer–microenvironment interactions. We plan to introduce fluorescently-labelled tumour cells (23-25) to be able to follow even few residual tumour cells. Recent data suggest that expression of fluorescent proteins enhances the efficiency of PDT (26). One can hypothesize, that due to utilization of far-red light activation of AlOH-PC, the treatment will not be affected by expression of green fluorescent protein.
Conclusion
We have shown that liposomal AlOH-PC prepared by a patented microfluidization procedure is potentially suitable for PDT of prostate carcinomas. As previously reported for other tumour models, only a very short interval between gel application and irradiation (10 min) is required for the efficient treatment after topical administration of liposomal AlOH-PC. AlOH-PC showed promising efficacy against both aggressive PC3 tumours with high metastatic potential (100% cure at a dose of 6 mg/ml) and for less aggressive LNCaP tumours with low metastatic potential (100% cure at a dose of 4.5 mg/ml).
Acknowledgements
This research was supported partly by the Czech Ministry of Industry and Commerce, grant No. 2A-1TP1/026, the Czech Ministry of Education, grant No. OE 09026 and the Czech Technological Agency, grant No. TA 01010781.
- Received January 17, 2013.
- Revision received February 26, 2013.
- Accepted February 26, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
