The median-effect equation derived from the mass-action law principle at equilibrium-steady state via mathematical induction and deduction for different reaction sequences and mechanisms and different types of inhibition has been shown to be the unified theory for the Michaelis-Menten equation, Hill equation, Henderson-Hasselbalch equation, and Scatchard equation. It is shown that dose and effect are interchangeable via defined parameters. This general equation for the single drug effect has been extended to the multiple drug effect equation for n drugs. These equations provide the theoretical basis for the combination index (CI)-isobologram equation that allows quantitative determination of drug interactions, where CI 1 indicate synergism, additive effect, and antagonism, respectively. Based on these algorithms, computer software has been developed to allow automated simulation of synergism and antagonism at all dose or effect levels. It displays the dose-effect curve, median-effect plot, combination index plot, isobologram, dose-reduction index plot, and polygonogram for in vitro or in vivo studies. This theoretical development, experimental design, and computerized data analysis have facilitated dose-effect analysis for single drug evaluation or carcinogen and radiation risk assessment, as well as for drug or other entity combinations in a vast field of disciplines of biomedical sciences. In this review, selected examples of applications are given, and step-by-step examples of experimental designs and real data analysis are also illustrated. The merging of the mass-action law principle with mathematical induction-deduction has been proven to be a unique and effective scientific method for general theory development. The median-effect principle and its mass-action law based computer software are gaining increased applications in biomedical sciences, from how to effectively evaluate a single compound or entity to how to beneficially use multiple drugs or modalities in combination therapies.

http://www.protocol-online.org/forums/uploads/monthly_09_2010/post-8165-008503000%201284677034.ipb

Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies

Photodynamic therapy (PDT) is a clinically approved, minimally invasive therapeutic procedure that can exert a selective cytotoxic activity toward malignant cells. The procedure involves administration of a photosensitizing agent followed by irradiation at a wavelength corresponding to an absorbance band of the sensitizer. In the presence of oxygen, a series of events lead to direct tumor cell death, damage to the microvasculature, and induction of a local inflammatory reaction. Clinical studies revealed that PDT can be curative, particularly in early stage tumors. It can prolong survival in patients with inoperable cancers and significantly improve quality of life. Minimal normal tissue toxicity, negligible systemic effects, greatly reduced long-term morbidity, lack of intrinsic or acquired resistance mechanisms, and excellent cosmetic as well as organ function-sparing effects of this treatment make it a valuable therapeutic option for combination treatments. With a number of recent technological improvements, PDT has the potential to become integrated into the mainstream of cancer treatment. CA Cancer J Clin 2011;61:250-281. V C

/pdf/photodynamic-therapy-of-cancer-an-update-4fcrlehb17.pdf

Photodynamic therapy of cancer: An update†‡

Sasidharan Swarnalatha Lucky,†,§ Khee Chee Soo,‡ and Yong Zhang*,†,§,∥ †NUS Graduate School for Integrative Sciences & Engineering (NGS), National University of Singapore, Singapore, Singapore 117456 ‡Division of Medical Sciences, National Cancer Centre Singapore, Singapore, Singapore 169610 Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore, Singapore 117576 College of Chemistry and Life Sciences, Zhejiang Normal University, Zhejiang, P. R. China 321004

Nanoparticles in photodynamic therapy.

Photodynamic therapy (PDT) uses non-toxic photosensitizers and harmless visible light in combination with oxygen to produce cytotoxic reactive oxygen species that kill malignant cells by apoptosis and/or necrosis, shut down the tumour microvasculature and stimulate the host immune system. In contrast to surgery, radiotherapy and chemotherapy that are mostly immunosuppressive, PDT causes acute inflammation, expression of heat-shock proteins, invasion and infiltration of the tumour by leukocytes, and might increase the presentation of tumour-derived antigens to T cells.

/pdf/photodynamic-therapy-and-anti-tumour-immunity-3h4u2nk6bb.pdf

Photodynamic therapy and anti-tumour immunity

Lipid-based drug delivery systems, or lipidic carriers, are being extensively employed to enhance the bioavailability of poorly-soluble drugs. They have the ability to incorporate both lipophilic and hydrophilic molecules and protecting them against degradation in vitro and in vivo. There is a number of physical attributes of lipid-based nanocarriers that determine their safety, stability, efficacy, as well as their in vitro and in vivo behaviour. These include average particle size/diameter and the polydispersity index (PDI), which is an indication of their quality with respect to the size distribution. The suitability of nanocarrier formulations for a particular route of drug administration depends on their average diameter, PDI and size stability, among other parameters. Controlling and validating these parameters are of key importance for the effective clinical applications of nanocarrier formulations. This review highlights the significance of size and PDI in the successful design, formulation and development of nanosystems for pharmaceutical, nutraceutical and other applications. Liposomes, nanoliposomes, vesicular phospholipid gels, solid lipid nanoparticles, transfersomes and tocosomes are presented as frequently-used lipidic drug carriers. The advantages and limitations of a range of available analytical techniques used to characterize lipidic nanocarrier formulations are also covered.

Impact of Particle Size and Polydispersity Index on the Clinical Applications of Lipidic Nanocarrier Systems.

Fluorescence lifetime of indocyanine green in molecular and nano-forms in the cellular model of a brain tumor in vitro

Cancer is the main problem of all developed and many developing countries of the world and the cause of death and disability of population. Today, the actual problem is receipt of reliable information about the boundaries of malignant neoplasms and the detection of pathology in the early stages.

The investigation of the photodynamic efficiency of chlorine e6 on a model of multicellular tumor spheroids using the developed video fluorescent equipment

The Spectroscopic Study of Indocyanine Green J-Aggregate Stability in Human Blood and Plasma

Introduction Since Sutherland developed the multicellular tumor spheroid (MTS) model to mimic the 3D-structure of small size solid tumors (R. Sutherland et al., 1970), MTS have been found to be useful in several aspects of tumor biology, including studies in the field of radiation biology and photodynamic therapy (PDT). Cellular organization of MTS allows to imitate in vivo small size tumors much better than 2D in vitro models (G. Hamilton, 1998). MTS were demonstrated to represent quite realistically the 3D growth and organization of solid tumors, and consequently to simulate well the cell-cell interactions and microenvironmental conditions found in tumor tissue. This similarities to a tumor xenograft let us apply MTS as a more rapid and valid in vitro model for anticancer drug screening compared to a monolayer culture. MTS could be formed from monolayer tumor cells grown by various in vitro classical methods, such as liquid-overlay, spinner flask and gyratory rotation systems. At the same time all classical methods are time consuming and can not provide the production of MTS with narrow spheroid size distribution within a range of 300 – 900 µm. More over, some tumor cells cannot form spheroids in suspension. The method proposed by Markvicheva et al. (Markvicheva et al., 2003) for microencapsulated MTS production provides several advantages over all classical techniques, such as generation of significant spheroid quantities, production of MTS of desired sizes, generation of MTS based on tumor and non-tumor cells which normally can't form aggregates in suspension culture. The objective of this research was to estimate the response of a novel in vitro model based on encapsulated MTS to PDT. Chlorine e6 was chosen as a model photosensitizer. Materials and Methods Chemicals: Sodium alginate (medium viscosity), EDTA and CaCl2 were from Sigma. All solutions for cell immobilization were prepared using 0.9% NaCl. Oligochitosan (MM 3500 Da, DD 98 %) was kindly provided by Prof. A.Bartkowiak (Poland). Chlorine e6 (Ce6) was supplied by Porphyrin Products (Logan, UT, USA). Ce6 stock solution (2 mM) was prepared in dimethyl sulfoxide (DMSO) and stored at -20oC. Before being added into the cell cultures, Ce6 was further diluted in the culture medium. All solutions for cell immobilization were prepared using 0.9% NaCl. Cells and cell cultivation media: In our study MCF-7 human adenocarcinoma cell line was used. The cells were cultured as a monolayer in DMEM medium supplemented with 10 mg/l human insulin, 10 % fetal calf serum (FCS) BioClot at 370C in a 5 % CO2 humidified atmosphere and were reseeded into fresh medium every 2-3 days. Bioencapsulation of tumor cells in microcapsules: Cell precipitate (107 cells), obtained by trypsinization of monolayer culture was mixed with 2 ml of a sterilized sodium alginate solution, and the mixture was extruded using an electrostatic bead generator into 0.5 % CaCl2 by peristaltic pump. The obtained hydrogel microbeads were incubated with 0.2 % oligochitosan solution for 10 min, in order to form alginate-oligochitosan membrane on microbeads surface. Then microbeads were washed 3 times with physiological saline. In order to get hollow microcapsules, the microbeads were incubated in 50 mM EDTA solution for 10 min and they were again washed and transferred into cultivation medium. Empty microcapsules were prepared as mentioned above. Cultivation of bioencapsulated cells to generate MTS: The resulted alginate-oligochitosan microcapsules with MCF-7 cells were cultivated in RPMI medium supplemented with 10 % FBS in 150 cm2 (Corning Inc.) flasks at 370C in 5 % CO2 for 2-4 weeks until MTS were formed. Ce6 non-specific adsorption on microcapsule surface: Microcapsules were incubated in Ce6 solution in the darkness at various final Ce6 concentrations for 24 h. Then supernatants and microcapsules were washed in the physiological solution, and were analysed using a computer-controlled luminescence spectrofluorimetre (Perkin-Elmer LS50B). The excitation wavelength was 410 nm, and spectra were collected at emission wavelength ranged between 600 – 800 nm. Ce6 solution in the same conditions but without microcapsules was used as a control. Ce6 cytotoxicity: Encapsulated MTS (100 µl) were incubated with Ce6 (0 - 34 nM) in 24-well plates for 24h. Each well contained 0.5 ml of RPMI medium supplemented with 2 % FBS. Cell viability was measured using MTT-assay. MCF-7 cells growing in monolayer were used as control. The cytotoxicity was expressed in the form of the viability using the following formula: Viability (%) = (Viable cells concentration in experiment / Viable cells concentration in control) x 100. Every experiment was repeated three times. Ce6 photoxicity: Encapsulated MTS (100 µl) were incubated with Ce6 (8.4 nM) in 24-well plates for 24h. Then MTS were washed with PBS 3 times, and 0.5 ml of RPMI was added in each well. The cells were irradiated by 650 nm diode laser (Coherent, France). Light energy densities were 0.5 – 70 J cm-2 at power density 30 mW cm-2. The cell viability was measured using MTT-assay in 24 h after irradiation. To study the structure of MTS before and after irradiation at various Ce6 concentrations, a set of MTS samples was selected. The samples were fixed in a 2 % (w/v) formaldehyde solution and embedded to the paraffin to prepare thin sectioned on slides. Results and Discussion Cell encapsulation was carried out using a special device, namely an electrostatic bead generator. The best calcium-alginate microbeads with a narrow bead size distribution (within the range of 300 – 600 µm, a mean size 340 ± 40 µm) were prepared using the voltage of 7.8 kV (Tabl.1). At the same time alginate-oligochitosan microcapsules mean diameter was much bigger (608 ± 50 µm) that that one of microbeads (Fig. 1). In order to understand the Ce6 non-specific adsorption on microcapsule surface, microcapsules were incubated with Ce6 solution for 24 h. The non-specific Ce6 sorption by empty microcapsules was 30 nmol per 1 ml of microcapsule slurry. Therefore the Ce6 sorption on the microcapsule surface could be ignored, and there was no necessity to remove the microcapsule membrane at photodynamic treatment of encapsulated MTS. To get MTS model, tumor cells (MCF-7) were microencapsulated and cultivated in 150 ml T-flasks in a 5 % CO2 atmosphere at 37 oC for 2 – 4 weeks. The cell proliferation has been easily observed by light microscope (Leitz, Germany). The cells grew in aggregates which have been increasing in their sizes with the cultivation time. The cell concentration in obtained encapsulated MTS was 5 x 106 cells/ml slurry. The cytotoxicity of Ce6 was estimated as an inhibition rate in cell viability. The cytotoxicity increased with increasing photosensitizer concentration in both encapsulated MTS and monolayer culture, but the viability of encapsulated MTS was higher than that one in monolayer culture (Fig. 2). Results obtained for MCF-7 monolayer were in agreement with data previously reported (JL. Merlin et al., 2003). Maximal non-toxic concentrations were 8.4 µM and 1.7 µM for encapsulated spheroids and for monolayer culture, respectively. These results revealed that the difference between monolayer culture and MTS was rather remarkable even for these previous experiments. These two concentrations were chosen for the next experiments. Parameter Value Voltage (Electrostatic bead generator) 7.8 kV Flow rate(peristaltic pump) 0.5 ml/min Tube diameter(peristaltic pump) 1.3 mm Needle diameter 0.3 mm Sodium alginate solution concentration 1.3 % (w/v) Table 1: Optimized conditions for microcapsule preparation technique using an electrostatic bead generator. Figure 1: Microcapsule size distribution in RPMI medium (a mean diameter is 608±50 µm; a membrane thickness is 70 ± 5 µm). Figure 2: Cell viability in monolayer model (▲) and MTS model (▄) after incubation with Ce6 for 24 h. Figure 3: Cell viability in monolayer model (▲) and MTS model (▄) 24h after PDT at light energy densities 1-70 J cm-2 As can be seen in Fig. 3, phototoxicity increased with light energy density enhance both for spheroids and monolayer culture. However, the cell viability of the encapsulated MTS was higher than that one of monolayer culture, in spite of the fifth-fold Ce6 concentration taken for MTS. For instance, a percentage of viable cells in MTS was tree times bigger compared to monolayer culture at light energy density 10 J cm-2. Conclusions Biocompatible polyelectrolyte microcapsules were used to generate MCF-7 cell based encapsulated MTS. Our results demonstrated that the proposed MTS model was much more resistant to the photodynamic treatment than monolayer model. We concluded that the encapsulated MTS model could mimic small size solid tumors more precisely, than commonly used classical monolayer model. Acknowledgments The authors are greatful to FEBS for support of D. Zaytseva-Zotova with FEBS Collaborative Experimental Scholarship for Central & Eastern Europe. References 1. R. Sutherland et al. (1970) Growth of nodular carcinomas in rodents compared with multi-cell spheroids in tissue culture. Growth 34 271-272 2. G. Hamilton. (1998) Multicellular tumor spheroids as an in vitro model. CancerLett 131 29 3. E. Markvicheva et al. (2003) Encapsulated multicellular tumor spheroids as a novel in vitro model to study small size tumors. ChemInd 57 585-588 4. JL. Merlin et al. (2003) The multidrug resistance modulator SDZ-PSC 833 potentiates the photodynamic activity of chlorine e6 independently on P-glycoprotein in multidrug resistant human breast adenocarcinoma cells. Int J Oncol 22 (4) 733-739

Photodynamic effects of chlorine e6 on bioencapsulated tumour spheroids

<h3>Purpose/Objective(s)</h3> Approximately 900 children/adolescents are treated with radiotherapy (RT) every year in France. However, among the 80% of survivors, the cumulative incidence of long-term morbidity – including second malignancies - reach 73.4% thirty years after the cancer diagnosis. Identifying a priori the subjects at risk for RT sequelae is a major challenge of pediatric oncology. Individual radiosensitivity (IRS) of children/adolescents is unknown at this time. The cellular response to radiation depends on the recognition and repair of radiation-induced DNA double-strand breaks (DSB). Unrepaired DSB as well as a delay in the intra nuclear activation of the pATM protein are common features to patients with RT toxicity. From these conclusions, we proposed and retrospectively validated a functional IRS immunofluorescence assay based on Radio-Induced ATM Nucleoshuttling (RIANS) (Granzotto et al, 2016). The purpose of this study was to define prospectively the predictive impact of RIANS assay in children. <h3>Materials/Methods</h3> Patients of the ARPEGE prospective open-label, non-randomized multicenter clinical trial were included in the study and prospectively evaluated for early toxicities. For each patient, skin samples were collected prior RT to raise a primary dermal fibroblast line and carry out in blind the RIANS assay. Different endpoints were measured: residual double-strand breaks at 24 h (γH2AX marker), pATM foci at 10 min and 1 h (pATM marker) and micronuclei at 24 h respectively after ex vivo radiation (2 Gy). The procedures were repeated in triplicates in blind in two independent expert centers (Neolys and Institut de Cancerologie de Lorraine). In parallel early toxicity was reported according to NCI-CTCAE v4.0 three months after the completion of RT. Patients with grade <2 or ≥2 toxicity were considered as radioresistant (RR) and radiosensitive (RS) respectively. We correlated the parameters of the RIANS assay according to toxicity with logistic regression. <h3>Results</h3> Among the 29 analyzable patients, 37 % exhibit brain cancers, 10% Hodgkin lymphomas, 10% nephroblastoma, 10% neuroblastoma, 10% Ewing cancer and 13% malignant mesenchymal cancer. The mean delivered dose was 32.5 Gy (range 1,5 - 60 Gy) with fractionation between 1.5 to 2 Gy. 16/29 (56%) patients were clinically considered as RS. There was no significant difference in total dose delivered between the two groups. Among the radiobiological factors tested, the number of Micronuclei at 24h post irradiation provided the best discrimination among RS patients (p-value= 0.0216). Overall, the RIANS assay provided the following performance: sensitivity=0.82, specificity=0.70 and AUC=0.75 (0.550-0.888). <h3>Conclusion</h3> ARPEGE is the first study to document the distribution of IRS in such a population. Screening hypersensitive patients would be a major step forward in the management of cancers, opening a way to personalized pediatric oncology. 

Lina Bezdetnaya

Papers

Fluorescence lifetime of indocyanine green in molecular and nano-forms in the cellular model of a brain tumor in vitro

The investigation of the photodynamic efficiency of chlorine e6 on a model of multicellular tumor spheroids using the developed video fluorescent equipment

The Spectroscopic Study of Indocyanine Green J-Aggregate Stability in Human Blood and Plasma

Photodynamic effects of chlorine e6 on bioencapsulated tumour spheroids

Evaluation of a Functional Assay for Radiosensitivity in the Pediatric Prospective Cohort ARPEGE