Photoactive metabolite mediated photodynamic therapy of Rhabdomyosarcoma cell lines using medicinal plants and Doxorubicin co-treatments.

BACKGROUND: Medicinal plant-mediated combinational therapies have gained importance globally due to minimal side effects and enhanced treatment outcomes compared to single-drug modalities. We aimed to analyze the cytotoxic potential of each conventional treatment i.e., photodynamic therapy (PDT), chemotherapy (doxorubicin hydrochloride; Dox-HCl) with or without various concentrations of medicinal plant extracts (PE) on soft tissue cancer Rhabdomyosarcoma (RD) cell line. METHODS: The Rhabdomyosarcoma (RD) cell line was cultured and treated with Photosensitizer (Photosense (AlPc4)), Chemo (Dox-HCl), and their combinations with different concentrations of each plant extract i.e., Thuja occidentalis, Moringa oleifera, Solanum surattense. For the source of illumination, a Diode laser (λ = 630 nm ± 1 nm, Pmax = 1.5 mW) was used. Photosensitizer uptake time (∼ 45 min) was optimized through spectrophotometric measurements (absorption spectroscopy). Drug response of each treatment arm was assessed post 24 h of administration using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5- 5-diphenyl-2 H- tetrazolium bromide (MTT) assay. RESULTS: PE-mediated Chemo-Photodynamic therapy (PDT) exhibited synergistic effects (CI < 1). Moreover, Rhabdomyosarcoma culture pretreated with various plant extracts for 24 h exhibited significant inhibition of cell viability however most effective outcomes were shown by low and high doses of Moringa oleifera compared to other plant extracts. Post low doses treated culture with all plant extracts followed by PDT came up with more effectiveness when compared to all di-therapy treatments. CONCLUSION: The general outcome of this work shows that the ethanolic plant extracts (higher doses) promote the death of cancerous cells in a dose-dependent way and combining Dox-HCl and photo-mediated photodynamic therapy can yield better therapeutic outcomes.

An Overview of Potential Natural Photosensitizers in Cancer Photodynamic Therapy.

Cancer is one of the main causes of death worldwide. There are several different types of cancer recognized thus far, which can be treated by different approaches including surgery, radiotherapy, chemotherapy or a combination thereof. However, these approaches have certain drawbacks and limitations. Photodynamic therapy (PDT) is regarded as an alternative noninvasive approach for cancer treatment based on the generation of toxic oxygen (known as reactive oxygen species (ROS)) at the treatment site. PDT requires photoactivation by a photosensitizer (PS) at a specific wavelength (λ) of light in the vicinity of molecular oxygen (singlet oxygen). The cell death mechanisms adopted in PDT upon PS photoactivation are necrosis, apoptosis and stimulation of the immune system. Over the past few decades, the use of natural compounds as a photoactive agent for the selective eradication of neoplastic lesions has attracted researchers' attention. Many reviews have focused on the PS cell death mode of action and photonanomedicine approaches for PDT, while limited attention has been paid to the photoactivation of phytocompounds. Photoactivation is ever-present in nature and also found in natural plant compounds. The availability of various laser light setups can play a vital role in the discovery of photoactive phytocompounds that can be used as a natural PS. Exploring phytocompounds for their photoactive properties could reveal novel natural compounds that can be used as a PS in future pharmaceutical research. In this review, we highlight the current research regarding several photoactive phytocompound classes (furanocoumarins, alkaloids, poly-acetylenes and thiophenes, curcumins, flavonoids, anthraquinones, and natural extracts) and their photoactive potential to encourage researchers to focus on studies of natural agents and their use as a potent PS to enhance the efficiency of PDT.

In-vitro co-delivery of decarbazine and photosense using poly lactic-co-glycolic acid nanocarrier for combinational therapy.

PLGA (Poly lactic-co-glycolic acid) nanoparticles are a new trend for drug delivery due to their good biodegradability properties. In this study, we have synthesized PLGA nanoparticles by solvent evaporation method and loaded decarbazine (DTIC, 5-3,3-(dimethyl-ltriazeno)imidazole-4-carboxamide) and photosense (AlPc4) drug alone as well as combined with two different concentrations i-e 25 nM and 250 nM. No cytotoxicity (viability ∼ 100%) was observed for different treatment arms either alone or in co-delivery of nano-formulation for Rhabdomyosarcoma (RD) cell culture which showed the biocompatibility of carrier. On comparison, the Photodynamic therapy (PDT) alone showed more significant cell death then the combinational therapy (PDT + chemotherapy) at 2 joule /cm2 and 5 joule /cm2. Lower doses co-delivery showed light dose dependent toxicity to culture i.e., 0% death @ 2 joule /cm2, ∼ 40% death @ 5 joule /cm2. Gene expressions of four apoptosis related genes (CASP3, CASP9, PARP1 and P53) were quantified by RT-PCR which shows down regulation for all the treatment arms indicating the absence of apoptosis for the cell death during PDT and combinational therapy. It was concluded that apoptosis related genes were down-regulated and morphological changes i.e., swelling and disruption suggest that the mode of cell death was necrosis.

Pegylated Eu-enabled submicron alumina spheres as potential theranostics agent RD cell line as model.

OBJECTIVES: This study is aimed to synthesis and evaluate PEGylated Eu enabled spherical alumina submicron particles (s-Al2O3:Eu) for potential theranostic applications. METHODS: This study is bisected into two parts, a) synthesis of PEGylated Eu enabled spherical alumina submicron particles (s-Al2O3:Eu), and b) characterization of the synthesized particles to determine their efficacy for potential theranostic applications.The synthesis of the particles involved the following steps. In the first step, s-Al2O3:Eu is synthesized using solvothermal synthesis. In the next step, the particles undergo post synthesis water-ethanol treatment and calcination. The surface of the synthesized s-Al2O3:Eu particles is then coated by PEG to increase its biocompatibility.Once the particles are prepared, they are characterized using different techniques. The microstructure, composition and structure of the particles is characterized using SEM, EDX and XRD techniques. The detection of the functional groups is done using FTIR analysis. The photoluminescence emission spectrum of s-Al2O3:Eu is studied using Photoluminescence spectroscopy. And, finally, the biocompatibility is studied using MTT assay on RD cell lines. RESULTS: The microstructure analysis, from the micrographs obtained from SEM, shows that the spherical alumina particles have a submicron size with narrow size distribution. The compositional analysis, as per EDX, confirms the presence of Oxygen, Aluminum and Europium in the particles. While, XRD analysis of s-Al2O3:Eu confirms the formation of alpha alumina phase after calcination at 700 °C. Emission peaks, obtained by Photoluminescence emission spectroscopy, show that the optimum emission intensities correspond to the transition from 5D0 to 7Fj orbital of Eu+3. FTIR analysis confirms the successful coating of PEG. Finally, a cell viability of more than 86% is observed when the biocompatibility of the particles is studied, using MTT assay on RD cell lines. CONCLUSIONS: s-Al2O3:Eu with narrow distribution are successfully synthesized. Structural and functional characterizations support the suitability of s-Al2O3:Eu as potential theranostic agent.

Study of synergistic effects of Ficus Carica leaves extract mediated chemo-photodynamic therapy on rhabdomyosarcoma cells.

BACKGROUND: Chemotherapy for rhabdomyosarcoma (RD) is effective, but it has critical side effects and unavoidable challenges. Photodynamic therapy (PDT) is an approach to treating cancer with relatively moderate side effects. Plant products are a rich source of polyphenols, which have potent antioxidant and anticancer activities. Therefore, their research has become an emerging field in recent decades. PURPOSE: This work aimed to evaluate the potential of hydrophobic extract of Ficus Carica (FC) to determine whether FC in the presence of low dose chemo and Aluminium Phthalocyanine (Photosense®) mediated photodynamic therapy synergistically enhances the treatment efficacy of RD cells. METHOD: FC with and without combination with individual therapeutic modalities like photosense mediated photodynamic therapy, chemotherapy, and their combinations were studied for cell viability and morphological changes in invitro RD cells. A semiconductor diode laser (630 nm) was used as a light source in PDT. The cytotoxic effect of FC on cell viability and cellular morphological changes were investigated by MTT reagent and a camera attached to an inverted visible light microscope. The effect of FC, followed by di-combination with low dose chemo (doxorubicin-HCl, and dacarbazine), Photosense® mediated PDT and chemo-Photosense® mediated PDT (tri-combination) at 630 nm diode laser and 10 J/cm2 fluency were also investigated by MTT reagent. The combination index method is used to identify the synergistic effect of combination therapy by using CompuSyn software based on the Chou-Talalay method. RESULTS: The dose-dependent effect of FC on cell viability and cellular morphological changes were observed in the RD cell line. It was found that the pre incubation of FC potentiated the anticancer effect as a neoadjuvant agent for doxorubicin-HCl and decarbazine based chemotherapy, Photosense® mediated PDT and chemo-PDT (tri-combination) with synergistic effect (CI<1). CONCLUSION: These results suggest a possible thread that the low dose combination of the aforementioned therapeutic modalities in the presence of FC remarkably enhances the treatment efficacy of RD in comparison with a single-agent treatment modality. The proposed sequence of FC with chemo and PDT might present better therapeutic outcomes in RD therapies and may provide result for RD metastasis. FC may also be used in the application of phyto-PDT to cancer in the future.

Polarimetric comparison of fresh and frozen skeletal muscle tissues of goat.

Optical properties can provide rich information about morphology and structure of tissues. Fresh and frozen muscle tissue samples of goat are investigated using imaging polarimetry to understand its structural nature. The outcomes demonstrate that the muscle tissues lose, to some extent, their integrity and organization on freezing. The fresh tissues offer very small circular retardance as compared to frozen samples. However, linear retardance is the main contributor in fresh muscle samples. Ultimately, linear and circular retardance can be used to differentiate fresh and frozen tissues. These investigations illustrate the capabilities of optical polarimetry for the characterization of muscle tissue structures. Specifically, the structure of biological tissue samples can be differentiated using real-time, cost effective and non-invasive optical polarimetry in the field of meat industry and biomedical diagnosis.

Machine assisted classification of chicken, beef and mutton tissues using optical polarimetry and Bagging model.

Optical polarimetry has been used to characterize muscle tissue samples of chicken, beef and mutton, exhibiting statistically significant (p <  0.01) differences in total depolarization and retardance of three tissue groups. Herein, the total depolarization and retardance were utilized to differentiate and classify the three tissue groups. Specifically, the Bagging classification algorithm was employed for this multi-class differentiation. The performance of the optical polarimetry in tandem with the Bagging model for machine-assisted classification of the three tissue groups was assessed in terms of a comprehensive set of evaluation metrics. The Bagging model correctly classified 47/48, 19/20 and 15/18, whereas the sensitivity (Sn = 97.9 %, 82.6 %, 100 %), specificity (Sp = 97.4 %, 98.4 %, 95.8 %), positive predictive values (PPV = 0.97, 0.95, 0.83) and negative predictive values (NPV = 0.97, 0.94, 1.0) were calculated for the chicken, beef and mutton tissue samples, respectively. This automatic classification of the three tissue samples indicates a novel application of the optical polarimetry in the meat industry.

Effective phthalocyanines mediated photodynamic therapy with doxorubicin or methotrexate combination therapy at sub-micromolar concentrations in vitro.

To improve a cancer patient's quality of life, short treatment duration resulting in rapid tumour removal while sparing normal tissue are highly desirable. Photodynamic therapy (PDT) commonly applied in a single treatment, while often effective can be limited at low photosensitizer or light doses. Combination therapies can overcome the efficacy limitations while not increasing treatment-associated morbidity. Here the efficacy of combination therapy comprised of doxorubicin (DOX) or methotrexate (MTX) with Photosens mediated PDT was investigated in three cell lines in vitro, employing multiple incubation sequences. Photosense is a mixture of aluminium phthalocyanines with different sulfonation. The results demonstrated higher synergistic effects when DOX or MTX-mediated chemotherapy preceded PDT light activation by 24 h. MTX is marginally more cytotoxic than DOX, when combined with Photosens (AlPcS2-4) mediated PDT. While MTX and DOX exposure prior to AlPcS2-4 incubation may enhance mitochondrial localisation photosensitizer, the simultaneous targeting of DNA, proteins, and lipids of the combination therapies leads to the observed high cytotoxicity at sub µM drug doses.

Effects of varying local temperature on the optical properties of cells in-vitro.

Increase in local temperature during light exposure of biological tissues plays an important role in determining the fate of most therapeutic modalities. Variations in the optical properties (absorption coefficient, scattering coefficient, anisotropy factor, optical depth etc.) of two cancer cell lines "Rhobdomyosarcoma and Cervical carcinoma" due to gradual increase in temperature were determine quantitatively with a double integrating sphere system. It was observed that all three coefficients showed decreasing tendency as the temperature increases for both the cell lines except for scattering coefficient of HeLa which remain constant within error limit. Anisotropy factor for both cell lines increased indicating temperature dependent subcellular density variations. Temperature dependent optical properties information may lead to precise dosimetry and could help clinicians for predicting the therapeutic modality outcome.

Study of low doses cisplatin synergistic effect on photodynamic outcome of aluminum phythalocyanine on soft tissue sarcoma (RD) cell line.

Photodynamic therapy (PDT) in combination with other treatment modality expects to overcome the drug resistance experienced in monotherapy. In this present work combination of chemo cum PDT is studied over the range of doses. It is found that treating cells/exposing cells to chemo drug (cisplatin, CDDP) and PDT individually results in minimal cell killing (∼7% and ∼16%) compared to the administration of chemo followed by PDT (∼50% cells were viable). These results showed that cell viability synergistically decreases in case of combination treatment as compared with individual treatment. Photodynamic therapy (PDT) in combination with CDDP chemotherapy expects to overcome the drug resistance experienced in monotherapy.

Ex vivo assessment of carbon tetrachloride (CCl(4))-induced chronic injury using polarized light spectroscopy.

The liver performs various functions, such as the production and detoxification of chemicals; therefore, it is susceptible to hepatotoxins such as carbon tetrachloride (CCl4), which causes chronic injury. Thus, assessment of injury and its status of severity are of prime importance. Current work reports an ex vivo study for probing the severance of hepatic injury induced by CCl4 with polarized light over the spectral range 400-800 nm. Different concentrations of CCl4 were used to induce varying severity of hepatic injury in a rat model. Linear retardance, depolarization rates, and diagonal Mueller matrix elements (m22, m33, and m44), were successfully used as the distinguishing criterion for normal and different liver injuries. Our results show that linear retardance for injured liver samples with lower doses of CCl4 tends to increase when compared with normal liver samples, while samples injured at higher doses of CCl4 offer almost no retardance. Total, linear, and circular depolarizations follow decreasing trends with increased liver injury severity over the entire investigated wavelength range. Linear polarization states were observed to be better maintained as compared to circular polarization states for all samples. Furthermore, numerical values of diagonal elements of the experimentally measured Mueller matrix also increase with increasing doses of CCl4. Liver fibroses, change in transport albedo, and the relative refractive index of the extracellular matrix caused by CCl4 are responsible for the observed differences. These results will provide a pathway to gauge the severity of injury caused by toxic chemicals.

Development and applications of photo-triggered theranostic agents.

Theranostics, the fusion of therapy and diagnostics for optimizing efficacy and safety of therapeutic regimes, is a growing field that is paving the way towards the goal of personalized medicine for the benefit of patients. The use of light as a remote-activation mechanism for drug delivery has received increased attention due to its advantages in highly specific spatial and temporal control of compound release. Photo-triggered theranostic constructs could facilitate an entirely new category of clinical solutions which permit early recognition of the disease by enhancing contrast in various imaging modalities followed by the tailored guidance of therapy. Finally, such theranostic agents could aid imaging modalities in monitoring response to therapy. This article reviews recent developments in the use of light-triggered theranostic agents for simultaneous imaging and photoactivation of therapeutic agents. Specifically, we discuss recent developments in the use of theranostic agents for photodynamic-, photothermal- or photo-triggered chemotherapy for several diseases.