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.

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.

In vitro anticancer activities of Withania coagulans against HeLa, MCF-7, RD, RG2, and INS-1 cancer cells and phytochemical analysis.

BACKGROUND: The Pakistani Salt Range has a rich floral diversity including Withania coagulans from the Solanaceae family. METHODS: The crude methanolic extracts of the root, leaf, leaf stalk, and fruit of this plant were screened for their cytotoxic activity against human (HeLa, MCF-7, RD) and rat (RG2 and INS-1) cancer cell lines at 20 µg/mL and compared to methotrexate. The IC50 values indicated that leaf stalk and fruit extracts exert an 80% or higher cytotoxic activity against all cell lines at 24 hours. RESULTS: The leaf stalk extract showed the highest cytotoxic efficacy against all tested cell lines, with IC50 values ranging from 0.96 ± 0.01 µg/mL to 4.73 ± 0.05 µg/mL followed by the fruit extract with IC50 values of 0.69 ± 0.01-6.69 ± 0.06 µg/mL after 48-72 hours incubation. The leaf stalk and seed extracts were analyzed for polyphenols and flavonoids using RP-HPLC. The total flavonoid content (TFC) was calculated for all tested samples, and the highest TFC was recorded for the root extract (394.34 ± 1.26 µg/g). The total phenolic content (TPC) was found in the seed extract (307.86 ± 9.42 µg/g) of W. coagulans. The highest contents of myricetin (358.46 ± 2.91 µg/g) were noted in the leaf extract, and highest quercetin was recorded in the seed extract (21.43 ± 0.13 µg/g). The highest gallic acid concentration (83.62 ± 0.71 µg/g) was recorded in leaf stalk extract and p-hydroxybenzoic acid in the seed extract (157.46 ± 1.43 µg/g). CONCLUSION: The present study gives a scientific insight and comparative analysis of various plant parts in this medicinally important plant species from the Salt Range of Pakistan against both human and rat cancer cells.

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.

Polarization image segmentation of radiofrequency ablated porcine myocardial tissue.

Optical polarimetry has previously imaged the spatial extent of a typical radiofrequency ablated (RFA) lesion in myocardial tissue, exhibiting significantly lower total depolarization at the necrotic core compared to healthy tissue, and intermediate values at the RFA rim region. Here, total depolarization in ablated myocardium was used to segment the total depolarization image into three (core, rim and healthy) zones. A local fuzzy thresholding algorithm was used for this multi-region segmentation, and then compared with a ground truth segmentation obtained from manual demarcation of RFA core and rim regions on the histopathology image. Quantitative comparison of the algorithm segmentation results was performed with evaluation metrics such as dice similarity coefficient (DSC = 0.78 ± 0.02 and 0.80 ± 0.02), sensitivity (Sn = 0.83 ± 0.10 and 0.91 ± 0.08), specificity (Sp = 0.76 ± 0.17 and 0.72 ± 0.17) and accuracy (Acc = 0.81 ± 0.09 and 0.71 ± 0.10) for RFA core and rim regions, respectively. This automatic segmentation of parametric depolarization images suggests a novel application of optical polarimetry, namely its use in objective RFA image quantification.

Polarimetry based partial least square classification of ex vivo healthy and basal cell carcinoma human skin tissues.

Optical polarimetry was employed for assessment of ex vivo healthy and basal cell carcinoma (BCC) tissue samples from human skin. Polarimetric analyses revealed that depolarization and retardance for healthy tissue group were significantly higher (p<0.001) compared to BCC tissue group. Histopathology indicated that these differences partially arise from BCC-related characteristic changes in tissue morphology. Wilks lambda statistics demonstrated the potential of all investigated polarimetric properties for computer assisted classification of the two tissue groups. Based on differences in polarimetric properties, partial least square (PLS) regression classified the samples with 100% accuracy, sensitivity and specificity. These findings indicate that optical polarimetry together with PLS statistics hold promise for automated pathology classification.

Polarimetric assessment of healthy and radiofrequency ablated porcine myocardial tissue.

Radiofrequency (RF) ablation offers a potential treatment for cardiac arrhythmia, where properly titrated energy delivered at critical sites can destroy arrhythmogenic foci. The resulting ablation lesion typically consists of a core (coagulative necrosis) surrounded by a rim of mixed viable and non-viable cells. The extent of the RF lesion is difficult to delineate with current imaging techniques. Here, we explore polarization signatures of ten ex-vivo samples from untreated (n = 5) and RF ablated porcine hearts (n = 5), in backscattered geometry through Mueller matrix polarimetry. Significant differences (p < 0.01) in depolarization, ΔT , were observed between the healthy, RF ablated and rim regions. Linear retardance, δ, was significantly lower in the core and rim regions compared to healthy regions (p < 0.05). The results demonstrate a novel application of polarimetry, namely the characterization of RF ablation extent in myocardium, including the visualization of the important lesion rim region. White light photo (top) of porcine myocardium tissue with radiofrequency ablation lesion and corresponding depolarization map (bottom). Depolarization is useful for visualizing the lesion core and rim.

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.

Ex vivo characterization of normal and adenocarcinoma colon samples by Mueller matrix polarimetry.

Mueller matrix polarimetry along with polar decomposition algorithm was employed for the characterization of ex vivo normal and adenocarcinoma human colon tissues by polarized light in the visible spectral range (425-725 nm). Six derived polarization metrics [total diattenuation (DT ), retardance (RT ), depolarization(ΔT ), linear diattenuation (DL), retardance (δ), and depolarization (ΔL)] were compared for normal and adenocarcinoma colon tissue samples. The results show that all six polarimetric properties for adenocarcinoma samples were significantly higher as compared to the normal samples for all wavelengths. The Wilcoxon rank sum test illustrated that total retardance is a good candidate for the discrimination of normal and adenocarcinoma colon samples. Support vector machine classification for normal and adenocarcinoma based on the four polarization properties spectra (ΔT , ΔL, RT ,and δ) yielded 100% accuracy, sensitivity, and specificity, while both DTa nd DL showed 66.6%, 33.3%, and 83.3% accuracy, sensitivity, and specificity, respectively. The combination of polarization analysis and given classification methods provides a framework to distinguish the normal and cancerous tissues.

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.

Colorization and automated segmentation of human T2 MR brain images for characterization of soft tissues.

Characterization of tissues like brain by using magnetic resonance (MR) images and colorization of the gray scale image has been reported in the literature, along with the advantages and drawbacks. Here, we present two independent methods; (i) a novel colorization method to underscore the variability in brain MR images, indicative of the underlying physical density of bio tissue, (ii) a segmentation method (both hard and soft segmentation) to characterize gray brain MR images. The segmented images are then transformed into color using the above-mentioned colorization method, yielding promising results for manual tracing. Our color transformation incorporates the voxel classification by matching the luminance of voxels of the source MR image and provided color image by measuring the distance between them. The segmentation method is based on single-phase clustering for 2D and 3D image segmentation with a new auto centroid selection method, which divides the image into three distinct regions (gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF) using prior anatomical knowledge). Results have been successfully validated on human T2-weighted (T2) brain MR images. The proposed method can be potentially applied to gray-scale images from other imaging modalities, in bringing out additional diagnostic tissue information contained in the colorized image processing approach as described.

Do different turbid media with matched bulk optical properties also exhibit similar polarization properties?

We here investigate polarimetric behavior of thick samples of porcine liver, Intralipid, and microsphere-based tissue phantoms whose absorption and scattering properties are matched. Using polarized light we measured reflection mode Mueller matrices and derived linear/circular/total depolarization rates, based on polar decomposition. According to our results, phantoms exhibit greater depolarization rates in the backscattering geometry than the liver sample. The enhanced tissue polarization preservation differs from previous reports of polarimetric transmission studies, with the likely cause of this difference being the angular dependence of the single-scattering phase function. Also, Intralipid approximated polarimetric liver behavior well, whereas the polystyrene phantoms did not.

Measurement of angle and axis of rotation in a carousel interferometer: a detailed analysis.

A detailed analysis of a carousel interferometer is presented for the measurement of an unknown angle and axis of rotation. The technique exploits a set of compensator glass plates and a right-angle prism that is placed in each of the two arms of the interferometer. The two sets are placed at the same rotational stage, while the end mirrors of the interferometer are static. When rotation takes place, individual and relative optical path differences are generated in the two beams of the interferometer. The generated phase differences contribute toward finding the angle and axis of rotation. The analysis is presented for any initial position of the interferometer, i.e., the radial vector from the axis of rotation to the apex of one of the prisms used. The results show the slight variations in the error and nonlinearity when different parameters are manipulated. Moreover, the trade-off between the maximum size of the prisms and the radial distances are also presented.

Optical measurements of angle and axis of rotation.

A carousel interferometer is designed to find the unknown angle and axis of rotation simultaneously. A set consisting of a compensator glass plate and a right-angle prism is placed in each of the two arms of the interferometer while located at the same rotational stage. When rotation takes place individual and relative optical path differences are generated in the beams that contribute toward finding out the angle and axis of rotation. Computer simulation shows that error remains less than a nanometer for a 1 m length of unknown radius and rotation range of +/-15 degrees .

Formulation of a Mueller matrix for modeling of depolarization and scattering of nitrobenzene in a Kerr cell.

We have studied the depolarization of light from nitrobenzene in a Kerr cell. We observed that absorption in nitrobenzene is electric-field dependent. For modeling a nitrobenzene device we formulated a Mueller matrix for the Kerr-cell assembly, and by operating it on a Stokes vector of the input light we obtained a corresponding Stokes vector for the output light. The first parameter of the output Stokes vector corresponds to the intensity transmittance. It was simulated and compared with the measured intensity transmittance for several orientations of the polarizer-analyzer pair with respect to the applied voltages. The measurement of all unknown coefficients in a Mueller matrix consisting of the superposition of nondepolarizing and depolarizing components predicts the depolarization, scattering, and absorption in the nitrobenzene electro-optic device. The output intensities of the orthogonally polarized and cross-coupled depolarizing coefficients are in good agreement for a semi-isotropic medium. The formulated Mueller matrix agrees with the experimentally measured transmittance.

Optimization of linearity by use of a glass plate in carousel interferometers.

Carousel interferometers are compact swinging interferometers, but their popularity is limited because of significant nonlinearity in rotational measurements. Introducing a glass plate in a rotating interferometer and optimizing its thickness has improved the linearity drastically. Computer simulations show reductions in nonlinearity of several orders of magnitude.