Tissue Penetration Of Light Research Articles

Comparison of red-shifted firefly luciferase Ppy RE9 and conventional Luc2 as bioluminescence imaging reporter genes for <italic>in vivo</italic> imaging of stem cells

One critical issue for noninvasive imaging of transplanted bioluminescent cells is the large amount of light absorption in tissue when emission wavelengths below 600 nm are used. Luciferase with a red-shifted spectrum can potentially bypass this limitation. We assessed and compared a mutant of firefly luciferase (Ppy RE9, PRE9) against the yellow luciferase luc2 gene for use in cell transplantation studies. C17.2 neural stem cells expressing PRE9-Venus and luc2-Venus were sorted by flow cytometry and assessed for bioluminescence in vitro in culture and in vivo after transplantation into the brain of immunodeficient Rag2-/- mice. We found that the luminescence from PRE9 was stable, with a peak emission at 620 nm, shifted to the red compared to that of luc2. The emission peak for PRE9 was pH-independent, in contrast to luc2, and much less affected by tissue absorbance compared to that of luc2. However, the total emitted light radiance from PRE9 was substantially lower than that of luc2, both in vitro and in vivo. We conclude that PRE9 has favorable properties as compared to luc2 in terms of pH independence, red-shifted spectrum, tissue light penetration, and signal quantification, justifying further optimization of protein expression and enzymatic activity.

High-sensitivity in vivo imaging for tumors using a spectral up-conversion nanoparticle NaYF4: Yb3+, Er3+ in cooperation with a microtubulin inhibitor

Fluorescein has been used for in vivo imaging to identify tumors. However, this technique presents several limitations, mainly due to its limited targeting efficiency, tissue autofluorescence and poor light penetration in tissue. In the present study, an alternative fluorescence imaging technique to localize tumors has been developed by using up-conversion nanoparticles (UCNs) and enhanced targeting approaches. A folic acid molecule is conjoined with UCNs (NaYF(4): Yb(3+), Er(3+)) to improve the tumor-specificity; the UCN is also loaded with the microtubule inhibitor CA4P, to further improve the local delivery of particles in the tumor. The proposed imaging technique combines several well-established individual concepts into one novel integrated procedure and significantly improves its tumor-imaging capability: the near-infrared excitation for UCNs minimizes tissue autofluorescence and allows imaging into deeper tissue; the improvement in the signal to noise ratio (SNR) is at least a magnitude better than that of a conventional fluorescence imaging technique, and the modification of UCNs with folic acid significantly improves the tumor targeting efficiency by utilizing its affinity for the folic acid receptor that is often over expressed in tumors. The loading of CA4P further helps UCNs to cross blood vessel walls to reach tumor cells by depolymerizing the microtubules of endothelial cells. The integrated nanoparticle possesses the near-infrared-identical optical properties of UCNs alone, thus achieving a highly effective fluorescence imaging probe. The results demonstrated that the proposed method provides an excellent alternative for tumor localization and a potential traceable vehicle for highly efficient drug delivery.

New Hydrophilic/Lipophilic Tetra-α-(4-carboxyphenoxy) Phthalocyanine Zinc-Mediated Photodynamic Therapy Inhibits the Proliferation of Human Hepatocellular Carcinoma Bel-7402 Cells by Triggering Apoptosis and Arresting Cell Cycle

Photodynamic therapy (PDT) is a novel and promising antitumor treatment. Phthalocyanine-mediated PDT has shown antitumor activity in some tumor cells, but the effect of new hydrophilic/lipophilic tetra-α-(4-carboxyphenoxy)phthalocyanine zinc (TαPcZn)-mediated PDT (TαPcZn-PDT) on human hepatocellular carcinoma Bel-7402 cells and underlying mechanisms have not been clarified. In the present study, therefore, the ultraviolet-visible (UV-vis) absorption spectrum and cellular localization of TαPcZn, and effect of TαPcZn-PDT on the proliferation, apoptosis, cell cycle, Bcl-2 and Fas in Bel-7402 cells were investigated by spectrophotometry, inverted microscope, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, electron microscopy, annexinV-FITC/propidium iodide double staining, DNA content and immunoblot assay, respectively. We found that an intense absorption in UV-vis absorption spectrum of TαPcZn was in the red visible region at 650–680 nm, where light penetration in tissue is efficient, that green TαPcZn localized to both plasma membrane and nuclear membrane of Bel-7402 cells, signifying that there was a selective uptake of TαPcZn in Bel-7402 cells and TαPcZn-PDT would be expected to directly damage DNA, and that TαPcZn-PDT significantly resulted in the proliferation inhibition, apoptosis induction, S cell cycle arrest, and down-regulation of Bcl-2 and Fas. Taken together, we conclude that TαPcZn-PDT inhibits the proliferation of Bel-7402 cells by triggering apoptosis and arresting the cell cycle.

Fiber Optic Microneedles for Transdermal Light Delivery: Ex Vivo Porcine Skin Penetration Experiments

Shallow light penetration in tissue has been a technical barrier to the development of light-based methods for in vivo diagnosis and treatment of epithelial carcinomas. This problem can potentially be solved by utilizing minimally invasive probes to deliver light directly to target areas. To develop this solution, fiber optic microneedles capable of delivering light for either imaging or therapy were manufactured by tapering step-index silica-based optical fibers employing a melt-drawing process. Some of the microneedles were manufactured to have sharper tips by changing the heat source during the melt-drawing process. All of the microneedles were individually inserted into ex vivo pig skin samples to demonstrate the feasibility of their application in human tissues. The force on each microneedle was measured during insertion in order to determine the effects of sharper tips on the peak force and the steadiness of the increase in force. Skin penetration experiments showed that sharp fiber optic microneedles that are 3 mm long penetrate through 2 mm of ex vivo pig skin specimens. These sharp microneedles had a minimum average diameter of 73 mum and a maximum tip diameter of 8 mum. Flat microneedles, which had larger tip diameters, required a minimum average diameter of 125 mum in order to penetrate through pig skin samples. Force versus displacement plots showed that a sharp tip on a fiber optic microneedle decreased the skin's resistance during insertion. Also, the force acting on a sharp microneedle increased more steadily compared with a microneedle with a flat tip. However, many of the sharp microneedles sustained damage during skin penetration. Two designs that did not accrue damage were identified and will provide a basis of more robust microneedles. Developing resilient microneedles with smaller diameters will lead to transformative, novel modes of transdermal imaging and treatment that are less invasive and less painful for the patient.

Enhancement of skin optical clearing efficacy using photo‐irradiation

Tissue optical clearing technique based on immersion of tissues into optical clearing agents (OCAs) can reduce the scattering and enhance the penetration of light in tissue. However, the barrier function of epidermis limits the penetration of OCAs, and hence is responsible for the poor optical clearing efficacy of skin by topical action. In this study, a variety of light irradiation was applied to increase permeability of agents in skin and improve the optical clearing efficacy. Different light sources with different dose, i.e, CO(2) laser, Nd:YAG laser (532 and 1,064 nm) with different pulse modes and Intense Pulsed Light (IPL) (400-700 and 560-950 nm) were used to irradiate rat skin in vivo, and then glycerol was applied onto the irradiated zone. VIS-NIR spectrometer was utilized to monitor the changes of reflectance. In vitro skin samples were also irradiated by Q-switched Nd:YAG laser (1,064 nm) and then treated by glycerol for 10-60 minutes. Based on the measurement of the reflectance and transmittance of the samples, the optical properties of skin and penetration depth of light were calculated. Results show that photo-irradiation with appropriate dose combining with the following glycerol treatment is able to reduce in vivo skin reflectance. Compared with the control group, the maximal changes in reflectance are ninefold at 575 nm and eightfold at 615 nm, respectively, which were caused by Q-switched 1,064-nm Nd:YAG laser irradiation and following glycerol treatment. The results for in vitro skin demonstrate that the joint action can significantly increase the optical penetration depth in samples. The combination of Q-switched Nd:YAG (1,064 nm) laser and glycerol could enhance optical skin clearing efficacy significantly. This study provides a non-invasive way to improve the optical clearing of skin, which will benefit the skin optical therapy.

Controling the scattering of Intralipid by using optical clearing agents

Optical clearing agents (OCAs) with high refractive indices and hyperosmolarity can enhance the penetration of light in tissues by reducing scattering in tissues. However, the mechanism of tissue optical clearing is not much clear for the complex interaction between tissues and OCAs. In this work, Intralipid was mixed with different concentrations of OCAs, i.e. dimethyl sulfoxide (DMSO), glycerol, 1,4-butanediol, 1,2-propanediol, poly-ethylene glycol 200 (PEG200) and poly-ethylene glycol 400 (PEG400). Except for PEG200 and PEG400 that make aggregation of particles, the others kept the mixture uniform. The reduced scattering coefficients of uniform mixtures were predicted with Mie theory and measured by a commercially available spectrophotometer with an integrating sphere. The results show that all of the OCAs used enhance the optical clearing effect of Intralipid. If OCAs do not change the structure of Intralipid, Mie theory prediction matches well with the measurements. And the higher the refractive index of OCA, the smaller the reduced scattering coefficient. A simple formula deduced can quantitatively predict the optical clearing effect caused by OCAs. This work is helpful for clarifying the mechanism of tissue optical clearing, which will make the effect of optical clearing of tissue predictable and controllable.

Multimodality Cardiovascular Molecular Imaging, Part I

In Part I of this consensus article, the imaging methodology, evolving imaging technology, and development of novel targeted molecular probes relevant to the developing field of cardiovascular molecular imaging were reviewed. Novel reporter gene and reporter probe imaging approaches for tracking of cardiac transgene expression were also discussed and have important future implications for evaluation of gene- and cell-based therapies for the failing heart. The current role of metabolic and receptor imaging was also briefly reviewed, as these represent the beginning of our clinical application of molecular imaging within the cardiovascular system. Part II will summarize the available targeted imaging probes as well as specific future applications of molecular imaging for identification and evaluation of critical pathophysiological processes of the cardiovascular system.

Photodynamic Therapy with Motexafin Lutetium for Rectal Cancer: A Preclinical Model in the Dog

Local recurrence of rectal cancer remains a significant clinical problem despite multi-modality therapy. Photodynamic Therapy (PDT) is a cancer treatment which generates tumor kill through the production of singlet oxygen in cells containing a photosensitizing drug when exposed to laser light of a specific wavelength. PDT is a promising modality for prevention of local recurrence of rectal cancer for several reasons: tumor cells may selectively retain photosensitizer at higher levels than normal tissues, the pelvis after mesorectal excision is a fixed space amenable to intra-operative illumination, and PDT can generate toxicity in tissues up to 1 cm thick. This study evaluated the safety, tissue penetration of 730 nm light, normal tissue toxicity and surgical outcome in a dog model of rectal resection after motexafin lutetium-mediated photodynamic therapy. Ten mixed breed dogs were used. Eight dogs underwent proctectomy and low rectal end to end stapled anastomosis. Six dogs received the photosensitizing agent motexafin lutetium (MLu, Pharmacyclics, Inc., Sunnyvale, CA) of 2 mg/kg preoperatively and underwent subsequent pelvic illumination of the transected distal rectum of 730 nm light with light doses ranging from 0.5 J/cm(2) to 10 J/cm(2) three hours after drug delivery. Two dogs received light, but no drug, and underwent proctectomy and low-rectal stapled anastomosis. Two dogs underwent midline laparotomy and pelvic illumination. Light penetration in tissues was determined for small bowel, rectum, pelvic sidewall, and skin. Clinical outcomes were recorded. Animals were sacrificed at 14 days and histological evaluation was performed. All dogs recovered uneventfully. No dog suffered an anastomotic leak. Severe tissue toxicity was not seen. Histological findings at necropsy revealed mild enteritis in all dogs. The excitation light penetration depths were 0.46 +/- 0.18, 0.46 +/- 0.15, and 0.69 +/- 0.39 cm, respectively, for rectum, small bowel, and peritoneum in dogs that had received MLu. For control dogs without photosensitizer MLu, the optical penetration depths were longer: 0.92 +/- 0.63, 0.67 +/- 0.10, and 1.1 +/- 0.80 cm for rectum, small bowel, and peritoneum, respectively. Low rectal stapled anastomosis is safe when performed with MLu-mediated pelvic PDT in a dog model. Significant tissue penetration of 730 nm light into the rectum and pelvic sidewall was revealed without generation of significant toxicity or histological sequelae. Penetration depths of 730 nm light in pelvic tissue suggest that microscopic residual disease of less than 5 mm are likely to be treated adequately with MLu-mediated PDT. This approach merits further investigation as an adjuvant to total mesorectal excision and chemoradiation for rectal cancer.

Optical Properties of Human Prostate at 732 nm Measured In Vivo During Motexafin Lutetium–mediated Photodynamic Therapy¶

ABSTRACTCharacterization of the tissue light penetration in prostate photodynamic therapy (PDT) is important to plan the arrangement and weighting of light sources so that sufficient light fluence is delivered to the treatment volume. The optical properties (absorption [μa], transport scattering [μ′s] and effective attenuation [μeff] coefficients) of 13 patients with locally recurrent prostate cancer were measured in situ using interstitial isotropic detectors. Measurements were made at 732 nm before and after motexafin lutetium (MLu)–mediated PDT in four quadrants. Optical properties were derived by applying the diffusion theory to the fluence rates measured at several distances (0.5–5 cm) from a point source. μa and μ′s varied between 0.07 and 1.62 cm−1 (mean 0.37 ± 0.24 cm−1) and 1.1 and 44 cm−1 (mean 14 ± 11 cm−1), respectively. μa was proportional to the concentration of MLu measured by an ex vivo fluorescence assay. We have observed, on average, a reduction of the MLu concentration after PDT, presumably due to the PDT consumption of MLu. μeff varied between 0.91 and 6.7 cm−1 (mean 2.9 ± 0.7 cm−1), corresponding to an optical penetration depth (δ= 1/μeff) of 0.1–1.1 cm (mean 0.4 ± 0.1 cm). The mean penetration depth at 732 nm in human prostate is at least two times smaller than that found in normal canine prostates, which can be explained by a four times increase of the mean value of μ′s in human prostates. The mean light fluence rate per unit source strength at 0.5 cm from a point source was 1.5 ± 1.1 cm−2, excluding situations when bleeding occurs. The total number of measurements was N= 121 for all mean quantities listed above. This study showed significant inter‐ and intraprostatic differences in the optical properties, suggesting that a real‐time dosimetry measurement and feedback system for monitoring light fluences during treatment should be considered for future PDT studies.

Optical Properties of Human Prostate at 732 nm Measured In Vivo During Motexafin Lutetium–mediated Photodynamic Therapy¶

Characterization of the tissue light penetration in prostate photodynamic therapy (PDT) is important to plan the arrangement and weighting of light sources so that sufficient light fluence is delivered to the treatment volume. The optical properties (absorption [μa], transport scattering [μs′] and effective attenuation [μeff] coefficients) of 13 patients with locally recurrent prostate cancer were measured in situ using interstitial isotropic detectors. Measurements were made at 732 nm before and after motexafin lutetium (MLu)–mediated PDT in four quadrants. Optical properties were derived by applying the diffusion theory to the fluence rates measured at several distances (0.5–5 cm) from a point source. μa and μs′ varied between 0.07 and 1.62 cm−1 (mean 0.37 ± 0.24 cm−1) and 1.1 and 44 cm−1 (mean 14 ± 11 cm−1), respectively. μa was proportional to the concentration of MLu measured by an ex vivo fluorescence assay. We have observed, on average, a reduction of the MLu concentration after PDT, presumably due to the PDT consumption of MLu. μeff varied between 0.91 and 6.7 cm−1 (mean 2.9 ± 0.7 cm−1), corresponding to an optical penetration depth (δ = 1/μeff) of 0.1–1.1 cm (mean 0.4 ± 0.1 cm). The mean penetration depth at 732 nm in human prostate is at least two times smaller than that found in normal canine prostates, which can be explained by a four times increase of the mean value of μs′ in human prostates. The mean light fluence rate per unit source strength at 0.5 cm from a point source was 1.5 ± 1.1 cm−2, excluding situations when bleeding occurs. The total number of measurements was N = 121 for all mean quantities listed above. This study showed significant inter- and intraprostatic differences in the optical properties, suggesting that a real-time dosimetry measurement and feedback system for monitoring light fluences during treatment should be considered for future PDT studies.

Carbogen breathing significantly enhances the penetration of red light in murine tumours in vivo

We report results of experiments that evaluated the influence of oxygenation on the penetration of red light in tissue, with particular emphasis on 630 and 650 nm laser wavelengths commonly used in photodynamic therapy (PDT) of solid tumours. Direct measurements in tissue-simulating phantoms comprised of intact human erythrocytes suspended in a scattering emulsion demonstrated significant enhancements in fluence rate at depths of 0.5–2 cm from the irradiated surface when the cells were fully oxygenated versus fully deoxygenated. The 630 and 650 nm fluence rates at depth in the homogeneous phantoms continued to increase when examined over a continuous range of oxygen partial pressures from 0 to 160 Torr. When considered as a function of haemoglobin oxygen saturation, the largest increases in fluence rate were observed as the saturation increased beyond 70%. Dramatic increases in optical fluence rate were measured at the base of 1-cm-thick subcutaneous EMT6 mammary carcinomas in vivo when the tumour-bearing mouse was subjected to carbogen through a nose cone. These results indicate that improved tumour oxygenation is important in PDT not only for the maintenance of the oxygen-dependent photochemistry but, through the effects reported here, may also enable more efficient treatment of thicker lesions.

A fluorescent orthotopic model of metastatic cervical carcinoma.

Currently, there is no mouse model of cervical cancer that allows for the study of the later stages of the disease, including metastasis. We report here the development of an orthotopic model of human cervical carcinoma in which tumor fragments are surgically implanted into the cervix of SCID mice. The human cervical carcinoma cell lines used in this study (CaSki, ME-180, and SiHa) have been engineered to stably express the fluorescent proteins enhanced green fluorescent protein (EGFP) or DsRed2, allowing for in vivo optical monitoring of tumor growth and metastasis. The cervical implants develop into large intraperitoneal masses involving the entire reproductive tract, with little local invasion of other abdominal structures. These tumors metastasize initially to local lymph nodes and later to lung, a pattern consistent with the clinical course of the disease. It was found that the use of the DsRed2 protein as a fluorescent marker has distinct advantages over EGFP due to the wavelength of its emission spectrum (575-625 nm vs 500-550 nm). Tissue penetration of light at this wavelength is greater, and the auto-fluorescence of mouse tissues is less intense, resulting in an enhanced signal to noise ratio compared to results obtained with EGFP. This model should allow for a more relevant investigation of the factors that affect the metastasis of cervical carcinoma and presents an opportunity to evaluate potential therapeutic strategies designed to prevent the spread of disease.

Optical Properties of Human Prostate at 732 nm Measured In Vivo during Motexafin Lutetium-Mediated Photodynamic Therapy

Characterization of the tissue light penetration in prostate photodynamic therapy (PDT) is important to plan the arrangement and weighting of light sources so that sufficient light fluence is delivered to the treatment volume. The optical properties (absorption [mu(a)], transport scattering [mu(s)'] and effective attenuation [mu(eff)] coefficients) of 13 patients with locally recurrent prostate cancer were measured in situ using interstitial isotropic detectors. Measurements were made at 732 nm before and after motexafin lutetium (MLu)-mediated PDT in four quadrants. Optical properties were derived by applying the diffusion theory to the fluence rates measured at several distances (0.5-5 cm) from a point source. mu(a) and mu(s)' varied between 0.07 and 1.62 cm(-1) (mean 0.37 +/- 0.24 cm(-1)) and 1.1 and 44 cm(-1) (mean 14 +/- 11 cm(-1)), respectively. mu(a) was proportional to the concentration of MLu measured by an ex vivo fluorescence assay. We have observed, on average, a reduction of the MLu concentration after PDT, presumably due to the PDT consumption of MLu. mu(eff) varied between 0.91 and 6.7 cm(-1) (mean 2.9 +/- 0.7 cm(-1)), corresponding to an optical penetration depth (delta = 1/micro(eff)) of 0.1-1.1 cm (mean 0.4 +/- 0.1 cm). The mean penetration depth at 732 nm in human prostate is at least two times smaller than that found in normal canine prostates, which can be explained by a four times increase of the mean value of mu(s)' in human prostates. The mean light fluence rate per unit source strength at 0.5 cm from a point source was 1.5 +/- 1.1 cm(-2), excluding situations when bleeding occurs. The total number of measurements was N = 121 for all mean quantities listed above. This study showed significant inter- and intraprostatic differences in the optical properties, suggesting that a real-time dosimetry measurement and feedback system for monitoring light fluences during treatment should be considered for future PDT studies.

Measurement of thermal effects on the optical properties of prostate tissue at wavelengths of 1,064 and 633 nm

Background and Objective The extent of thermal injury during laser prostatectomy is dependent on the light distribution in laser-irradiated tissue. As tissue is irradiated, the optical properties change as a function of temperature due to an alteration of molecular and cellular structure. The purpose of the present study was to determine how the exposure of both fresh and previously frozen canine prostate tissue to elevated temperatures affects the optical properties. Study Design/Materials and Methods Optical properties were measured by using a double integrating sphere spectrophotometer with an inverse adding-doubling algorithm. Measurements were made at two wavelengths (1,064 nm and 633 nm) on samples heated in a waterbath in 5°–10° increments for 10 min through a 50°C temperature range. Results Upon coagulation, the absorption coefficient of fresh tissue decreased from the baseline measurement for both wavelengths (0.027 ± 0.003 to 0.019 ± 0.002 for λ = 1,064 nm; 0.073 ± 0.007 to 0.061 ± 0.006 for λ = 633 nm). However, the scattering coefficient increased sharply from the baseline measurement following coagulation (3.06 ± 0.26 to 6.05 ± 0.29 for λ = 1,064 nm; 4.89 ± 0.23 to 7.22 ± 0.30 for λ = 633 nm). Thermal coagulation occurred during exposure to temperatures between 60°C and 70°C. Conclusion Data obtained in this study indicate that thermal coagulation of tissue alters the optical properties. The extent to which these changes occur was found to be dependent on wavelength and freshness of tissue. These results are significant because they suggest how thermally induced changes in the optical properties may limit the depth of light penetration in tissue thus compromising treatment. Lasers Surg. Med. 24:38–47, 1999. © 1999 Wiley-Liss, Inc.

Photodynamic therapy: a promising new modality for the treatment of cancer

The first reports on photodynamic therapy (PDT) date back to the 1970s. Since then, several thousands of patients, both with early stage and advanced stage solid tumours, have been treated with PDT and many claims have been made regarding its efficacy. Nevertheless, the therapy has not yet found general acceptance by oncologists. Therefore it seems legitimate to ask whether PDT can still be described as “a promising new therapy in the treatment of cancer”. Clinically, PDT has been mainly used for bladder cancer, lung cancer and in malignant diseases of the skin and upper aerodigestive tract. The sensitizer used in the photodynamic treatment of most patients is Photofrin®, (Photofrin®, the commercial name of dihematoporphyrin ether/ester, containing >80% of the active porphyrin dimers/oligomers (A.M.R. Fisher, A.L. Murphee and C.J. Gomer, Clinical and preclinical photodynamictherapy, Review Series Article, Lasers Surg. Med., 17 (1995) 2–31). It is a complex mixture of porphyrins derived from hematoporphyrin. Although this sensitizer is effective, it is not the most suitable photosensitizer for PDT. Prolonged skin photosensitivity and the relatively low absorbance at 630 nm, a wavelength where tissue penetration of light is not optimal, have been frequently cited as negative aspects hindering general acceptance. A multitude of new sensitizers is currently under evaluation. Most of these “second generation photosensitizers” are chemically pure, absorb light at around 650 nm or greater and induce no or less general skin photosensitivity. Another novel approach is the photosensitization of neoplasms by the induction of endogenous photosensitizers through the application of 5-aminolevulinic acid (ALA). This article addresses the use of PDT in the disciplines mentioned above and attempts to indicate developments of PDT which could be necessary for this therapy to gain a wider acceptance in the various fields.

Hypocrellins as photosensitizers for photodynamic therapy: a screening evaluation and pharmacokinetic study.

Hypocrellin compounds were selected as potential photosensitizers for photodynamic therapy (PDT) owing to their high quantum yields of singlet oxygen (1O2), and facility for site-directed chemical modification to enhance phototoxicity, pharmacokinetics, solubility, and light absorption in the red spectral region, among other properties. Parent hypocrellins A and B share an absorption peak at 658 nm. These molecules may therefore be considered useful progenitors of derivatives which absorb more strongly in the red, considering that the ideal sensitizer should absorb in the 650-800 nm range, beyond the absorption range of hemoglobin and melanin, and where light penetration in tissues is maximized through reduced scattering. A series of pure, monomeric hypocrellin derivatives was tested for properties of dark cytotoxicity and photosensitizing potential by clonogenic assay in monolayer cultures of EMT6/Ed murine tumor cells. Their respective toxicities are reported on a molar basis. The in vitro screening assay has, to date, resulted in the selection of four hypocrellin derivatives for further development as photosensitizers for PDT. Cellular uptake for photosensitizing doses of selected compounds was determined by fluorimetry. Dose escalation studies in rodents indicate that potentially photosensitizing doses promote no demonstrable systemic toxicity.

Near infra-red diode laser for photodynamic tumour therapy using bacteriochlorina

Clonogenic cell survivals were performed in order to assess the feasibility of tumour cell kill with an experimental diode laser emitting 250 mW of light at λ = 779 nm using the photosensitizer bacteriochlorina(BCA). The AlGaAs diode laser is based on organometallic vapour epitaxial crystal growth technology. The electrical to optical conversion efficiency amounts to 21% and the beam divergence is 47° by 7.0° full width at half maximum. BCA was proved to be an effective non-toxic photosensitizer in vitro and in vivo. It has a major absorption peak at 760 nm where tissue penetration of light is optimal. Clonogenic T24 human bladder carcinoma cell survivals were photosensitizer concentration and light dose dependent. A 0.1% survival rate was obtained with an illumination intensity of 50 mWcm−2 for 90 s (4.5 Jcm−2) and a BCA concentration of 6 μgml−1. Illumination without BCA at energy levels exceeding the PDT levels with a factor 10, or BCA alone without illumination had no effect on the cells in the clonogenic cell survivals. The combination of BCA with a near infra-red diode laser is most promising for photodynamic tumour therapy as a result of the reliability, compactness and relatively low price of the illumination device, the high transmittance of near infra-red light in tissue and the tumour killing potential of BCA.

Sensitizers of photoradiation and ionizing radiation in the management of head and neck cancer

PDT and IUdR-sensitized radiation therapy represent potential advances in the treatment of tumors of the head and neck. Light-activated photosensitizers have definite antitumor activity in both in vitro and in vivo experimental systems. Much of the early clinical work in head and neck cancer involved treatment of patients with advanced, recurrent disease who had not responded to conventional therapy. Because of the limited light penetration in tissue and infiltrative nature of most recurrent lesions, little effective palliation was seen in these advanced cases. More success has been achieved in the treatment of earlier, more superficial lesions, and active investigation continues in this area. Current research is aimed at defining the most appropriate sites and applications for the technique. HpD and DHE are currently only approved for use as investigational compounds in clinical studies. If ongoing trials of PDT in the treatment of superficial bladder cancer, obstructing esophageal cancer, and non-small cell lung cancer show encouraging results, an application will be made to the Food and Drug Administration for approval of DHE as a photosensitizer for general clinical use for these indications. Laboratory work to better understand the mechanism of action of HpD also continues, as well as investigations into alternative photosensitizers with improved tumor localization, less cutaneous photosensitivity, and absorption peaks at deeper penetrating wavelengths of light. A pilot program evaluating IUdR-sensitized radiation therapy for treatment of advanced head and neck cancer is in progress. If encouraging early results continue to be observed, a randomized trial comparing IUdR-sensitized radiation therapy with conventional radiation therapy can be conducted. Hopefully, these developments in the field will improve the therapy for patients with head and neck cancers.

Measurement of in vivo tumorous/normal tissue pH by localized spectroscopy using a fluorescent marker

The fluorescein derivatives are now commonly used for measuring intracellular pH. Cytoplasmic pH is estimated from the shape of the pH-dependent excitation spectrum of the trapped fluorescent marker. The ability of 2',7'-bis-(2 carboxyethyl)-5-(and-6)-carboxyfluorescein as a fluorescent probe for pH monitoring in vivo is evaluated and the in vitro pH sensitivity ofthis fluorescent probe is investigated. In vivo experiments were carried out on tumor-grafted mice. Dyes were injected intraperitoneally and fluorescence emission spectra were collected at different excitation wavelengths by an adapted system bearing optic fibers. Ratios were calculated on the basis of maximum emission wavelength fluorescence intensities at retained excitation wavelengths. The choice of wavelength excitation was restricted to 465 nm because the 450-nm excitation wavelength leads to problems of autofluorescence and tissue penetration of light. At a 500/465 ratio, the pH sensitivity is greatly reduced. Kinetic profiles of fluorescence intensities show low tissue and tumoral uptake with a plateau phase 100 mm after dye injection. Ratio values reach a steady state 30 mm after dye injection and remain stable during the whole experiment. The results clearly indicate differences in ratio values for tumoral and normal tissues (p < 0.005). The tumoral ratio (1.88 ± 0.07) is always lower than normal tissue ratio (2.45 ± 0.09).

Synergism between electrolysis and methylene blue photodynamic action in Escherichia coli.

There is interest in the use of photodynamic therapy for the treatment of certain diseases, including cancer. However, weak penetration of visible light in tissues has restricted its use. In this study the possibility of enhancing photodynamic effects by the use of energies that penetrate more deeply in tissues was investigated. Weak electric currents (1.0 mA) applied to Escherichia coli cells for short periods, producing little or no lethal damage, was found to act synergistically with the photodynamic action of methylene blue, significantly enhancing the effects of this treatment. This synergism exists also between electrolysis and X-rays but not between electrolysis and UV-254 nm. It is suggested that this synergism might eventually be used to improve the results obtained in therapeutic practice based on the utilization of photodynamic action.