Steen J. Madsen

Photothermal treatment of glioma; an in vitro study of macrophage-mediated delivery of gold nanoshells

One of the major factors that limits the treatment effectiveness for gliomas is the presence of the blood–brain barrier (BBB) which protects infiltrating glioma cells from the effects of anti-cancer agents. Circulating monocytes/macrophages (Ma) have a natural ability to traverse the intact and compromised BBB and loaded with anti cancer agents could be used as vectors to target tumors and surrounding tumor infiltrated tissue. Nanoshells (NS) are composed of a dielectric core (silica) coated with an ultrathin gold layer which converts absorbed near-infrared light (NIR) to heat with an extremely high efficacy and stability. We have investigated the effects of exposure to laser NIR on multicell human glioma spheroids infiltrated with empty (containing no nanoshells) or nanoshell loaded macrophages. Our results demonstrated that; (1) macrophages could efficiently take up bare or coated (PEGylated) gold NS: (2) NS loaded macrophages infiltrated into glioma spheroids to the same or, in some cases, to a greater degree than empty Ma; (3) NIR laser irradiation of spheroids incorporating NS loaded macrophages resulted in complete growth inhibition in an irradiance dependent manner, and (4) spheroids infiltrated with empty macrophages had growth curves identical to untreated control cultures. The results of this study provide proof of concept for the use of macrophages as a delivery vector of NS into gliomas for photothermal ablation and open the possibility of developing such regimens for patient treatment.

PORTABLE, HIGH-BANDWIDTH FREQUENCY-DOMAIN PHOTON MIGRATION INSTRUMENT FOR TISSUE SPECTROSCOPY

We describe a novel frequency-domain photon migration instrument employing direct diode laser modulation and avalanche photodiode detection, which is capable of noninvasively determinating the optical properties of biological tissues in near real time. An infinite medium diffusion model was used to extract absorption and transport scattering coefficients from 300-kHz to 800-MHz photon-density wave phase data. Optical properties measured in tissue-simulating solutions at 670 nm agreed to within 10% of those expected.

A mathematical model for light dosimetry in photodynamic destruction of human endometrium

We are involved in the development of photodynamic therapy (PDT) as a minimally invasive method for treating dysfunctional uterine bleeding, one of the primary clinical indications for hysterectomy. In this paper, we analyse light propagation through the uterus in order to specify the requirements for a light delivery system capable of effectively performing endometrial PDT. Our approach involves developing an analytical model based on diffusion theory to predict optical fluence rate distributions when cylindrical and spherical optical applicators are placed in the uterine cavity. We apply the results of our model calculations to estimate the thermal effects of optical irradiation and the effective photodynamic optical dose. Theoretical fluence rate calculations are compared to fluence rate measurements made in fresh, surgically removed human uteri. Our results show that a trifurcated cylindrical optical applicator inserted into the human uterus can provide a light dose that is sufficient to cause photodynamic destruction of the entire endometrium. When the optical power per unit length of each cylindrical applicator is 100 mW cm-1 (at 630 nm), a fluence rate of 40 mW cm-2 is delivered to the boundary layer between the endometrium and the myometrium (a depth of about 4-6 mm). The optical fluence delivered to the boundary layer after 20 min of exposure is 50 J cm-2, a level that is generally accepted to cause tissue damage throughout the endometrium in most patients.

Effects of Combined Photodynamic Therapy and Ionizing Radiationon Human Glioma Spheroids

Abstract The effects of combined photodynamic therapy (PDT) and ionizing radiation are studied in a human glioma spheroid model. The degree of interaction between the two modalities depends in a complex manner on factors such as PDT irradiation fluence, fluence rate and dose of ionizing radiation. It is shown that gamma radiation and PDT interact in a synergistic manner only if both light fluence and gamma radiation dose exceed approximately 25 J cm−2 and 8 Gy, respectively. Synergistic interactions are observed only for the lower fluence rate (25 mW cm−2) investigated. The degree of interaction appears to be independent of both sequence and the PDT or ionizing radiation time intervals investigated (1 and 24 h). Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling assays show that low-fluence rate PDT is very efficient at inducing apoptotic cell death, whereas neither high-fluence rate PDT nor ionizing radiation produces significant apoptosis. Although the mechanisms remain to be elucidated, the data imply that the observed synergism is likely not due to gamma-induced cell cycle arrest or to PDT-induced inhibition of DNA repair.

Determination of the optical properties of the human uterus using frequency-domain photon migration and steady-state techniques

The optical properties (absorption and transport scattering coefficients) of freshly excised, bulk human uterine tissues were measured at 630 nm using frequency-domain and steady-state photon migration techniques. Measurements were made on both normal (pre- and post-menopausal) and non-neoplastic fibrotic tissues. The absorption coefficient of normal post-menopausal tissue (approximately 0.06 mm(-1)) was found to be significantly greater than that of normal pre-menopausal tissue (0.02-0.03 mm(-1)) and pre-menopausal fibrotic tissue (0.008 mm(-1)). The transport scattering coefficient was similar in all three tissue types considered (0.6-0.9 mm(-1)). From the preliminary results presented here, we conclude that optical properties can be reliably calculated either from the frequency-dependent behaviour of diffusely propagating photon density waves or by combining the frequency-independent photon density wave phase velocity with steady-state light penetration depth measurements. Instrument bandwidth and tissue absorption relaxation time ultimately determine the useful frequency range necessary for frequency-domain photon migration (FDPM) measurements. Based on the optical properties measured in this study, we estimate that non-invasive FDPM measurements of normal uterine tissue require modulation frequencies in excess of 350 MHz.

Bypassing the blood brain barrier: delivery of therapeutic agents by macrophages

Introduction: Failure to eradicate infiltrating glioma cells using conventional treatment regimens results in tumor recurrence and is responsible for the dismal prognosis of patients with glioblastoma multiforme (GBM). This is due to the fact that these migratory cells are protected by the blood-brain barrier (BBB) and the blood brain tumor barrier (BBTB) which prevents the delivery of most anti-cancer agents. We have evaluated the ability of monocytes/macrophages (Mo/Ma) to cross the BBB in rats. This will permit access of anti-cancer agents such as nanoparticles to effectively target the infiltrating tumor cells, and potentially improve the treatment effectiveness for malignant gliomas. Materials and Methods: The infiltration of Mo/Ma into brain tumor spheroids in vitro was determined using fluorescent stained Mo/Ma. Tumors were also established in the brains of inbred rats and ALA-PDT was given 18 days following tumor induction. The degredation of the BBTB and quantification of the number of infiltrating Mo/Ma was examined on histological sections from removed brains. Results & Conclusion: PDT was highly effective in locally opening the BBTB and inducing macrophage migration into the irradiated portions of brain tumors.

Evaluation of Motexafin gadolinium (MGd) as a contrast agent for intraoperative MRI.

OBJECTIVE The characteristics of an ideal contrast agent for use in the intraoperative MRI would be tumor-specificity and intracellular localization, combined with extended tumor enhancement, but with rapid elimination from the blood. The radiation sensitizing properties of Motexafin gadolinium (MGd) have been investigated in a number of clinical trials involving patients with brain metastases. These studies clearly show that MGd is detectable in magnetic resonance images many days following administration. The aim of this experimental study was to test whether Motexafin gadolinium (MGd) could serve as an efficient intraoperative contrast agent avoiding problems that arise with surgically induced intracranial enhancement. METHODS F98 orthotopic brain tumors or surgical lesions were induced in Fisher rats. T1-weighted MRI studies were performed with either a single or multiple daily doses of MGd. The last contrast dose was administered either 7 or 24 hours prior to scanning in both tumor-bearing or surgically-treated animals. All scans were T1-weighted nce (TR=495 ms; TE=1 ms.) with a slice thickness of 1.0 mm. Three tubes containing 2.3, 0.23 and 0.023 mg/mL of MGd (in physiological saline) respectively, were used as standards to calibrate the scans. RESULTS Animals receiving either 30 or 60 mg/kg MGd i.v. developed clinical signs of impaired motor activity, and increasing lethargy and were euthanized 48 hours after MGd administration due to their poor and deteriorating condition. MGd given i.p. was tolerated up to a dose of 140 mg/kg. Despite multiple dosages and several administration modes (i.p., i.v.) no significant enhancement was observed if the scans were performed 7 or 24 hours following the last MGd dose. Clear enhancement was seen though when the scans were performed 30 min following MGd administration, indicating that the agent was being taken up by the tumor. Scans of necrotic lesions though were positive though 7 hours following MGd injection. MGd scans had no significant enhancement following surgically-induced lesions while scans with conventional contrast agents showed both meningeal and intraparenchymal enhancement. CONCLUSION This study suggests that MGd is not sequestered in viable tumor for the necessary time interval required to allow delayed imaging in this model. The agent does seem to remain in necrotic tissue for longer time intervals. MGd therefore would not be suitable as a contrast agent in intraoperative MRI for the detection of remaining tumor tissue during surgery.

Repetitive 5-aminolevulinic acid mediated photodynamic therapy of rat glioma

The probability of achieving local control with current single-shot, intraoperative PDT treatments of intracerebral gliomas seems improbable due to the length of time required to deliver adequate light fluences to depths of 1 - 2 cm in the resection margin. The results of in vitro experiments indicated that PDT, repeated at weekly intervals, was substantially more effective at inhibiting glioma spheriod growth than single treatment regimes. This prompted the initiation of in vivo studies of repetitive PDT in a rat glioma model. BT4C cell line tumors were established in the brains of inbred BD-IX rats. Three days following tumor induction, the animals were injected with 250 mg/kg ALA i.p. and four hours later, after the introduction of an optical fiber, light treatment at various radiant energies was given over a 10- to 30-minute interval. Two additional treatments were given at weekly intervals. In vitro experiments verified that spheroids derived from the cell line were sensitive to ALA PDT. Microfluorometry of frozen tissue sections showed that PpIX is produced with a 10 - 20:1 tumor to normal tissue selectivity ratio four hours after 250 mg/kg ALA i.p. Toxic radiant energy levels for ALA PDT have been determined.