Chemotherapeutic agents, either in the form of systemically injected free drug or encapsulated in nanoparticles transport vehicles, must overcome three main obstacles prior to reaching and interacting with their intended target inside tumor cells. Drugs must leave the circulation, overcome the tissue-tumor barrier and penetrate the cell's plasma membrane. Since, many agents enter the cell by endocytosis, they must avoid entrapment and degradation by the intracellular endolysosome complex. Ultrasound has demonstrated potential to enhance the efficacy of chemotherapy by reducing these barriers. The purpose of this review is to highlight the potential of ultrasound in combination with sonosensitizers to enhance the efficacy of chemotherapy by optimizing the anticancer agent's intracellular ability to engage and interact with its target.

Gold based nanoparticles with strong near infra-red (NIR) absorption are ideally suited for photothermal therapy (PTT) of brain tumors. The goal of PTT is to induce rapid heating in tumor tissues while minimizing thermal diffusion to normal brain. PTT efficacy is sensitively dependent on both nanoparticle concentration and distribution in tumor tissues. Nanoparticle delivery via passive approaches such as the enhanced permeability and retention (EPR) effect is unlikely to achieve sufficient nanoparticle concentrations throughout tumor volumes required for effective PTT. A simple approach for improving tumor biodsitribution of nanoparticles is the use of cellular delivery vehicles. Specifically, this review focuses on the use of monocytes/macrophages (Mo/Ma) as gold nanoparticle delivery vectors for PTT of brain tumors. Although the efficacy of this delivery approach has been demonstrated in both in vitro and animal PTT studies, its clinical potential for the treatment of brain tumors remains uncertain.

Macrophages (Ma) loaded with gold based nanoparticles, which convert near infrared light to heat, have been studied as targeted transport vectors for photothermal therapy (PTT) of tumors. The purpose of the experiments reported here was to compare the efficacy of gold-silica nanoshells (AuNS) and gold nanorods (AuNR) in macrophage mediated PTT. PTT efficacy was evaluated in hybrid glioma spheroids consisting of human glioma cells and either AuNS or AuNR loaded Ma, designated MaNS and MaNR respectivly. Spheroids were irradiated for 10 min. with light from an 810 nm diode laser at irradiances ranging from 0 to 28 W/cm2. PTT efficacy was determined from spheroid growth over a 14-day period. The uptake by Ma of pegylated AuNR (3.9 ± 0.9 %) was twice that of pegylated AuNS, (7.9 ± 0.7 %). Hybrid spheroids consisting of a 5:1 ratio of glioma cells to loaded Ma exhibited significant growth inhibition with MaNS when subjected to irradiances of 7 W/cm2 or greater. In contrast, no significant growth inhibition was observed for the MaNR hybrid spheroids at this 5:1 ratio, even at the highest irradiance investigated (28 W/cm2). Although AuNR were taken up by Ma in larger numbers then AuNS, MaNS were shown to have greater PTT efficacy compared to MaNR for equivalent numbers of loaded Ma.

Background

Photodynamic therapy (PDT) efficacy is determined in part by the concentration of photosensitizer (PS) at the treatment site. The blood-brain barrier (BBB) poses a significant limitation on the transport of PS into the post-operative resection region where brain tumors most often recur. Macrophages (Ma), as opposed to free or nanoparticle bound agents, are known to actively migrate to and around tumors, and can therefore be used as delivery vectors for both drugs and photosensitizers.

Methods

Mouse Ma (RAW264.7) and F98 rat glioma cells were used in all experiments along with the photosensitizer AlPcS_2a. Mitomycin-treated Ma were loaded with photosensitizer (PS) and mixed with glioma cells, forming hybrid spheroids. F98 spheroids were incubated with supernatants derived from PS-loaded Ma (Ma^PS). Light treatment (PDT) was administered at various radiant exposures from a 670 nm diode laser. The growth of both types of spheroids was evaluated by measurement of spheroid volume after 14 days in culture.

Results

PDT on F98 cell spheroid cultures, mediated by either free or PS-released from Ma, demonstrated a significant growth inhibition with supernatants harvested after 4 and 24 h. A significant PDT-induced growth inhibition was demonstrated in the Ma^PS/F98 hybrid spheroid experiments.

Conclusion

Since the efficacy of PDT, mediated by either free or released photosensitizer was comparable, the uptake and released photosensitizer was not degraded. Ma^PS, incorporated in hybrid tumor spheroids also mediated effective PDT. These results indicate that Ma have potential as an effective vector for photosensitizer delivery to resected brain tumors.

Gold-based nanoparticles have been used in a number of therapeutic and diagnostic applications. The purpose of this study was to investigate the efficacy of gold-silica nanoshells (AuNS) in photothermal therapy (PTT) of rat gliomas. Rat alveolar macrophages (Ma) were used as nanoparticle delivery vectors. Uptake of AuNS (bare and PEGylated) was investigated in Ma. AuNS were incubated with Ma for 24 h. Phase contrast microscopy was used to visualize the distribution of loaded Ma in three-dimensional glioma spheroids. PTT efficacy was evaluated for both empty (Ma) and AuNS-loaded Ma (Ma(NS)) in both monolayers and spheroids consisting of C6 rat glioma cells and Ma. Monolayers/spheroids were irradiated for 5 min with light from an 810-nm diode laser at irradiances ranging from 7 to 28 W cm(-2). Monolayer survival was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay while PTT efficacy in spheroids was determined from growth kinetics and live/dead fluorescence microscopy. PTT efficacy was investigated in vivo using a Sprague-Dawley rat glioma model. Five rats received direct intracranial injection of a mixture of 10(4) C6 glioma cells and, 2 days later, an equal number of Ma(NS). Three rats received laser treatment (810 nm; 10 min; 1 W) while the remaining two served as controls (no laser treatment). The uptake ratio of bare to PEGylated AuNS by Ma was 4:1. A significant photothermal effect was observed in vitro, albeit at relatively high radiant exposures (2.1-4.2 kJ cm(-2)). PTT proved effective in vivo in preventing or delaying tumor development in the PTT-treated animals.

The effects of photodynamic therapy (PDT) in human glioma spheroids incubated in 5-aminolevulinic acid (ALA), or ALA esters, are investigated. Spheroid survival and growth are monitored following PDT at representative drug concentrations, light doses, and dose rates. The primary finding of this study is that the response of human glioma spheroids to PDT with lipophilic ester derivatives, such as benzyl-ALA and hexyl-ALA, is equivalent to that observed with ALA, however, this equivalency is obtained for ester concentrations 10-20 times lower than the parent compound. The enhanced efficiency of the esters is likely due to their increased membrane penetrance. Potential clinical advantages of using lipophilic esters in PDT of gliomas are discussed.

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.

Background

Chemotherapy has had disappointing results in the treatment of glioblastoma multiforme (GBM). This is in part due to limited systemic drug penetration through the blood-brain barrier. This limitation can be overcome by implantation of drug-loaded hydrogels, such as fibrin glue (FG), directly into the tumor resection cavity. Photochemical internalization (PCI) has been shown to enhance the efficacy of a large number of chemotherapeutic agents, including bleomycin (BLM). This study examined the ability of loaded FG to release BLM and photosensitizer to enable PCI-induced growth inhibition of glioma spheroids in vitro.

Materials and methods

FG layers, loaded with drug and photosensitizer, were formed in wells of a 24-well plate. Supernatants covering the FG layers were harvested after 48 h. F98 glioma spheroids were co-incubated with harvested supernatants for 24 h, followed by light exposure. Spheroid growth was monitored for an additional 14 days.

Results

100% of the drug bleomycin and 90% of the photosensitizer (AlPcS_2a) was released from the FG over a 48 h interval. Spheroid growth was significantly inhibited or completely suppressed by PCI of released drug and photosensitizer in many of the concentration combinations tested. PCI-induced growth inhibition increased with increasing light levels.

Conclusions

The results demonstrate that both drug and photosensitizer were loaded into and released in a non-degraded form for an extended time period. The growth inhibition caused by FG-released BLM was significantly enhanced by FG-released AlPcS_2a-mediated PCI.

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.

The effects of fluence rate are investigated in human glioma spheroids incubated in 5-aminolevulinic acid (ALA). It is shown that the response of glioma spheroids to ALA-mediated PDT depends strongly on the rate at which the light dose is delivered. At low doses, lower fluence rates are more effective. For example, at a dose of 50 J/cm2, near total spheroid kill is observed at fluence rates of as low as 10 mW/cm2. Below 10 mW/cm2, however, treatments are not as effective. The fluence rate effect is not as pronounced at higher doses where a favorable response is observed throughout the range of fluence rates investigated. The clinical implications of these findings are discussed.