Fibrin glue mediated direct delivery of radiation sensitizers results in enhanced efficacy of radiation treatment.

PURPOSE: Radiation therapy (RT) plays an important role in the treatment of glioblastoma multiforme (GBM). However, inherent intrinsic resistance of tumors to radiation, coupled with the need to consider the tolerance of normal tissues and the potential effects on neurocognitive function, impose constraints on the amount of RT that can be safely delivered. A strategy for augmenting the effectiveness of RT involves the utilization of radiation sensitizers (RS). Directly implanting RS-loaded fibrin glue (FG) into the tumor resection cavity would by-pass the blood brain barrier, potentially enhancing the impact of RT on tumor recurrence. This study investigated the ability of FG to incorporate and release, in non-degraded form, the radiation sensitizers 5-Fluorouracil (5FU) and Motexafin gadolinium (MGd). METHODS: FG layers were created in a 24-well plate by combining thrombin, fibrinogen, and 5FU or MGd. Supernatants from these layers were collected at various intervals and added to F98 glioma spheroid cultures in 96-well plates. Radiation was applied either before or after RS application as single or fractionated dosages. Spheroid growth was monitored for 14 days. RESULTS: Combined treatment of FG-released 5FU and RT significantly inhibited spheroid growth compared to RS or RT as a single treatment. As a free drug, MGd demonstrated its efficacy in reducing spheroid volume, but had diminished potency as a released RS. Fractionated radiation was more effective than single dose radiation. CONCLUSION: Non-degraded RS was released from the FG for up to 72 h. FG-released 5FU greatly increased the efficacy of radiation therapy.

Macrophages as a photosensitizer delivery system for photodynamic therapy: Potential for the local treatment of resected glioblastoma.

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 AlPcS2a. 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 (MaPS). 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 MaPS/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. MaPS, 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.

Fibrin glue as a local drug and photosensitizer delivery system for photochemical internalization: Potential for bypassing the blood-brain barrier.

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 (AlPcS2a) 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 AlPcS2a-mediated PCI.

Sonochemical Internalization of Bleomycin Inhibits Adenocarcinoma Breast Tumor Development in an Orthotopic Rat Model.

One approach to reducing post-operative tumor recurrence and alleviate debilitating side effects of systemic chemotherapy, is work centered on the development of drug activation by focused and targeted externally applied physical energy thus providing site and temporal specificity. One such technique, light mediated photochemical internalization (PCI), has been shown to be a method to obtain enhanced chemotherapy efficacy for a wide variety of anti-cancer agents. A related technology, sonochemical internaization (SCI), is an extension of the PCI concept developed to overcome the limitations of poor light penetration in tissue. SCI utilizes ultrasonic energy, to activate sonosensitizers, co-administered with anti cancer agents. The purpose of the study reported here was to evaluate the inhibitory effects of SCI of bleomycin (BLM), both in vitro and in vivo, on the adenocarcinoma breast tumor rat cell line Mat B III. In vitro, the two aspects of sonication, sonoporation (SP) and sonochemical internalization (SCI) of BLM were examined. In vivo, BLM-SCI significantly inhibited tumor development, following Mat B III implantation, in an orthotopic breast tumor animal model using Fisher rats.

Inhibition of glioma development by doxorubicin-photochemical internalization generated macrophage vaccine: A survival study in rats.

BACKGROUND: The process known as immunogenic cell death (ICD) is characterized by dead and dying cancer cells exposing and releasing so-called damage associated molecular patterns (DAMPs). ICD has been shown to enhance the efficacy of antigen presenting cell (APC) immunotherapy. Both anthracycline drugs such as doxorubicin (DOX), and photodynamic therapy (PDT) have been shown to be inducers of ICD. It was therefore hypothesized that combined PDT and DOX i.e. photochemical internalization of DOX (DOX-PCI) would increase ICD compared to DOX acting as a single agent. MATERIALS AND METHODS: F98 glioma cells were treated with DOX-PCI in vitro and the ICD markers HMGB1, HSP70, and HSP90 were determined by ELISA assay. Peritoneal macrophages (Ma), obtained from Fisher rats acting as APCs, were co-incubated with dead F98 glioma cells killed via DOX or DOX-PCI treatment ex vivo. The pulsed Ma (Ma DOX or Ma DOX-PCI) were used to inoculate the animals either before (preventive) or after (curative) intra-cranially implantation of the glioma cells. RESULTS: F98 cells, treated with DOX-PCI in vitro, induced a significantly higher level of HGMB1, HSP70, and HSP90 than DOX acting alone. Ma DOX-PCI inoculated animals, in both preventive and curative protocols, had a pronounced survival benefit compared to either the non-treatment or MaDOX control groups. In the curative protocol, a second booster inoculation significantly improved survival, with 60% of the animals alive at day 60. CONCLUSION: Macrophages primed with DOX-PCI treated glioma cells appeared to be highly effective as APCs and, when injected into host animals, could delay and, in some cases, prevent tumor development.

An improved in vitro photochemical internalization protocol for 3D spheroid cultures.

Photochemical internalization (PCI) is a modified form of photodynamic therapy (PDT) that enhances the efficacy of therapeutic agents in a site and temporal specific manner in both in vitro and in vivo publications. The purpose of the study reported here was to evaluate the benefits of a modified PCI protocol in a 3D rat glioma spheroid model. In the modified protocol, F98 glioma cells were incubated with photosensitizer (AlPcS2a) prior to spheroid generation, as opposed to post-spheroid formation photosensitizer exposure commonly used in conventional protocols. The efficacy of both bleomycin and doxorubicin PCI was evaluated using either the conventional or modified protocols. The formed spheroids were then exposed to light treatment from a diode laser, λ= 670 nm. Spheroid growth was monitored for a period of 14 days. The results of spheroid growth assays showed that there was no statistically significant difference in PCI efficacy between the conventional and modified protocols for both of the drugs tested. The direct PDT effect was significantly reduced using the modified protocol. Therefore, due to its several advantages, the modified protocol is recommended for evaluating the efficacy of PCI in tumor spheroid models.

Photosensitizer delivery by fibrin glue: potential for bypassing the blood-brain barrier.

Fibrin glue (FG) has potential as a delivery vehicle for photosensitizer directly to the resection cavity, so it may bypass the blood-brain barrier (BBB) and increase the concentration of successfully delivered photosensitizer. A specialized form of photodynamic therapy (PDT), photochemical internalization (PCI), which involves both photosensitizer and chemotherapeutic agent internalization, can locally inhibit the growth of cells. This will allow the reduction of recurrence of malignant gliomas around surgical resection. This study will look at the efficacy of FG loaded with drugs in mediating both PDT and PCI in inhibiting 3-dimensional tumor spheroid growth in vitro. Experiments were conducted on spheroids comprised of F98 glioma cells using photosensitizer AlPcS2a and chemotherapeutic drug bleomycin (BLM). At 2-, 24-, 48-, and 72-h increments, supernatant covering an FG layer within a well was collected and replaced by fresh medium, then added to spheroid-containing wells, which contained the respective chemicals for PDT and PCI. The wells were then exposed to light treatment from a diode laser, and after, spheroid growth was monitored for a period of 14 days. Significant spheroid growth inhibition was observed in both PDT and PCI modalities, but was far greater in PCI. Additionally, complete growth suppression was achieved via PCI at the highest radiant exposure. Achieving a slow photosensitizer release, significant F98 spheroid inhibition was observed in FG-mediated PDT and PCI. The present study showed BLM-PCI was the most efficacious of the two modalities.

Enhanced gene transfection of macrophages by photochemical internalization: Potential for gene-directed enzyme prodrug therapy of gliomas.

BACKGROUND: Drawn by tumor synthesis of chemo-attractive factors, macrophages are frequently found in and around glioblastomas and play an important role both in augmenting as well as inhibiting tumor growth. Patient-derived macrophages have the potential, therefore, to act as targeted delivery vectors for a variety of anti-cancer treatments. Among these is ex vivo gene transfection and re-injection back into the patient of macrophages to target residual tumors. In this study, photochemical internalization (PCI) is investigated as a technique for the non-viral transfection of the cytosine deaminase (CD) prodrug activating gene into macrophages. The CD gene encodes an enzyme that converts the nontoxic antifungal agent, 5-fluorocytosine (5-FC), into 5-fluorouracil (5-FU) - a potent chemotherapeutic agent. MATERIALS: PCI (photosensitizer + light treatment) mediated CD gene transfection of rat alveolar Ma cells was carried out in vitro. CD gene transfected NR8383 macrophages were co-cultured with F98 rat glioma cells in the presence or absence of 5-FC. Cell viability was assayed using the MTS colorimetric assay. RESULTS: Compared to the glioma cells, NR8383 demonstrated enhanced resistance to the toxic effects of 5-FU. PCI greatly increased the transfection efficiency of the CD gene in NR8383 cells. The viability of F98 cells was significantly inhibited by coculture with CD transfected NR8383 macrophages and 5-FC. CONCLUSION: Although gene insertion into macrophages has proven difficult, the results presented here show that non-viral transfection of the CD gene into these immune cells can be enhanced via PCI. CD transfected NR8383 cells could efficiently convert 5-FC to 5-FU and export the drug, producing a pronounced bystander toxic effect on adjacent non-transfected glioma cells. Compared to single treatment, repetitive PCI-induced transfection was more efficient at low CD plasmid concentration.

Biomimetic Gold Nanoshell-Loaded Macrophage for Photothermal Biomedicine.

The purpose of this study was to investigate the effect of photothermal treatment (PTT) with gold nanoshell (ANS) using a macrophage-mediated delivery system in a head and neck squamous cell carcinoma (HNSCC) cell line. To achieve this, ANS-loaded rat macrophages (ANS-MAs) were prepared via the coculture method with ANS. The human HNSCC (FaDu cell) and macrophage (rat macrophage; NR8383 cell) hybrid spheroid models were generated by the centrifugation method to determine the possibility of using ANS-MAs as a cancer therapy. These ANS-MAs were set into the tumor and macrophage hybrid spheroid model to measure PTT efficacy. Kinetic analysis of the spheroid growth pattern revealed that this PTT process caused a decreasing pattern in the volume of the hybrid model containing ANS-MAs (p < 0.001). Comparison with empty macrophages showed harmony between ANS and laser irradiation for the generation of PTT. An annexin V/dead cell marker assay indicated that the PTT-treated hybrid model induced increasing apoptosis and dead cells. Further studies on the toxicity of ANS-MAs are needed to reveal whether it can be considered biocompatible. In summary, the ANS was prepared with a macrophage as the delivery method and protective carrier. The ANS was successfully localized to the macrophages, and their photoabsorption property was stationary. This strategy showed significant growth inhibition of the tumor and macrophage spheroid model under NIR laser irradiation. In vivo toxicology results suggest that ANS-MA is a promising candidate for a biocompatible strategy to overcome the limitations of fabricated nanomaterials. This ANS-MA delivery and PTT strategy may potentially lead to improvements in the quality of life of patients with HNSCC by providing a biocompatible, minimally invasive modality for cancer treatment.

Photochemical Internalization for Intracellular Drug Delivery. From Basic Mechanisms to Clinical Research.

Photochemical internalisation (PCI) is a unique intervention which involves the release of endocytosed macromolecules into the cytoplasmic matrix. PCI is based on the use of photosensitizers placed in endocytic vesicles that, following light activation, lead to rupture of the endocytic vesicles and the release of the macromolecules into the cytoplasmic matrix. This technology has been shown to improve the biological activity of a number of macromolecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus and oligonucleotides and certain chemotherapeutics, such as bleomycin. This new intervention has also been found appealing for intracellular delivery of drugs incorporated into nanocarriers and for cancer vaccination. PCI is currently being evaluated in clinical trials. Data from the first-in-human phase I clinical trial as well as an update on the development of the PCI technology towards clinical practice is presented here.

The effects of low irradiance long duration photochemical internalization on glioma spheroids.

BACKGROUND: Photodynamic therapy (PDT), if given over extended time periods (i.e. hours or days) and at very low irradiance in the µW/cm2 range, has been shown to be more effective than acute PDT (aPDT) administered over minutes. This has led to the concept of metronomic PDT (mPDT), which consists of ultra-low irradiance light illumination for extended periods of time along with either continuous or repetitive delivery of photosensitizer. Since the drug activating technology photochemical internalization (PCI) is based on PDT it seemed reasonable to expect that ultra-low irradiance, if administered over an extended period of time, could nevertheless result in effective metronomic PCI (mPCI) comparable to or more effective than that obtained with relatively high and short irradiance i.e. acute PCI (aPCI). METHODS: Tumor spheroids consisting of F98 cells were used as in-vitro tumor models. The amphiphilic photosensitizer Al phthalocyanine disulfonate (AlPcS2a) was used for all PCI experiments. Light treatment was administered from a diode laser at λ=670 nm at various irradiance exposures of 2 mW/cm2 for aPCI and 0.05 - 0.2 mW/cm2 for mPCI with durations ranging from 3 to 12 min for aPCI and 120 min for mPCI. RESULTS: AlPcS2a fluorescence was seen throughout the cytosol following short or long light treatment, corresponding to aPCI and mPCI respectively. Spheroid growth was significantly inhibited or completely suppressed at a mPCI radiance of 0.05 or 0.72 J/cm2 respectively, with all bleomycin (BLM) concentrations used, compared to either BLM alone or aPCI at radiant exposure at these levels. The effects of BLM-aPCI and mPCI were comparable at radiance levels of 0.96 and 1.44 J/cm2. CONCLUSIONS: Results show that mPCI could effectively cause significant spheroid growth inhibition with the delivery of extremely low light irradiance rates delivered over an extended period of time. These findings suggest that effective implementation of mPCI can deliver adequate drug efficacy at depths necessary to reach infiltrating glioma cells in the surgical resection cavity wall.

Macrophages as delivery vehicles for anticancer agents.

The delivery of anticancer agents via passive approaches such as the enhanced permeability and retention effect is unlikely to achieve sufficient concentrations throughout the tumor volume for effective treatment. Cell-based delivery approaches using tumor tropic cells have the potential to overcome the limitations of passive approaches. Specifically, this review focuses on the use of monocytes/macrophages for the delivery of a variety of anticancer agents, including nanoparticles, chemotherapeutics and gene constructs. The efficacy of this delivery approach, both as monotherapy and in combination with light-based phototherapy modalities, has been demonstrated in numerous in vitro and animal studies, however, its clinical potential remains to be determined.

Photochemical Internalization Enhanced Nonviral Suicide Gene Therapy.

Nonviral gene transfection overcomes some of the disadvantages of viral vectors, such as undesired immune responses, safety concerns, issues relating to bulk production, payload capacity, and quality control, but generally have low transfection efficiency. Here we describe the effects of a modified form of photodynamic therapy (PDT), i.e., photochemical internalization (PCI) to: (1) greatly increase nonviral cytosine deaminase gene (CD) transfection into tumor cells, significantly increasing the conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), and (2) enhance the toxic efficacy of the locally produced 5-FU to induce cell death on both transfected and non-transfected bystander cells.

Photodynamic therapy mediated immune therapy of brain tumors.

Photodynamic therapy of tumors requires the topical, systemic or oral administration of a photosensitizing compound, illumination of the tumor area by light of a specific wavelength and the presence of oxygen. Light activation of the photosensitizer transfers energy to molecular oxygen creating singlet oxygen, a highly reactive and toxic species that rapidly reacts with cellular components causing oxidative damage, ultimately leading to cell death. Tumor destruction caused by photodynamic therapy is not only a result of direct tumor cell toxicity via the generation of reactive oxygen species but there is also an immunological and vascular component involved. The immune response to photodynamic therapy has been demonstrated to significantly enhance its efficacy. Depending on a number of factors, including type of photosensitizer, light dose and dose rate, photodynamic therapy has been shown to induce cell death via apoptosis, necrosis, autophagy and in particular immunogenic cell death. It is the purpose of this review to focus mainly on the role photodynamic therapy could play in the generation of specific anti-tumor immunity and vaccines for the treatment of brain tumors.

Enhancing the effects of chemotherapy by combined macrophage-mediated photothermal therapy (PTT) and photochemical internalization (PCI).

Light-based treatment modalities such as photothermal therapy (PTT) or photochemical internalization (PCI) have been well documented both experimentally and clinically to enhance the efficacy of chemotherapy. The main purpose of this study was to examine the cytotoxic effects of silica-gold nanoshell (AuNS)-loaded macrophage-mediated (MaNS) PTT and bleomycin BLM-PCI on monolayers of squamous cell carcinoma cells. The two modalities were applied separately and in simultaneous combination. Two different wavelengths of light were employed simultaneously, one to activate a highly efficient PCI photosensitizer, AlPcS2a (670 nm) and the other for the MaNS-mediated PTT (810 nm), to evaluate the combined effects of these modalities. The results clearly demonstrated that macrophages could ingest sufficient numbers of silica-gold nanoshells for efficient near infrared (NIR) activated PTT. A significant synergistic effect of simultaneously applied combined PTT and PCI, compared to each modality applied separately, was achieved. Light-driven therapies have the advantage of site specificity, non-invasive and non-toxic application, require inexpensive equipment and can be given as repetitive treatment protocols.

Limiting glioma development by photodynamic therapy-generated macrophage vaccine and allo-stimulation: an in vivo histological study in rats.

Immunotherapy of brain tumors involves the stimulation of an antitumor immune response. This type of therapy can be targeted specifically to tumor cells thus sparing surrounding normal brain. Due to the presence of the blood-brain barrier, the brain is relatively isolated from the systemic circulation and, as such, the initiation of significant immune responses is more limited than other types of cancers. The purpose of this study was to show that the efficacy of tumor primed antigen presenting macrophage (MaF98) vaccines can be increased by: (1) photodynamic therapy (PDT) of the priming tumor cells and (2) intracranial injection of allogeneic glioma cells directly into the tumor site. Experiments were conducted in an in vivo brain tumor development model using Fischer rats and F98 (syngeneic) and BT4C (allogeneic) glioma cells. The results showed that immunization with Ma (acting as antigen-presenting cells), primed with PDT-treated tumor cells (MaF98), significantly slowed but did not prevent the growth of F98-induced tumors in the brain. Complete suppression of tumor development was obtained via MaF98 inoculation combined with direct intracranial injection of allogeneic glioma cells. No deleterious effects were noted in any of the animals during the 14-day observation period.

Photochemical internalization enhanced macrophage delivered chemotherapy.

BACKGROUND: Macrophage (Ma) vectorization of chemotherapeutic drugs has the advantage for cancer therapy in that it can actively target and maintain an elevated concentration of drugs at the tumor site, preventing their spread into healthy tissue. A potential drawback is the inability to deliver a sufficient number of drug-loaded Ma into the tumor, thus limiting the amount of active drug delivered. This study examined the ability of photochemical internalization (PCI) to enhance the efficacy of released drug by Ma transport. METHODS: Tumor spheroids consisting of either F98 rat glioma cells or F98 cells combined with a subpopulation of empty or doxorubicin (DOX)-loaded mouse Ma (RAW264.7) were used as in vitro tumor models. PCI was performed with the photosensitizer AlPcS2a and laser irradiation at 670 nm. RESULTS: RAW264.7 Ma pulsed with DOX released the majority of the incorporated DOX within two hours of incubation. PCI significantly increased the toxicity of DOX either as pure drug or derived from monolayers of DOX-loaded Ma. Significant growth inhibition of hybrid spheroids was also observed with PCI even at subpopulations of DOX-loaded Ma as low as 11% of the total initial hybrid spheroid cell number. CONCLUSION: Results show that RAW264.7 Ma, pulsed with DOX, could effectively incorporate and release DOX. PCI significantly increased the ability of both free and Ma-released DOX to inhibit the growth of tumor spheroids in vitro. The growth of F98 + DOX loaded Ma hybrid spheroids were synergistically reduced by PCI, compared to either photodynamic therapy or released DOX acting alone.

Photothermal Therapy Employing Gold Nanoparticle- Loaded Macrophages as Delivery Vehicles: Comparing the Efficiency of Nanoshells Versus Nanorods.

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.

Synergistic efficacy of ultrasound, sonosensitizers and chemotherapy: a review.

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.

Photodynamic therapy enhances the efficacy of gene-directed enzyme prodrug therapy.

INTRODUCTION: Gene-directed enzyme prodrug therapy (GDEPT) employing the cytosine deaminase (CD) gene, which encodes an enzyme that converts the nontoxic agent 5-fluorocytosine (5-FC) into the chemotherapeutic drug 5-fluorouracil (5-FU), has shown promise both in experimental animals and in clinical trials. Nevertheless, with the transfection systems available presently the percentage of tumor cells incorporating the desired gene is usually too low for successful therapy. We have examined the ability of photodynamic therapy (PDT) to enhance the efficacy of the metabolites, converted from 5-FC by CD gene transfected rat glioma cells. METHODS: Hybrid tumor cell spheroids consisting of CD poitive and CD negative F98 glioma cells in varying ratios were used as in vitro tumor models. PDT was performed with the photosensitizer AlPcS2a and λ=670nm laser irradiance, both before and after confrontation with 5-FC. RESULTS: PDT increased the toxicity of 5-FU either as pure drug or derived from monolayers of CD positive cells chalanged with 5-FC. PDT in combination with 5-FC resulted in a significantly enhanced inhibition of hybrid spheroid growth compared to non light treated controls. This was the case even at tumor to producer cell ratios as high as 40:1. CONCLUSION: The results of the present study show that GDEPT and PDT interact in a synergistic manner over a range of prodrug concentration and tumor to transfected cell ratios. The degree of synergy was significant regardless if PDT treatment was given before or after 5-FC administration. The highest degree of interaction was observed though, when PDT was delivered prior to prodrug exposure.