Increased sensitivity of glioma cells to 5-fluorocytosine following photo-chemical internalization enhanced nonviral transfection of the cytosine deaminase suicide gene.

Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80% of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments.PCI was performed with the photosensitizer AlPcS2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect.

Glioma cell growth inhibition following photochemical internalization enhanced non-viral PTEN gene transfection.

BACKGROUND AND OBJECTIVE: One of many limitations for cancer gene therapy is the inability of the therapeutic gene to transfect a sufficient number of tumor cells. Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. The utility of PCI for the delivery of the GFP reporter gene on the same plasmid as a tumor suppressor gene (PTEN) was investigated in monolayers of U251 human glioma cells and muticell U87 glioma spheroids. MATERIALS AND METHODS: U251 monolayers or U87 spheroids were incubated in AlPcS(2a) and non-viral vector polyplexes for 18 hours. In all cases, light treatment was performed with a diode laser at a wavelength of 670 nm. The non-viral transfection agents, branched polyethylenimine (bPEI), or protamine sulfate (PS), were used with the plasmid constructs GFP/PTEN or GFP. RESULTS: PS/GFP polyplexes were much less toxic to the glioma cells compared to bPEI/GFP polyplexes but were highly inefficient at gene transfection if used alone. PCI resulted in a 5- to 10-fold increase in GFP protein expression compared to controls. PCI-bPEI/PTEN or PCI-PS/PTEN transfection of either U251 monolayers or U87 spheroids significantly inhibited their growth. but had no effect on MCF-7 cells containing a wild-type PTEN gene. In addition PCI-GFP transfection of gliomas cells had no effect on their growth pattern. CONCLUSIONS: Collectively, the results suggest that AlPcS(2a) -mediated PCI can be used to enhance cell growth inhibition via transfection of tumor suppressor genes in glioma cells containing mutant PTEN genes.

Comparing the Effects of Light- or Sonic-Activated Drug Delivery: Photochemical/Sonochemical Internalization.

Photochemical internalization (PCI) is a technique that uses the photochemical properties of photodynamic therapy (PDT) for the enhanced delivery of endolysosomal-trapped macromolecules into the cell cytoplasm. The released agent can therefore exert its full biological activity, in contrast to being degraded by lysosomal hydrolases. Activation of photosensitizers via ultrasound (US), called sonodynamic therapy (SDT), has been proposed as an alternative to light-activated PDT for the treatment of cancerous tumors. The use of focused US (FUS) to activate photosensitizers allows treatment at tumor sites buried deep within tissues, overcoming one of the main limitations of PDT/PCI. We have examined ultrasonic activation of photosensitizers together with the anticancer agent bleomycin (BLM) using sonochemical internalization (SCI), as an alternative to light-activated PCI. Our results indicate that, compared to drug or US treatment alone, US activation of the photosensitizer AlPcS2a together with BLM significantly inhibits the ability of treated glioma cells to form clonogenic colonies.

Photochemical internalization-mediated nonviral gene transfection: polyamine core-shell nanoparticles as gene carrier.

The overall objective of the research was to investigate the utility of photochemical internalization (PCI) for the enhanced nonviral transfection of genes into glioma cells. The PCI-mediated introduction of the tumor suppressor gene phosphatase and tensin homolog (PTEN) or the cytosine deaminase (CD) pro-drug activating gene into U87 or U251 glioma cell monolayers and multicell tumor spheroids were evaluated. In the study reported here, polyamine-DNA gene polyplexes were encapsulated in a nanoparticle (NP) with an acid degradable polyketal outer shell. These NP synthetically mimic the roles of viral capsid and envelope, which transport and release the gene, respectively. The effects of PCI-mediated suppressor and suicide genes transfection efficiency employing either "naked" polyplex cores alone or as NP-shelled cores were compared. PCI was performed with the photosensitizer AlPcS 2a and λ=670-nm laser irradiance. The results clearly demonstrated that the PCI can enhance the delivery of both the PTEN or CD genes in human glioma cell monolayers and multicell tumor spheroids. The transfection efficiency, as measured by cell survival and inhibition of spheroid growth, was found to be significantly greater at suboptimal light and DNA levels for shelled NPs compared with polyamine-DNA polyplexes alone.

Photochemical internalization-mediated delivery of chemotherapeutic agents in human breast tumor cell lines.

Breast-conservation surgery (BCS) is now utilized in patients with stage I and II invasive breast cancer. However, positive surgical margins are associated with recurrence, and therefore some form of localized postoperative therapy (radiation/chemotherapy) is necessary to eliminate remaining cancer cells. Existing modalities have significant treatment-limiting side effects; therefore, alternative forms of localized therapy need to be explored. We studied the ex vivo effects of photochemical internalization (PCI) using 4 chemotherapeutic agents: cisplatin, cisplatin analog [D prostanoid, DP], doxorubicin, and bleomycin) on 3 breast cancer cell lines: MCF-7, MDA-MB-435, and MDA-MB-231. Illumination was carried out using a 670-nm diode laser at 5 mW/cm2 following incubation in the photosensitizer with aluminum phthalocyanine disulfonate. Toxicity was investigated using colony-forming assays and the mechanism of cell death was determined using Annexin flow-cytometry. We found that toxicity of DP and bleomycin was significantly enhanced by PCI compared with drug alone but was unchanged for cisplatin and doxorubicin. PCI treatment caused a decrease in the percentage of viable cells, predominantly by enhancing apoptosis. The action was synergistic across all 3 cell lines tested for DP and bleomycin. Thus, with appropriate delivery devices and choice of chemotherapeutic agents, PCI holds the promise of enhancing tumor cell toxicity surrounding the cavity of BCS resection sites and thereby decreasing local recurrence.

Photochemical internalization of bleomycin for glioma treatment.

We study the use of photochemical internalization (PCI) for enhancing chemotherapeutic response to malignant glioma cells in vitro. Two models are studied: monolayers consisting of F98 rat glioma cells and human glioma spheroids established from biopsy-derived glioma cells. In both cases, the cytotoxicity of aluminum phthalocyanine disulfonate (AlPcS2a)-based PCI of bleomycin was compared to AlPcS(2a)-photodynamic therapy (PDT) and chemotherapy alone. Monolayers and spheroids were incubated with AlPcS(2a) (PDT effect), bleomycin (chemotherapy effect), or AlPcS(2a)+bleomycin (PCI effect) and were illuminated (670 nm). Toxicity was evaluated using colony formation assays or spheroid growth kinetics. F98 cells in monolayer/spheroids were not particularly sensitive to the effects of low radiant exposure (1.5 J/cm(2) @ 5 mW/cm(2)) AlPcS(2a)-PDT. Bleomycin was moderately toxic to F98 cells in monolayer at relatively low concentrations-incubation of F98 cells in 0.1 µg/ml for 4 h resulted in 80% survival, but less toxic in human glioma spheroids respectively. In both in vitro systems investigated, a significant PCI effect is seen. PCI using 1.5 J/cm(2) together with 0.25 µg/ml bleomycin resulted in approximately 20% and 18% survival of F98 rat glioma cells and human glioma spheroids, respectively. These results show that AlPcS(2a)-mediated PCI can be used to enhance the efficacy of chemotherapeutic agents such as bleomycin in malignant gliomas.