Novel loading protocol combines highly efficient encapsulation of exogenous therapeutic toxin with preservation of extracellular vesicles properties, uptake and cargo activity.

Extracellular vesicles (EVs) have mostly been investigated as carriers of biological therapeutics such as proteins and RNA. Nevertheless, small-molecule drugs of natural or synthetic origin have also been loaded into EVs, resulting in an improvement of their therapeutic properties. A few methods have been employed for EV cargo loading, but poor yield and drastic modifications of vesicles remain unsolved challenges. We tested a different strategy based on temporary pH alteration through incubation of EVs with alkaline sodium carbonate, which resulted in conspicuous exogenous molecule incorporation. In-depth characterization showed that vesicle size, morphology, composition, and uptake were not affected. Our method was more efficient than gold-standard electroporation, particularly for a potential therapeutic toxin: the plant Ribosome Inactivating Protein saporin. The encapsulated saporin resulted protected from degradation, and was efficiently conveyed to receiving cancer cells and triggered cell death. EV-delivered saporin was more cytotoxic compared to the free toxin. This approach allows both the structural preservation of vesicle properties and the transfer of protected cargo in the context of drug delivery.

The cell type dependent sorting of CD9- and CD81 to extracellular vesicles can be exploited to convey tumor sensitive cargo to target cells.

Extracellular vesicles (EVs) are lipid membrane-bound particles involved in cell-to-cell communication through a delivery of regulatory molecules essential for physiological processes. Since EVs efficiently vectorize specific cargo molecules, they have been proposed as suitable vehicles for therapeutic agents. Drug loading into EVs can be achieved by active, exogenous strategies or by genetic modifications of vesicle-producing cells. With the aim to produce EVs conveying therapeutic proteins, we genetically engineered and compared HEK293 to tumor cells. Tetraspanin-based RFP fusions were found to be more stable and preferentially sorted into EVs in HEK293. EVs isolated from genetically modified HEK293 cells media were captured by cancer cells, efficiently delivering their cargo. Cathepsin B cleavage site introduced between CD9/CD81 and RFP was recognized by tumor specific proteases allowing the release of the reporter protein. Our results indicate HEK293 cells as a preferential system for the production of EVs and pave the way to the development of nano-platforms for the efficient delivery of therapeutic proteins and prodrugs to tumor cells.

A Novel RGD-4C-Saporin Conjugate Inhibits Tumor Growth in Mouse Models of Bladder Cancer.

Although toxin may have some advantages compared to chemotherapeutic drugs in cancer therapy, e.g. a potent cytotoxic activity and a reduced risk of resistance, their successful application in the treatments to solid tumors still remains to be fully demonstrated. In this study, we genetically modified the structure of the plant-derived single-chain ribosome inactivating protein saporin (SAP) by fusing its N-terminus to the ACDCRGDCFCG peptide (RGD-4C), an αv-integrin ligand, and explored the anti-tumor activity of the resulting protein (called RGD-SAP) in vitro and in vivo, using a model of muscle invasive bladder cancer. We found that the RGD-4C targeting domain enhances the cytotoxic activity of SAP against various tumor cell lines, in a manner dependent on αv-integrin expression levels. In a subcutaneous syngeneic model of bladder cancer, RGD-SAP significantly reduced tumor growth in a dose-dependent manner. Furthermore, systemic administration of RGD-SAP in combination with mitomycin C, a chemotherapeutic drug currently used to treat patients with bladder cancer, increased the survival of mice bearing orthotopic bladder cancer with no evidence of systemic toxicity. Overall, the results suggest that RGD-SAP represents an efficient drug that could be exploited, either alone or in combination with the state-of-the-art therapies, for the treatment of bladder cancer and, potentially, of other solid tumors.

Pre-clinical Research on Bladder Toxicity After Radiotherapy for Pelvic Cancers: State-of-the Art and Challenges.

Despite the dramatic advancements in pelvic radiotherapy, urinary toxicity remains a significant side-effect. The assessment of clinico-dosimetric predictors of radiation cystitis (RC) based on clinical data has improved substantially over the last decade; however, a thorough understanding of the physiopathogenetic mechanisms underlying the onset of RC, with its variegated acute and late urinary symptoms, is still largely lacking, and data from pre-clinical research is still limited. The aim of this review is to provide an overview of the main open issues and, ideally, to help investigators in orienting future research. First, anatomy and physiology of bladder, as well as the current knowledge of dose and dose-volume effects in humans, are briefly summarized. Subsequently, pre-clinical radiobiology aspects of RC are discussed. The findings suggest that pre-clinical research on RC in animal models is a lively field of research with growing interest in the development of new radioprotective agents. The availability of new high precision micro-irradiators and the rapid advances in small animal imaging might lead to big improvement into this field. In particular, studies focusing on the definition of dose and fractionation are warranted, especially considering the growing interest in hypo-fractionation and ablative therapies for prostate cancer treatment. Moreover, improvement in radiotherapy plans optimization by selectively reducing radiation dose to more radiosensitive substructures close to the bladder would be of paramount importance. Finally, thanks to new pre-clinical imaging platforms, reliable and reproducible methods to assess the severity of RC in animal models are expected to be developed.

A non-invasive ultrasound imaging method to measure acute radiation-induced bladder wall thickening in rats.

BACKGROUND: Methods for the non-invasive quantification of changes in bladder wall thickness as potential predictors of radiation cystitis in pre-clinical research would be desirable. The use of ultrasound for this aim seems promising, but is still relatively unexplored. A method using ultrasound for bladder wall thickness quantification in rats was developed and applied to measure early radiation-induced bladder wall thickness changes. METHODS: Two groups (n = 9 each) of female Fischer rats were treated with a single radiation dose of 25-30 and 35-40 Gy respectively, using an image-guided micro-irradiator; six untreated rats were monitored as a control group. Empty, half-filled and fully-filled bladder volumes were determined for four non-irradiated rats by measuring axes from ultrasound 3D-images and applying the ellipsoid formula. Mean bladder wall thickness was estimated for both ventral and dorsal bladder sides through the measurement of the bladder wall area along a segment of 4 mm in the central sagittal scan, in order to minimize operator-dependence on the measurement position. Ultrasound acquisitions of all fully-filled rat bladders were also acquired immediately before, and 4 and 28 days after irradiation. Mean bladder wall thickness normalized to the baseline value and corrected for filling were then used to evaluate acute bladder wall thickening and to quantify the dose-effect. RESULTS: The relationship between mean bladder wall thickness and volume in unirradiated rats showed that for a bladder volume > 1.5 mL the bladder wall thickness is almost constant and equal to 0.30 mm with variations within ± 15%. The average ratios between post and pre irradiation showed a dose-effect relationship. Bladder wall thickening was observed for the 25-30 Gy and 35-40 Gy groups in 2/9 (22%) and 5/9 (56%) cases at day 4 and in 4/9 (44%) and 8/9 (89%) cases at day 28, respectively. The two groups showed significantly different bladder wall thickness both relative to the control group (p < 0.0001) and between them (p = 0.022). The bladder wall thickness increment was on average 1.32 ± 0.41, and was 1.30 ± 0.21 after 25-30 Gy and 1.47 ± 0.29 and 1.90 ± 0.83 after 35-40 Gy at days 4 and 28 respectively. CONCLUSIONS: The feasibility of using ultrasound on a preclinical rat model to detect bladder wall thickness changes after bladder irradiation was demonstrated, and a clear dose-effect relationship was quantified. Although preliminary, these results are promising in addressing the potential role of this non-invasive approach in quantifying radiation cystitis.

Hosts for Hostile Protein Production: The Challenge of Recombinant Immunotoxin Expression.

For the recombinant expression of toxin-based drugs, a crucial step lies not only in the choice of the production host(s) but also in the accurate design of the protein chimera. These issues are particularly important since such products may be toxic to the expressing host itself. To avoid or limit the toxicity to productive cells while obtaining a consistent yield in chimeric protein, several systems from bacterial to mammalian host cells have been employed. In this review, we will discuss the development of immunotoxin (IT) expression, placing special emphasis on advantages and on potential drawbacks, as one single perfect host for every chimeric protein toxin or ligand does not exist.

Nanoparticle-mediated delivery of suicide genes in cancer therapy.

Conventional chemotherapeutics have been employed in cancer treatment for decades due to their efficacy in killing the malignant cells, but the other side of the coin showed off-target effects, onset of drug resistance and recurrences. To overcome these limitations, different approaches have been investigated and suicide gene therapy has emerged as a promising alternative. This approach consists in the introduction of genetic materials into cancerous cells or the surrounding tissue to cause cell death or retard the growth of the tumor mass. Despite promising results obtained both in vitro and in vivo, this innovative approach has been limited, for long time, to the treatment of localized tumors, due to the suboptimal efficiency in introducing suicide genes into cancer cells. Nanoparticles represent a valuable non-viral delivery system to protect drugs in the bloodstream, to improve biodistribution, and to limit side effects by achieving target selectivity through surface ligands. In this scenario, the real potential of suicide genes can be translated into clinically viable treatments for patients. In the present review, we summarize the recent advances of inorganic nanoparticles as non-viral vectors in terms of therapeutic efficacy, targeting capacity and safety issues. We describe the main suicide genes currently used in therapy, with particular emphasis on toxin-encoding genes of bacterial and plant origin. In addition, we discuss the relevance of molecular targeting and tumor-restricted expression to improve treatment specificity to cancer tissue. Finally, we analyze the main clinical applications, limitations and future perspectives of suicide gene therapy.