Development of different nanosystems for drugs and siRNA delivery

Abstract Cancer is one of the leading causes of death in the world. Over the past several decades, the development of engineered nanosystems for targeted drug delivery have received great attention thanks to their possibility to overcome the limitations of classical cancer chemotherapy including poor solubility, targeting incapability, nonspecific action and, consequently, systemic toxicity. In this contest, four different models of nanocarriers have been analysed and compared for their capacity to target tumour tissue and to release the therapeutic agent in a controlled way: INU-EDA-P,C-DOXO; PHEA-EDA-P,C-DOXO; PVP-siRNA and RGO-siRNA. Inulin and PHEA were conjugated to the antineoplastic drug doxorubicin through a citraconylamide bridge used as a pH sensitive spacer and their different action in normal and tumour cells was compared by viability assay, fluorescence microscopy and flow cytometric analysis. The data revealed a higher effect against the cancer cells probably due to the higher capability to enter the cancer cells respect to the healthy ones, but also due to a partial drug release in tumour microenvironment, that presents a lower pH (6.5) respect to normal tissue (7.4). Because of the better efficiency of PHEA-EDA-P,C-DOXO, its preferential uptake into tumour cells was further demonstrated through co-culture experiments. Moreover, cellular internalization mechanism studies suggested a model in which the polymer would enter the cells through a caveolae-mediated endocytosis and would be steered toward lysosomal compartment where the drug would be released and become free to go into the nucleus. On the other hand, siRNA delivery was investigated comparing other two kind of nanosystems: PVP and Graphene nano-complexes conjugated with siRNA against Egr-1 (a transcription factor involved in the progress of the tumour) and Bcl-2 (a protein related with the apoptotic pathway in cancer cells). They are very different complexes, each with specific properties that would influence differently their ability as siRNA delivery system. Data relative to PVP nanogel showed it as a good candidate for siRNA delivery because it binds siRNA without any functional alteration or degradation. Moreover, the addition of a glutathione sensitive linker permitted a higher amount of siRNA released through a controlled way. Even graphene nanosystems have displayed good features as nanocarriers thanks to their biocompatibility and their ability to be internalized by cells. Furthermore, their bidimensional nature would permit to conjugate a large amount of oligonucleotides. All the complexes analysed showed specific features that make them good candidates for drug or siRNA delivery. The choice of the best nanosystem is depending on the target site and on the therapeutic agent conjugated. PHEA nanopolymers present better characteristics for doxorubicin delivery compared with those of Inulin. On the other hand, the hydrogel nature of PVP and the bidimensional structure of graphene can minimize the typical degradation problems of the oligonucleotides and, therefore, offer the best conditions for siRNA delivery. Moreover, the possibility to release biological molecules in controlled way (mediated by pH or Glutathione) and to recognize the specific tumour target allow to overcome the typical limits of the classic cancer therapy.

Cancer is one of the leading causes of death in the world. Over the past several decades, the development of engineered nanosystems for targeted drug delivery have received great attention thanks to their possibility to overcome the limitations of classical cancer chemotherapy including poor solubility, targeting incapability, nonspecific action and, consequently, systemic toxicity. In this contest, four different models of nanocarriers have been analysed and compared for their capacity to target tumour tissue and to release the therapeutic agent in a controlled way: INU-EDA-P,C-DOXO; PHEA-EDA-P,C-DOXO; PVP-siRNA and RGO-siRNA. Inulin and PHEA were conjugated to the antineoplastic drug doxorubicin through a citraconylamide bridge used as a pH sensitive spacer and their different action in normal and tumour cells was compared by viability assay, fluorescence microscopy and flow cytometric analysis. The data revealed a higher effect against the cancer cells probably due to the higher capability to enter the cancer cells respect to the healthy ones, but also due to a partial drug release in tumour microenvironment, that presents a lower pH (6.5) respect to normal tissue (7.4). Because of the better efficiency of PHEA-EDA-P,C-DOXO, its preferential uptake into tumour cells was further demonstrated through co-culture experiments. Moreover, cellular internalization mechanism studies suggested a model in which the polymer would enter the cells through a caveolae-mediated endocytosis and would be steered toward lysosomal compartment where the drug would be released and become free to go into the nucleus. On the other hand, siRNA delivery was investigated comparing other two kind of nanosystems: PVP and Graphene nano-complexes conjugated with siRNA against Egr-1 (a transcription factor involved in the progress of the tumour) and Bcl-2 (a protein related with the apoptotic pathway in cancer cells). They are very different complexes, each with specific properties that would influence differently their ability as siRNA delivery system. Data relative to PVP nanogel showed it as a good candidate for siRNA delivery because it binds siRNA without any functional alteration or degradation. Moreover, the addition of a glutathione sensitive linker permitted a higher amount of siRNA released through a controlled way. Even graphene nanosystems have displayed good features as nanocarriers thanks to their biocompatibility and their ability to be internalized by cells. Furthermore, their bidimensional nature would permit to conjugate a large amount of oligonucleotides. All the complexes analysed showed specific features that make them good candidates for drug or siRNA delivery. The choice of the best nanosystem is depending on the target site and on the therapeutic agent conjugated. PHEA nanopolymers present better characteristics for doxorubicin delivery compared with those of Inulin. On the other hand, the hydrogel nature of PVP and the bidimensional structure of graphene can minimize the typical degradation problems of the oligonucleotides and, therefore, offer the best conditions for siRNA delivery. Moreover, the possibility to release biological molecules in controlled way (mediated by pH or Glutathione) and to recognize the specific tumour target allow to overcome the typical limits of the classic cancer therapy.