Calcium phosphate nanoparticles for biomedical applications
This thesis covers different syntheses of CaP nanoparticles (triple-shell systems, silica-shelled CaP nanoparticles for surface functionalization or PLGA encapsulated CaP nanoparticles for gene silencing), the complete colloid-chemical and molecular biological characterization and possible applications of these nanoparticles. The investigated applications included the in vivo particle biodistribution of the different routes of administration, gene silencing, immunization and vaccination in mice, cell transfection and sub-cellular targeting within the process of endocytosis. The previously cloned plasmids were fully intact, used to transfect different cell lines in vitro and produced fluorescent proteins by using the T7 polymerase system of E. coli. The transport of BSAFAM mediated with triple-shell CaP nanoparticles showed fluorescent foci in cells which underwent a vesicular rupture and subsequently a cytoplasmic distribution. A closer look on the transfection of HeLa, Caco-2 and A549 cells with CaP nanoparticles loaded with a plasmid encoding for Tandem revealed the expected cytoplasmic distribution of the fluorescent fusion protein as well as the fluorescent PEIATTO490LS. Additionally, in context of the mentioned BSAFAM transport, the signal of PEIATTO490LS hints at the postulated proton sponge effect for an endolysosomal escape. Unfortunately, this phenomenon was not observed in the transport of the fusion protein directly loaded onto CaP nanoparticles. Nevertheless, the transport of the Tandem protein efficiently visualized the endocytic pathway. Tandem was accumulated significantly in lysosomes. This co-localization was analyzed using the measured signals of the high-resolution CLSM of HeLa cells. The biodistribution of CaP nanoparticles in mice was examined by applying radioisotope (68Ga) labeled DOTA-functionalized particles. An intravenous administration into the bloodstream had the consequence of a fast systemic distribution into the lung, spleen, liver and subsequentially into the bone marrow and hip bones. To a lesser extent the radioactive signal was found in the heart, kidney and lymph nodes. The intramuscular, intratumoral and soft tissue administration demonstrated a local distribution remaining near the injection site, which is beneficial when a stimulation of the immune system is intended. The biomedical application of functionalized CaP nanoparticles in vivo proved therapeutic effects in treating pulmonary inflammations, inflammation-derived carcinogenesis and retroviral infections. Thus, CaP nanoparticles functionalized with immunotherapeutic molecules represent an efficient tool to target and treat diseases. Besides the diagnostic and therapeutic CaP nanoparticles presented in this thesis, there are studies on similar nanoparticular systems like polymer nanospheres, gold nanoparticles or functionalized dendrimers on treating hepatitis B virus (HBV), herpes simplex virus 1 (HSV 1), and human immunodeficiency virus (HIV). HBV infection was treated efficiently with chitosan-functionalized gene silencing PLGA nanocapsules. An approach of a prophylactic viral treatment was presented in mercaptoethane sulfonate BSA nanospheres which effectively inhibited an infection with HSV-1 by binding to the virus and blocking the cellular entrance. Another in vitro study on treating the infection with HIV-1 showed promising results by using glucose-coated gold nanoparticles functionalized with reverse-transcriptase inhibitors abacavir and lamivudine. It was proven that HIV-1 infects regulatory T cells (Treg) to impair their suppressor activity in the immune homeostasis. A treatment with cationic and anionic carbosilane dendrimers preserved the Treg cells phenotype and prevented the viral replication and the spread of the infection to other cells.
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