Toxicity evaluation of poly(anhydride) nanoparticles as carriers for oral administration of drugs
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OJER OJER, Patricia. “Toxicity evaluation of poly(anhydride) nanoparticles as carriers for oral administration of drugs”. Irache Garreta, Juan M. (dir.), López de Cerain Salsamendi, Adela (codir.). Tesis doctoral. Universidad de Navarra, Pamplona, 2013
Nanoparticles designed from the copolymer of methyl vinyl ether and maleic anhydride are ideal candidates as vehicles for oral delivery. Thus, it was considered interesting to study its toxicity in relation to their physico-chemical characteristics by using appropriate protocols and guidelines. Conventional nanoparticles (NP), containing 2-hydroxypropyl-â-cyclodextrin (NP-HPCD) and coated with polyethylene glycol 6000 (PEG-NP)) can be obtained by desolvation of the poly(anhydride), followed by a purification and lyophilization. To simplify this method, spray-drying procedure was evaluated. Lactose, in a saccharide/polymer ratio of 2, was found to be the most suitable excipient regarding physico-chemical characteristics and stability of the resulting nanoparticles. The capabilities of this procedure were studied by the characterization of nanoparticles loaded with an antigenic extract (HS from Brucella ovis) or atovaquone. The new formulations displayed similar physico-chemical characteristics that those obtained by lyophilization. For cytotoxicity studies two different cell lines were chosen: HepG2 cells (liver model) and Caco-2 cells (gastrointestinal epithelium model). Although the results from the MTS and LDH assays showed a concentration-dependent cytotoxic effect, cytotoxicity appeared only at very high concentrations and long incubation times in HepG2 cells (1-2 mg/mL at 48-72 h). Thus, the in vitro safety of the different nanoparticles was demonstrated. Nanoparticle-cells interaction studies were performed in Caco-2 cell line after labeled the nanoparticles with BSA-FITC. Nanoparticles showed cytoadhesion but not internalization; PEG-NP was the most bioadhesive followed by NP-HPCD and NP as demonstrated by flow cytometry. Cellular localization of particles by fluorescence confocal microscopy confirmed the association of these nanoparticles with cells. Evaluation of poly(anhydride) nanoparticles safety as carriers for oral delivery studies were performed in rats in accordance to the OECD 425 and 407 guidelines. Biodistribution studies were carried out after radiolabelling nanoparticles with 99mTechnetium (99mTc). The LD50 for all nanoparticles tested was higher than 2000 mg/kg bw. In the sub-acute oral toxicity studies at two different doses (30 and 300 mg/kg bw), no evident signs of toxicity were found. Biodistribution studies demonstrated that these carriers remained in the gut with no evidences of particle translocation or distribution to other organs. Thus, poly(anhydride) nanoparticles might be a safe strategy for oral delivery of therapeutics. Poly(anhydride) nanoparticles hemocompatibility was studied in vitro at different concentrations (0.25, 0.5 and 1 mg/mL). To evaluate the acute toxicity of intravenously administered nanoparticles rats were exposed to two different doses (50 and 150 mg/kg bw). Biodistribution were assessed at a dose of 50 mg/kg bw using radiolabelling nanoparticles with 99mTc. Nanoparticles did not induce any agglutination effect neither hemolytic activity in the hemocompatibility studies carried out. In the acute toxicity study animals dosed with 150 mg/kg bw showed acute episodes of respiratory distress. In fact, 2/6 animals dosed with NP and NP-HPCD died. The histopathological examinations revealed apparent pathological changes characterized by the presence of foamy cells in the liver, spleen and lungs (Phospholipidosis). Finally, biodistribution studies showed that nanoparticles accumulation occurred predominantly in organs of the RES. Thus, although all nanoparticle formulations demonstrated to be hemocompatible in vitro the intravenous toxicity was dose dependent, with the presence of foamy cells in spleen, liver and lungs as the most relevant finding observed. Therefore, this research has permitted to establish the basis for assessing the toxicity of polymeric nanoparticles intended for oral delivery.
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