Nanoparticles are potentially interesting drug delivery systems due their selectivity for certain... more Nanoparticles are potentially interesting drug delivery systems due their selectivity for certain tissues (e.g., cancer tissue) and the ability of releasing drugs in the optimum dosage range, with the consequence of increased therapeutic efficacy and reduce side effects. Proteins unfortunately display many instability problems when exposed to even rather mild processing conditions. The objective herein was to develop a method for the encapsulation of proteins in biodegradable polymer nanospheres for drug delivery applications. Lysozyme nanoparticles were obtained by using a nanoprecipitation method employing acetonitrile as solvent. The particle diameter was 245 ± 38 nm and under optimized conditions 100% activity could be recovered. These nanoparticles were encapsulated in PLGA spheres of about 1 µm in diameter. Encapsulation efficiency, formation of aggregates, and recovered activity were optimized. The particles obtained showed excellent release properties with a very low burst r...
Amyloid-beta peptides (Abeta) and the protein human serum albumin (HSA) interact in vivo. They ar... more Amyloid-beta peptides (Abeta) and the protein human serum albumin (HSA) interact in vivo. They are both localised in the blood plasma and in the cerebrospinal fluid. Among other functions, HSA is involved in the transport of the essential metal copper. Complexes between Abeta and copper ions have been proposed to be an aberrant interaction implicated in the development of Alzheimer's disease, where Cu is involved in Abeta aggregation and production of reactive oxygen species (ROS). In the present work, we studied copper-exchange reaction between Abeta and HSA or the tetrapeptide DAHK (N-terminal Cu-binding domain of HSA) and the consequence of this exchange on Abeta-induced ROS production and cell toxicity. The following results were obtained: 1) HSA and DAHK removed Cu(II) from Abeta rapidly and stoichiometrically, 2) HSA and DAHK were able to decrease Cu-induced aggregation of Abeta, 3) HSA and DAHK suppressed the catalytic HO(.) production in vitro and ROS production in neuroblastoma cells generated by Cu-Abeta and ascorbate, 4) HSA and DAHK were able to rescue these cells from the toxicity of Cu-Abeta with ascorbate, 5) DAHK was more potent in ROS suppression and restoration of neuroblastoma cell viability than HSA, in correlation with an easier reduction of Cu(II)-HSA than Cu-DAHK by ascorbate, in vitro. Our data suggest that HSA is able to decrease aberrant Cu(II)-Abeta interaction. The repercussion of the competition between HSA and Abeta to bind Cu in the blood and brain and its relation to Alzheimer's disease are discussed.
Proteins often possess highly specific biological activities that make them potential therapeutic... more Proteins often possess highly specific biological activities that make them potential therapeutics, but their physical and chemical instabilities during formulation, storage, and delivery have limited their medical use. Therefore, engineering of nano-sized vehicles to stabilize protein therapeutics and to allow for targeted treatment of complex diseases, such as cancer, is of considerable interest. A micelle-like nanoparticle (NP) was designed for both, tumor targeting and stimulus-triggered release of the apoptotic protein cytochrome c (Cyt c). This system is composed of a Cyt c NP stabilized by a folate-receptor targeting amphiphilic copolymer (FA-PEG-PLGA) attached to Cyt c through a redox-sensitive bond. FA-PEG-PLGA-S-S-Cyt c NPs exhibited excellent stability under extracellular physiological conditions, whereas once in the intracellular reducing environment, Cyt c was released from the conjugate. Under the same conditions, the folate-decorated NP reduced folate receptor positive HeLa cell viability to 20% while the same complex without FA only reduced it to 80%. Confocal microscopy showed that the FA-PEG-PLGA-S-S-Cyt c NPs were internalized by HeLa cells and were capable of endosomal escape. The specificity of the folate receptor-mediated internalization was confirmed by the lack of uptake by two folate receptor deficient cell lines: A549 and NIH-3T3. Finally, the potential as anti-tumor therapy of our folate-decorated Cyt c-based NPs was confirmed with an in vivo brain tumor model. In conclusion, we were able to create a stable, selective, and smart nanosized Cyt c delivery system.
Nanoparticles are potentially interesting drug delivery systems due their selectivity for certain... more Nanoparticles are potentially interesting drug delivery systems due their selectivity for certain tissues (e.g., cancer tissue) and the ability of releasing drugs in the optimum dosage range, with the consequence of increased therapeutic efficacy and reduce side effects. Proteins unfortunately display many instability problems when exposed to even rather mild processing conditions. The objective herein was to develop a method for the encapsulation of proteins in biodegradable polymer nanospheres for drug delivery applications. Lysozyme nanoparticles were obtained by using a nanoprecipitation method employing acetonitrile as solvent. The particle diameter was 245 ± 38 nm and under optimized conditions 100% activity could be recovered. These nanoparticles were encapsulated in PLGA spheres of about 1 µm in diameter. Encapsulation efficiency, formation of aggregates, and recovered activity were optimized. The particles obtained showed excellent release properties with a very low burst r...
Amyloid-beta peptides (Abeta) and the protein human serum albumin (HSA) interact in vivo. They ar... more Amyloid-beta peptides (Abeta) and the protein human serum albumin (HSA) interact in vivo. They are both localised in the blood plasma and in the cerebrospinal fluid. Among other functions, HSA is involved in the transport of the essential metal copper. Complexes between Abeta and copper ions have been proposed to be an aberrant interaction implicated in the development of Alzheimer's disease, where Cu is involved in Abeta aggregation and production of reactive oxygen species (ROS). In the present work, we studied copper-exchange reaction between Abeta and HSA or the tetrapeptide DAHK (N-terminal Cu-binding domain of HSA) and the consequence of this exchange on Abeta-induced ROS production and cell toxicity. The following results were obtained: 1) HSA and DAHK removed Cu(II) from Abeta rapidly and stoichiometrically, 2) HSA and DAHK were able to decrease Cu-induced aggregation of Abeta, 3) HSA and DAHK suppressed the catalytic HO(.) production in vitro and ROS production in neuroblastoma cells generated by Cu-Abeta and ascorbate, 4) HSA and DAHK were able to rescue these cells from the toxicity of Cu-Abeta with ascorbate, 5) DAHK was more potent in ROS suppression and restoration of neuroblastoma cell viability than HSA, in correlation with an easier reduction of Cu(II)-HSA than Cu-DAHK by ascorbate, in vitro. Our data suggest that HSA is able to decrease aberrant Cu(II)-Abeta interaction. The repercussion of the competition between HSA and Abeta to bind Cu in the blood and brain and its relation to Alzheimer's disease are discussed.
Proteins often possess highly specific biological activities that make them potential therapeutic... more Proteins often possess highly specific biological activities that make them potential therapeutics, but their physical and chemical instabilities during formulation, storage, and delivery have limited their medical use. Therefore, engineering of nano-sized vehicles to stabilize protein therapeutics and to allow for targeted treatment of complex diseases, such as cancer, is of considerable interest. A micelle-like nanoparticle (NP) was designed for both, tumor targeting and stimulus-triggered release of the apoptotic protein cytochrome c (Cyt c). This system is composed of a Cyt c NP stabilized by a folate-receptor targeting amphiphilic copolymer (FA-PEG-PLGA) attached to Cyt c through a redox-sensitive bond. FA-PEG-PLGA-S-S-Cyt c NPs exhibited excellent stability under extracellular physiological conditions, whereas once in the intracellular reducing environment, Cyt c was released from the conjugate. Under the same conditions, the folate-decorated NP reduced folate receptor positive HeLa cell viability to 20% while the same complex without FA only reduced it to 80%. Confocal microscopy showed that the FA-PEG-PLGA-S-S-Cyt c NPs were internalized by HeLa cells and were capable of endosomal escape. The specificity of the folate receptor-mediated internalization was confirmed by the lack of uptake by two folate receptor deficient cell lines: A549 and NIH-3T3. Finally, the potential as anti-tumor therapy of our folate-decorated Cyt c-based NPs was confirmed with an in vivo brain tumor model. In conclusion, we were able to create a stable, selective, and smart nanosized Cyt c delivery system.
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