Ex vivo evaluation of prolidase loaded chitosan nanoparticles for the enzyme replacement therapy.
Prolidase loaded chitosan nanoparticles were set up in order to suggest an innovative therapeutic approach for Prolidase Deficiency (PD), a rare autosomal inherited disorder of the connective tissue. The satisfactory drug loading efficiency (42.6+/-2.1%) as well as the suitable physical characteristics (mean diameter of 365.5+/-35.1 nm and a positive zeta-potential of 17.94+/-0.12 mV) was achieved. In order to verify the compatibility of the chitosan nanoparticles with cells, the influence of the nanoparticles on the growth and the viability (MTT assay) of cultured skin fibroblasts were determined: the nanoparticles showed a good biocompatibility up to 5 microg of chitosan/10,000 fibroblasts. Uptake of chitosan nanoparticles by fibroblasts was verified by confocal microscopy using FITC-labelled chitosan nanoparticles. The ex vivo experiments were performed by incubating different amounts of prolidase loaded chitosan nanoparticles with skin human fibroblasts from PD patients for scheduled times. The restored prolidase activity was quantitatively monitored by a capillary electrophoretic method and confirmed by cells morphological observations. Standing from the nanoparticles internalization, the enzymatic activity was progressively restored reaching the best value (about 66%) after 5 days of co-incubation. Moreover, prolidase loaded chitosan nanoparticles permitted to restore prolidase activity in PD fibroblasts for a prolonged period of time (8 days).
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Pavia, Viale Taramelli 12, Pavia, Italy.
Claudia
C
Colonna
Bice
B
Conti
Paola
P
Perugini
Franca
F
Pavanetto
Tiziana
T
Modena
Rossella
R
Dorati
Paolo
P
Iadarola
Ida
I
Genta
D000592:Amino Acid Metabolism, Inborn Errors / Q000188:drug therapy* / Q000201:enzymology; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002626:Chemistry, Pharmaceutical; D048271:Chitosan / Q000737:chemistry* / Q000633:toxicity; D004150:Dipeptidases / Q000737:chemistry / Q000172:deficiency / Q000494:pharmacology* / Q000627:therapeutic use; D004305:Dose-Response Relationship, Drug; D004337:Drug Carriers; D019075:Electrophoresis, Capillary; D004705:Endocytosis; D004795:Enzyme Stability; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D018613:Microscopy, Confocal; D053758:Nanoparticles; D010316:Particle Size; D012995:Solubility; D013997:Time Factors
D004337:Drug Carriers; D048271:Chitosan; D004150:Dipeptidases; C022449:proline dipeptidase