Sustained release nanoparticles containing acyclovir prodrugs for ocular herpes simplex keratitis and characterization of folate transport proteins in a corneal epithelial cell line
Dissertation advisor: Ashim K. MitraTitle from PDF of title page, viewed on December 21, 2011VitaIncludes bibliographic references (p. 148-160)Thesis (Ph.D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri-Kansas City, 2011Ocular herpes is a persistent viral infection caused by the herpes simplex virus-1. It is one of
the most common infectious diseases causing corneal blindness in the United States. In this
study, polymeric nanoparticles of stereoisomeric di-peptide prodrugs of acyclovir (L-valine-
L-valine-ACV, L-valine-D-valine-ACV, D-valine-L-valine-ACV, and D-valine-D-valine-ACV)
were developed and characterized for the treatment of ocular herpes keratitis. L-valine-Lvaline-
ACV and L-valine-D-valine-ACV were determined to be optimum in terms of
enzymatic stability, uptake and cytotoxicity. Uptake and docking results indicated that Lvaline
in the terminal position increases the affinity of prodrug to the peptide transporter
protein. Entrapment efficiency values of L-valine-L-valine-ACV and L-valine-D-valine-ACV
were optimal with PLGA 75:25 and PLGA 65:35 polymers, respectively. In vitro release of
prodrugs from nanoparticles exhibited a biphasic release pattern with initial burst phase
followed by sustained release. Dispersion of nanoparticles in thermosensitive gels eliminated
the burst release phase. Novel nanoparticulate systems of dipeptide prodrugs of ACV
suspended in thermosensitive gels may provide sustained delivery following topical administration. The uptake of nanoparticles into corneal cells can be enhanced by attachment
of cell-specific ligands such as folate and biotin. Folate carrier-mediated system is widely
used in the targeted delivery of drugs. Thus Staten's Seruminstitut rabbit corneal (SIRC)
epithelial cell line was investigated for the expression of folate transport proteins that can be
utilized for targeted drug delivery of folate-conjugated nanoparticles and prodrugs to cornea.
Linear increase in [3H] Folic acid uptake was observed over 30min. The process followed
saturation kinetics with apparent Km of 14.2 nM, Vmax of 1.5x10-5 micro.moles/min/mg
protein and Kd of 2.1x10-6 min.-1 Uptake was inhibited in the presence of structural analogs
(cold folic acid, MTF and MTX) but structurally unrelated vitamins did not show any effect.
RT-PCR and Western blot analysis confirmed the presence of folate receptor-α (FR-α) and
proton-coupled folate transporter (PCFT). This work demonstrated the functional and
molecular presence of FR-α and PCFT in SIRC cells that can be utilized for enhanced uptake
of folate conjugated nanoparticles and prodrugs. In a different study the differential
expression of FR-α, sodium dependent multivitamin transporter (SMVT) and amino acid
transporter B (0, +) in retinoblastoma (Y-79) and retinal pigment epithelial (ARPE-19) cells. Higher expression of FR-α, SMVT and B (0, +) at mRNA level was observed in cancerous Y-
79 cells compared to normal ARPE-19 cells.Introduction -- Rationale for investigation -- Ocular sustained release nonoparticles containing stereoisomeric di-peptide prodrugs of acyclovir -- Rationale for investigation -- Functional characterization of folate transport proteins in staten's seruminstitut rabbit corneal epithelial cell line -- Rationale for investigation -- Differential expressions of folate receptor-alpha (FR-A), sodium dependent multivitamin transporter (SMVT), and neutral and catonic amino acid transporter B [super] (0, +) in human retinoblastoma (Y-79) and normal human retinal (ARPE-19) cell lines -- Summary and recommendations -- Appendi
