Hydroxyl PAMAM dendrimer-based gene vectors for transgene delivery to human retinal pigment epithelial cells

Abstract

Ocular gene therapy holds promise for the treatment of numerous blinding disorders. Despite the significant progress in the field of viral and non-viral gene delivery to the eye, significant obstacles remain in the way of achieving high-level transgene expression without adverse effects. The retinal pigment epithelium (RPE) is involved in the pathogenesis of retinal diseases and is a key target for a number of gene-based therapeutics. In this study, we addressed the inherent drawbacks of non-viral gene vectors and combined different approaches to design an efficient and safe dendrimer-based gene-delivery platform for delivery to human RPE cells. We used hydroxyl-terminated polyamidoamine (PAMAM) dendrimers functionalized with various amounts of amine groups to achieve effective plasmid compaction. We further used triamcinolone acetonide (TA) as a nuclear localization enhancer for the dendrimer-gene complex and achieved significant improvement in cell uptake and transfection of hard-to-transfect human RPE cells. To improve colloidal stability, we further shielded the gene vector surface through incorporation of PEGylated dendrimer along with dendrimer-TA for DNA complexation. The resultant complexes showed improved stability while minimally affecting transgene delivery, thus improving the translational relevance of this platform.

Intracellular delivery of dendrimer triamcinolone acetonide conjugates into microglial and human retinal pigment epithelial cells

“…Triamcinolone acetonide-21-glutarate (TA-Linker, 1) was synthesized using a previously established procedure [28]. A detailed synthesis description is provided as a part of supplementary information.…”

“…Upon systemic administration, dendrimer N-acetyl cysteine conjugates (D-NAC) targeted activated microglia in the brains of newborn rabbits with cerebral palsy, and significantly improved motor function, and reduced neurological injury [26,27]. We reported dendrimer-TA based gene delivery platform for improved delivery of genes and enhanced transfection in human retinal pigment epithelial cells [28]. Such targeting and enhanced permeation is desirable for intracellular delivery of corticosteroids to activated microglia that are located in deep layers of retina and RPE cells that have low permeability due to their tight junctions.…”

Biomateriais para formulações de base nanotecnológica visando terapia gênica ocular

“…122 No caso de modificações hidróxi-terminais a transfecção de células RPE não foi alterada para dendrímeros de geração 4 que sofreram posterior PEGuilação, mesmo que a estabilidade coloidal tenha sido significativamente incrementada. 123 Uma alternativa proposta ao uso de dendrímeors PAMAM são dendrímeros de poli-L-lisina. Esses dendrímeros foram capazes de associar AS-ON anti-VEGF, reduzindo a expressão gênica tanto in vitro (células RPE) quanto in vivo em modelo de neovascularização da coroide em ratos, inibindo dessa maneira neovascularização coroide relacionada à degeneração macular por até dois meses.…”

Nanoparticle‐motivated gene delivery for ophthalmic application

“…In another attempt, PEI condensed shRNA plasmid DNA to target melanopsin in Balb/c mice, and was able to knock down melanopsin in RGC cells at 16 hrs of intravitreal injection, which lasted for 2 months 61 . In 2015, Mastorakos and colleagues showed the combined effect of hydroxyl-terminated PAMAM and triamcinolone acetonide (TA) in enhancing transfection of dendrimer-gene complex into the most challenging human RPE cells in vitro 62 . In 2012, Sunshine and colleagues synthesized a novel poly (β-amino ester, Fig.…”