Original ArticleNoninvasive delivery of oligonucleotide by penetratin-modified polyplexes to inhibit protein expression of intraocular tumor
Graphical Abstract
We have developed an antisense oligonucleotide delivery polyplex (PG5/ASO/Pene) by using PAMAM G5 as the cationic carrier and penetratin as the permeation enhancer. The self-assembled polyplex was stabilized via non-covalent bonding. In vitro assays revealed that PG5/ASO/Pene polyplex exhibited efficient down-regulation of luciferase expression, which was further demonstrated in subcutaneous tumor model. Notably, in orthotopic tumor model, PG5/ASO/Pene polyplex inhibited protein expression of intraocular tumor via topical instillation.
Section snippets
Materials
PAMAM G5 dendrimer (M.W. ~28.82Ā kDa) was obtained from Sigma-Aldrich (USA). Antisense oligonucleotides (ASOs) targeting luciferase gene with sequence 5ā²-CCUCUUACCUCAGUUACA-3ā² were synthesized by Sangon Biotech (Shanghai, China). ASO labeled with 6-carboxyfluorescein (6-FAM) at 5ā²-end was assigned as ASO-FAM. Penetratin (RQIKIWFQNRRMKWKK) was provided by ChinaPeptides Co., Ltd. (Shanghai, China). Bicinchoninic acid (BCA) protein quantification kit was obtained from Pierce Biotech Inc. (Rockford,
Optimization of polyplexes formulation
Utilizing the self-quenching characteristic of fluorescence, fluorescence intensity of ASO-FAM was applied as the index to optimize composition of the polyplexes.23 ASO was condensed by PAMAM at different weight ratios, which led to changes in fluorescence intensity. As shown in Figure 1, A, when the weight ratio of PAMAM to ASO was higher than 6, the fluorescence intensity of the mixture exhibited about 80% decrease compared with that of naked ASO, indicating ASO was completely condensed.
Discussion
As potential gene therapeutic agents, oligonucleotides have currently become research highlights for their specific and effective silencing of target genes.24, 25 With the understanding of tumorigenesis, interfering tumor signal transduction and inhibiting tumor growth by using antisense technology have become an inevitable trend.26 Combination with nucleotides in oncotherapy can reduce toxicity and improve precision of chemotherapy. RB is a primary malignant intraocular tumor and its current
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2022, Journal of Controlled ReleaseCitation Excerpt :Recently, Tai et al. utilized PAMAM dendrimers to deliver fibroblast growth factor receptor-specific ASOs to treat intraocular retinoblastoma. The penetratin-PAMAM/ASO polyplex showed significantly suppressed luciferase expression in subcutaneous and orthotopic tumors [195]. Penetratin is a homeodomain-derived cell-penetrating peptide, which has the potential to reach posterior eye segments.
Unleashing cell-penetrating peptide applications for immunotherapy
2022, Trends in Molecular MedicineCitation Excerpt :As an approach to exploit the advantages of CPP, studies have used CPPs for drug delivery to control cancer in eye tissues where drug delivery is difficult. For topical delivery, antisense oligonucleotides (ASOs) were conjugated to the CPP penetratin and used for intraocular instillation in the U87āLuc orthotopic ocular tumor model [62]. Administration of penetratin conjugated with ASOs targeting the luciferase gene silenced luciferase activity in U87 cells, suggesting that CPPs can be used for drug delivery into ocular cells.
Topical instillation of cell-penetrating peptide-conjugated melphalan blocks metastases of retinoblastoma
2022, BiomaterialsCitation Excerpt :Unfortunately, it is difficult for small molecules like melphalan to be delivered into the posterior ocular segment via topical instillation. Penetratin was previously reported as a more safe and powerful ocular permeation enhancer than several other cell-penetrating peptides and could promote ocular delivery of both small molecules and nucleic acids [13ā18]. By hydrophobic amino acids mutation, various penetratin derivatives were obtained with even better ocular permeability, and tryptophan mutation at the eighth (glutamine) and ninth (asparagine) amino acid residues resulted in an excellent ocular permeable peptide (Q8W, N9W-Penetratin, 89WP) [19].
The authors declare that there are no commercial associations, current and within the past five years, that might pose a potential, perceived or real conflict of interest.