Original Article
Noninvasive delivery of oligonucleotide by penetratin-modified polyplexes to inhibit protein expression of intraocular tumor

https://doi.org/10.1016/j.nano.2017.04.011Get rights and content

Abstract

Our present study aimed to develop an antisense oligonucleotide (ASO) delivery system to achieve gene silencing in intraocular tumor via topical instillation. ASO specific for luciferase was chosen as model drug, polyamidoamine (PG5) was employed to condense ASO, and penetratin (Pene) was used to enhance cellular uptake. Nanoscale PG5/ASO/Pene polyplex was stabilized via noncovalent bonding. In vitro evaluations indicated that PG5/ASO/Pene exhibited improved cell-penetrating and gene silencing ability compared with naked ASO and PG5/ASO. Subcutaneous and orthotopic tumor models expressing luciferase were established in nude mice. After treated by PG5/ASO/Pene, immunohistochemical results of subcutaneous tumors showed significant inhibition of luciferase expression via peritumoral injection, and bioluminescence from orthotopic tumor was obviously weakened via topical instillation. To date, few works were successful in noninvasive treatment of intraocular diseases using antisense strategy, this penetratin-modified polyplex could be a promising vector to inhibit protein expression by effectively delivering ASOs into the eye.

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.

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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 5ā€²-CCUCUUACCUCAGUUACA-3ā€² were synthesized by Sangon Biotech (Shanghai, China). ASO labeled with 6-carboxyfluorescein (6-FAM) at 5ā€²-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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    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.

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