Cyclodextrin-Based Nanostructure Efficiently Delivers siRNA to Glioblastoma Cells Preferentially via Macropinocytosis.

Darío Manzanares,Carmen Ortiz Mellet,María Dolores Pérez-Carrión,Valentín Ceña,José Manuel García Fernández,José Luis Jiménez Blanco

doi:10.3390/ijms21239306

Abstract

Small interfering ribonucleic acid (siRNA) has the potential to revolutionize therapeutics since it can knockdown very efficiently the target protein. It is starting to be widely used to interfere with cell infection by HIV. However, naked siRNAs are unable to get into the cell, requiring the use of carriers to protect them from degradation and transporting them across the cell membrane. There is no information about which is the most efficient endocytosis route for high siRNA transfection efficiency. One of the most promising carriers to efficiently deliver siRNA are cyclodextrin derivatives. We have used nanocomplexes composed of siRNA and a β-cyclodextrin derivative, AMC6, with a very high transfection efficiency to selectively knockdown clathrin heavy chain, caveolin 1, and p21 Activated Kinase 1 to specifically block clathrin-mediated, caveolin-mediated and macropinocytosis endocytic pathways. The main objective was to identify whether there is a preferential endocytic pathway associated with high siRNA transfection efficiency. We have found that macropinocytosis is the preferential entry pathway for the nanoparticle and its associated siRNA cargo. However, blockade of macropinocytosis does not affect AMC6-mediated transfection efficiency, suggesting that macropinocytosis blockade can be functionally compensated by an increase in clathrin- and caveolin-mediated endocytosis.

Highlights

RNAi represents an effective mechanism that operates in most eukaryotic cells [1] regulating the activity of miRNAs and so being involved in the regulation of cellular metabolism, replication or malignant transformation [2]
We have found that the major endocytic pathway in T98G glioblastoma cells is macropinocytosis, the three major pathways contribute to AMC6 efficient Small interfering ribonucleic acid (siRNA) transfection functionally compensating the blockade of one of them
Once we established that we could selectively and efficiently block either clathrin-mediated endocytosis (CME), caveolin-mediated endocytosis (CVME) or macropinocytosis, we explored whether the decrease in AMC6-mediated Alexa Fluor 488-labelled siRNA cellular uptake following the observed knockdown of p21 (Rac1) Activated Kinase 1 (PAK1) and macropinocytosis blockade was correlated with a decrease in AMC6 transfection ability

Summary

Introduction

RNAi represents an effective mechanism that operates in most eukaryotic cells [1] regulating the activity of miRNAs and so being involved in the regulation of cellular metabolism, replication or malignant transformation [2]. RNAi has a high potential to be useful in therapeutics since it is able to knockdown proteins involved in the pathogenesis of different diseases [3], including HIV [4], by targeting cellular mRNA [5]. Several exogenous activators of the RNAi system can knockdown specific sequences involved in cellular signaling, small interfering RNAs (siRNAs) being the most widely used [9]. SiRNAs can knockdown very efficiently the proteins encoded by the target mRNAs, but naked siRNAs are unable to get into the cell, requiring the use of carriers capable of forming nanostructures (nanoparticles; NPs), protecting the siRNA molecules from degradation and transporting them across the cell membrane [11]. Several endocytosis mechanisms have been described, but the major pathways for endocytic uptake followed by NPs and their cargo to enter the cell are macropinocytosis, clathrin-mediated endocytosis (CME), and caveolin-mediated endocytosis (CVME) [14]

Methods

Results

Conclusion