The activation of hepatic stellate cells play a pivotal role in the pathogenesis of hepatic fibrosis. However, the current lack of specifically identified targets on these cells poses a significant challenge in developing targeted delivery tools for effective anti-hepatic fibrosis therapeutics in clinical practice.

Cell-systematic evolution of ligands by exponential enrichment method was conducted on HSC-T6 cell line to screen out activated hepatic stellate cell-specific aptamers. The specificity of the selected aptamers in targeting hepatic stellate cells was confirmed after truncation optimization. Furthermore, the optimal aptamer was conjugated with miR-23b-5p via C6 linkage to evaluate the targeting specificity of this complex and assess its potential in downregulating liver fibrosis-related proteins and slowing down the progression of liver fibrosis.

The present study successful identified 11 highly enriched single-stranded DNA sequences (APT1-11) that specifically target activated hepatic stellate cells. Subsequent affinity detection and optimization truncation led to the selection of APT8(16-34), which effectively targeted activated hepatic stellate cells both in vivo and in vitro. Moreover, when conjugated with miR-23b-5p, APT8(16-34) also exhibited internalization ability into activated hepatic stellate cells. The delivered cargo miR-23b-5p by APT8 (16-34) effectively targeted to mRNA, leading to translational inhibition and subsequent downregulation of related proteins.

We have identified APT8 (16- 34), which exhibits specific targeting and internalization capabilities into activated hepatic stellate cells. Moreover, when conjugated with miR-23b-5p, APT8 (16-34) also internalizes into activated hepatic stellate cells, enabling miR-23b-5p exert their respective functions.

Schematic representation of aptamer selection for activated hepatic stellate cells using Cell-SELEX technology. Initially, the DNA library was incubated with the cells isolated from rats liver, including primary hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells, and Kupffer cell, as negative controls to eliminate negative cell-binding sequences. The unbound DNAs were collected and subsequently incubated with pcDNA3.1-ALK5 treated HSC-T6 cell line for positive selection. Following thorough washing, the bound DNAs were eluted and subjected to PCR amplification for subsequent rounds of selection. After five rounds of selection, DNA sequencing was conducted to identify individual aptamer sequences. The top 11 sequences, named APT1-11, exhibiting significant enrichment were selected using high-throughput sequencing techniques. APT8(16–34) was finally identified after two rounds of truncation, followed by specificity and affinity analysis. Subsequently, miR-23b-5p was conjugated with APT8(16–34) using C6 as the linker and the resulting APT8(16–34)-miR-23b-5p complex was administered intravenously into hepatic fibrosis mice. The experimental evidence demonstrated the effective delivery of miR-23b-5p into activated HSC-T6 cells by APT8(16–34). Furthermore, the delivered miR-23b-5p downregulated the expression of related proteins.