In order to provide an experimental basis and theoretical foundation for the development of new strategies for efficient and multi-targeted hepatic fibrosis treatment, we constructed and optimized an engineered exosome based on the synergistic action of dual miRNAs (miR-455-3p and miR-148a-5p) and systematically evaluated its interventional effect on hepatic fibrosis.

Specific sub-objectives include:
1. To successfully construct engineered human umbilical cord mesenchymal stem cell exosomes (hUC-MSC-Exos) loaded with miR-455-3p and miR-148a-5p, respectively.
2. To clarify the inhibitory effects on the proliferation, activation and extracellular matrix secretion of activated hepatic stellate cells (HSCs) when the two engineered exosomes were applied singly and in combination in different ratios.
3. To validate and screen out the combination ratio of dual miRNA engineered exosomes with the best anti-fibrotic effect in an in vitro model of liver fibrosis.

To develop therapeutic strategies for end-stage liver disease, we designed an engineered exosome system that co-delivers two anti-fibrotic miRNAs (miR-455-3p and miR-148a-5p) derived from human umbilical cord mesenchymal stem cells (hUC-MSCs). The goal was to construct exosomes with enhanced miRNA loading capacity and validate their physical and molecular characterization.

We acquired hUC-MSCs (CTE-052) exosomes from ECHO AIOTECH. Synthetic miRNA mimics (miR-455-3p and miR-148a-5p) were then loaded into exosomes using two methods: commercial loading kit, ExoLoad® and direct co-incubation. Negative control (NC) mimics were also loaded for comparison.

We used electron microscopy and nanoparticle tracking analysis (NTA) to characterize the morphology and size distribution of exosomes. Exosome markers (e.g. CD9, CD63) were confirmed using protein blotting. Quantification of miRNA content in engineered exosomes by fluorescence detection.

We successfully constructed engineered exosomes with high miRNA loading efficiency. By comparing the kit method and the co-incubation method, we determined the best method for subsequent functional experiments.

To assess the anti-fibrotic effects of engineered exosomes, we designed an in vitro model of liver fibrosis using a human liver stellate cell line (LX-2) activated by TGF-β1. The model mimics key pathological features of liver fibrosis.

LX-2 cells were cultured and treated with TGF-β1 (10 ng/mL) for 24 hours to induce their activation. The cells were then divided into seven experimental groups:

No.	Group name	Whether induced by TGF-β1	Exosome type	Purpose
1	Negative blank control	No	None	Observation of LX-2 cells in their natural state
2	Positive Blank Control	Yes	None	Observation of changes following LX-2 modelling at any manual intervention
3	Natural exosome control		Natural exosomes	Observation of the effect of natural
4	NC mimic loading exosome group		Ev transferred to rna	Exclusion of artificial RNA backbones from LX-2 cells
5	miR-455-3p exosome control (group A)		ev to 455	Observation of the effect of miR-455-3p loaded exosomes on active LX-2
6	miR-148a-5p exosome control group (Group B)		Transferred to 148 ev	Observation of the effect of miR-148a-5p loaded exosomes on active LX-2
7	Experimental group ratio 1 (miR-455-3p :miR-148a-5p )		1:1	Observation of the effect of ratio 1 engineered exosomes on active LX-2
8	Experimental group ratio 2		2:1	Observation of the effect of ratio 2 engineered exosomes on active LX-2
9	Experimental group ratio 3		1:2	Observation of the effect of ratio 3 engineered exosomes on active LX-2

After intervention (e.g., 48 or 72 hours), cells were collected. Protein extracts are prepared and tested for fibrosis markers such as α-SMA by protein blot analysis.

We found that engineered exosomes would significantly inhibit HSC activation and extracellular matrix production. Preliminary data which we haven’t acquired can show whether single miRNA exosomes are (or are not) superior to natural exosomes and whether co-delivery is (or is not) showing enhanced effects.