This section falls under the exosome delivery efficacy validation module within the overall project design. The design encompasses miRNA-loaded exosome delivery methods, with the specific workflow and procedures as follows:
The experimental section is mainly divided into three parts:construction of engineered exosomes,establishment of and intervention with an in vitro hepatic fibrosis model,evaluation and validation of anti-fibrotic effects.

2025.8.14	2025.8.25	2025.9.5
Exosomes and RNA Ordering	RNA transfection into EVs and Hepatic Stellate Cell Culture	miRNA Quantification and Functional Validation

Director：Nuo Xu,Boliang Liu
Begin at：2025.8.14
Estimated end date ：2025.8.24
Current completion status：finished（ 2025.8.24）

Part One: Hepatic Stellate Cell Culture
Director：Nuo Xu,BoLiang Liu
Begin at：2025.8.15
Estimated end date ：2025.9.1
Current completion status： Finished（ 2025.9.16）
Part Two: In Vitro Validation of Anti-fibrotic Effects
Director：Nuo Xu，BoLiang Liu
Begin at：2025.9.18
Estimated end date ：2025.9.20
Current completion status ：Finished（ 2025.9.21）
Record of project:
Step 1: Establishment of a Hepatic Fibrosis Model
LX-2 cells were cultured in 24-well plates.
To induce cell activation and establish a hepatic fibrosis model, the cells were treated with TGF-β1 (10 ng/mL) for 24 hours.
Step 2: Experimental Group Setup
The following seven experimental groups were established for intervention:
1.Negative Control Group: Untreated normal LX-2 cells.
2.Positive Model Group: LX-2 cells induced by TGF-β1 only.
3.Natural Exosome Group: Treated with TGF-β1 and natural exosomes.
4.NC Exosome Group: Treated with TGF-β1 and exosomes loaded with NC mimic.
5.miR-455-3p Exosome Group (Group A): Treated with TGF-β1 and exosomes loaded with miR-455-3p mimics.
6.miR-148a-5p Exosome Group (Group B): Treated with TGF-β1 and exosomes loaded with miR-148a-5p mimics.
7.Co-delivery Exosome Group (Group A+B): Treated with TGF-β1 and a mixture of exosomes from Group A and B, at three different miRNA ratios (1:1, 1:2, 2:1).
Step 3: Exosome Intervention
Based on pre-determined actual miRNA content in each exosome preparation, the concentrations for different groups were precisely calculated and prepared.
This ensured a constant total exosome delivery concentration (50 µg/mL) while maintaining accurate and controllable miRNA ratios.

Director：Nuo Xu,Boliang Liu,Zewen Zhang
Begin at：2025.9.21
Estimated end date ：2025.9.30
Current completion status： Finished（ 2025.10.4） Record of project:
Step 1: Cell Collection
After the specified intervention time (e.g., 48h, 72h), cells from all groups were collected.
Step 2: Protein Extraction and Western Blot
Total protein was extracted from the collected cells.
The protein expression levels of α-SMA and Collagen I were detected by Western Blot analysis.
Step 3: Data Analysis
Grayscale analysis was performed on the Western Blot results.
The expression differences of α-SMA and Collagen I among the groups were compared to evaluate the synergistic anti-fibrotic effect of the dual-miRNA co-delivery and to determine the optimal effective molecular ratio.

kit method
Based on the results of fluorescence detection, we can infer that miR-148a-5p has been almost entirely transfected into the exosomes. A portion of the miRNAs in the miR-455-3p group were transfected into the exosomes, thus obtaining exosomes loaded with these two miRNAs.

Measuring the OD value to determine whether miRNA has been transferred into exosomes. The fluorescence report is as follows:

miR-455-3p:

miR-148a-5p:

co-incubation method
The exosomes loaded with miRNA provided by the company are as follows

Exosomes loaded with miRNA:

After being cultured in complete medium, resuscitated hepatic stellate cells (HSCs) exhibit the characteristics of a significant increase in density and a morphological transition from the quiescent state to the activated state under an optical microscope. At this stage, the cells have passed the initial adaptation phase and entered the late logarithmic growth phase; in terms of density, they can cover 70%-90% of the bottom area of the culture dish, with a slightly sparse distribution at the edges and a uniform distribution in the central area. Morphologically, the cells no longer have the round or oval shape (in the state of incomplete adherence or just adherent) observed in the early resuscitation stage, but transform into a spindle-shaped, polygonal, or long spindle-shaped morphology similar to myofibroblasts. Their cell bodies are significantly stretched with increased volume and abundant cytoplasm; the cell processes increase in number and length, and interconnect with the processes of adjacent cells to form a network structure.

Changes in Cell Density During Cell Culture (Taking Cell Resuscitation to the First Passage as an Example)

Day1

Day2

Day3

Day4

Day5

Day6

Before induction, quiescent hepatic stellate cells (HSCs) adhere to the substrate and grow. They exhibit a typical "spindle-shaped" or "polygonal" morphology, with small cell bodies. Additionally, the cells extend multiple long, slender cytoplasmic protrusions that interconnect to form a network-like structure.After induction with TGF-β, cell spacing decreases, and cell stacking even occurs in some regions. Meanwhile, the cell morphology transitions from "spindle-shaped" to "fusiform" (similar to fibroblasts). The cell bodies increase significantly in size, and the cytoplasmic staining deepens—this is due to increased expression of cytoskeletal proteins such as α-smooth muscle actin (α-SMA) in the cytoplasm. Furthermore, the original long, slender protrusions shorten and thicken, and the intercellular connections decrease.

LX-2 cells in normal condition

LX-2 cells induced for 24 hours

LX-2 cells induced for 48 hours

LX-2 cells induced for 72 hours

Both the ExoLoad® Exosome Nucleic Acid Loading Kit and co-incubation method successfully engineered exosomes.

Based on the above results, we will further carry out multi-metric validation experiments for more delivery ratios. This includes testing multiple fibrosis markers at different time points after drug delivery.