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1.1 Design and Amplification of the TAT Gene Sequence

First, we synthesized a 33 bp TAT peptide fragment (Table 1) through a biotech company, ensuring the accuracy and functionality of its sequence. Next, we utilized PCR technology to amplify this TAT fragment, obtaining a target segment that includes homologous recombination sequences, preparing for subsequent plasmid construction.

Table 1: Sequence of TAT Gene

1.2 Gene Amplification

First, we connected LAMP2A Signal Peptide and TAT fragment through homologous recombination and verified the construct(Figure 2). Then, the product of this reaction was further connected to LAMP2A Mature Peptide through another homologous recombination step, followed by verification of the final construct(Figure 3).

Figure 2: The connection of the LAMP2A signal peptides and the TAT fragment resulted in a successful fusion

Figure 3: The connection of the LAMP2A signal peptides, TAT fragments , and LAMP2A mature peptides resulted in a successful fusion. LAMP2A mature peptide:1168bp

Next, we used the existing LAMP2A mature peptide and IDS-Exosignal genes from our laboratory as templates to amplify segments containing homologous recombination sequences through PCR. After amplification, we validated the PCR products using agarose gel electrophoresis (Figure 4, Figure 5) to ensure that their length and purity met the experimental requirements.

Figure 4: LAMP2A mature peptide:1168bp

Figure 5: IDS-Exosignal:1816bp

In the gene amplification, both the length and intensity of the target bands matched the estimated theoretical length, indicating that the amplified segments possess high purity and integrity.

1.3 Linearization of the Plasmid Vector

We performed double digestion of the pLVX-N1-ACGFP vector using EcoR I and Xba I to linearize the vector in preparation for subsequent recombination reactions. After completing the double digestion, we used electrophoresis recovery and purification steps to ensure high-quality linearized vector fragments(Figure 6), creating favorable conditions for the following homologous recombination.

Figure 6: Linearized Vector:8012bp

In the vector digestion, both the length and intensity of the target bands matched the estimated theoretical length, indicating high purity and integrity of the amplified segments.

Homologous Recombination Reaction

During the homologous recombination process, we combined the amplified TAT, Lamp2a, and IDS fragments with the linearized vector, preparing the recombination reaction mix on ice using the 2 × ClonExpress Mix provided by the ClonExp-ress® Ultra kit. The optimal amounts of each fragment followed the calculation: optimal cloning vector amount = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol); optimal amount for each fragment = [0.02 × number of base pairs of each fragment] ng (0.03 pmol).

1.5 Transformation and Identification

Finally, we transformed the recombinant plasmids into DH5α competent cells and selected using plates containing Ampicillin. After overnight culture, several colonies formed on the transformation plates. We picked multiple colonies, streaked them on blank plates with Ampicillin for storage, and performed colony PCR identification. Using CMV-F and PGK-R universal primers (Table 2), we confirmed the correctness and integrity of the plasmid construction through PCR identification and sequencing. This series of experimental steps laid a solid foundation for subsequent functional validation and application research, while also providing new possibilities for efficient drug delivery using exosomes.

Table 2 Universal Primer Sequences for Vector

2.1 Lentivirus Packaging

In this experiment, we first constructed the pLVX-TAT-IDS plasmid and combined it with pMD2.G and psPAX2 plasmids to form a three-plasmid system. Next, we used polyethyleneimine (PEI) as the transfection reagent to transfect these plasmids into HEK293T cells to produce the lentiviral vector. After transfection, we replaced the culture medium 24 hours later to remove unbound plasmids and transfection reagents. After 48 hours of incubation, we collected the cell culture supernatant and removed cell debris through centrifugation and filtration, ultimately obtaining a high-purity viral solution.

Table3 Three-Plasmid Lentivirus Packaging System

2.2 Cell Transfection and Selection

During cell transfection, we seeded HEK293T cells in a 6-well plate and added the prepared viral solution when the cell density reached approximately 40%. After 48 hours of incubation, the viral infection rate within the cells significantly increased. We then added culture medium containing 3 µg/ml puromycin for selection, aiming to gradually eliminate cells that were not successfully transfected. After several rounds of selection, we ultimately obtained a stable cell line expressing TAT-IDS Exosignal.

3.1 Exosome Extraction and Morphological Verification

We extracted exosomes from the conditioned medium of the stable cell line using differential centrifugation. Initially, we performed stepwise centrifugation at different speeds to remove cell debris and larger particles, ultimately isolating purified exosomes through ultracentrifugation (Figure 7). The extracted exosomes were characterized using transmission electron microscopy (TEM) (Figure 8) to confirm their morphology and purity.

Figure 7: Centrifugation Steps Diagram

Figure 8: Exosome Morphology Diagram

3.2 Western Blot Analysis

In this experiment, we employed Western Blot technology to verify the expression of IDS protein in HEK293T cells (Figure 9). Specifically, we extracted protein samples from the stable TAT-IDS Exosignal-transfected cell line and conducted comparative analysis with the untransfected control group. The results demonstrated that the IDS protein expression level in the stable TAT-IDS Exosignal-transfected cell line was significantly higher than that in the untransfected cell group. Additionally, we assessed the IDS protein content in the exosomes and found a marked increase in the expression level of IDS protein in the exosomes from the transfected group. This result further confirmed that the elevated expression level of LAMP2A not only did not interfere with the normal secretion of IDS protein but also promoted its expression in exosomes.

Figure 9: Western Blot Results Diagram

4.1Fluorescent Labeling of Exosomes

We labeled the collected exosomes using DiI dye. The preparation involved first creating a dye dilution solution, followed by incubation in the dark at room temperature. Finally, we obtained a suspension of labeled exosomes through ultrafiltration (Figure 10).

Figure 10: Fluorescently Labeled Exosome Diagram

4.2 Construction of In Vitro Blood-Brain Barrier Model

To further explore the delivery capability of TAT-modified exosomes, we constructed an in vitro blood-brain barrier model. In this model, exosomes must penetrate the endothelial cells of the upper chamber (HCMEC/D3 cells) before being taken up by the astrocytoma cells (U87MG cells) in the lower chamber, as illustrated in Figures 11 and 12.

Figure 11: Construction of the In Vitro Blood-Brain Barrier Model

Figure 12: In Vitro Blood-Brain Barrier Model Diagram

4.3. Detection of the In Vitro Blood-Brain Barrier

Through the RB-Dextran leakage experiment, we assessed the in vitro dual-layer co-culture BBB model. As shown in Figure 13, starting from day four, the leakage of RB-Dextran in the lower chamber culture medium decreased to below 10% and stabilized, indicating successful construction of the in vitro BBB model.

Figure 13: Detection of RB-Dextran Leakage in the In Vitro BBB Model

4.4 Flow Cytometry Analysis

We evaluated the uptake of exosomes by U87MG cells in the lower chamber of the in vitro BBB model using flow cytometry and confocal fluorescence imaging techniques. Flow cytometry analysis （Figure 14） revealed that the uptake efficiency of exosomes in the TAT-IDS Exosignal group was 21.15%, significantly higher than that of the IDS group (3.30%) and the IDS Exosignal group (4.79%).

Figure 14: Flow Cytometry Analysis of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model

4.5 Confocal Fluorescence Imaging

Confocal fluorescence imaging （Figure 15）further demonstrated that the red signal (indicating TAT-IDS Exosignal sEVs) in the TAT-IDS Exosignal group was markedly stronger than in the IDS and IDS Exosignal groups, as illustrated in the corresponding bar graph on the right.

Figure 15: Confocal Fluorescence Imaging of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model

These results indicate that exosomes produced by cells stably transfected with TAT-IDS Exosignal exhibited significantly enhanced efficiency in crossing the BBB compared to those from the previous generations of plasmid-transfected cells. Moreover, TAT-modified exosomes displayed higher fluorescence signals in the model than unmodified exosomes, suggesting that TAT modification plays a positive role in the translocation of exosomes across the blood-brain barrier.