Ang/TAT-sEVs are loaded with specific drugs for the treatment of gliomas

Professor Cheng Guosheng from the School of Nanotechnology and Nanobiomimicry of the University of Science and Technology of China and others used Ang and TAT double peptide-modified sEVs as carriers to prepare efficient drug delivery systems targeting BBBs and gliomas.
It has great application prospects
for the treatment of central nervous system tumors.

Article published online in the August 2022 issue of J Extracell Vesicles
.

- Excerpted from the article chapter

【Ref: Zhu Z, et al.
J Extracell Vesicles.
2022 Aug; 11(8):e12255.
doi: 10.
1002/jev2.
12255.
】

Research background

Current studies confirm that small extracellular vesicles (sEVs) are natural endogenous cellular chemocestrial vector systems that can effectively cross biological barriers.
Angiopep-2 (Ang) is a polypeptide associated with low-density lipoprotein receptor protein-1 (LRP1) with high affinity and efficient brain penetration that can cross the blood-brain barrier (BBB) and deliver drugs to the brain; Transcriptional transactivator (TAT) peptides are highly efficient cell-penetrating peptides that penetrate not only the cell and nuclear membranes of most living cells, but also dense tumor tissue
.

Professor Guosheng Cheng from the School of Nanotechnology and Nanobiomimicry of University of Science and Technology of China and others used Ang and TAT double peptide-modified sEVs as carriers to prepare efficient drug delivery systems (Ang/TAT-sEVs) targeting BBBs and gliomas.
It has great application prospects
for the treatment of central nervous system tumors.

Article published online in the August 2022 issue of J Extracell Vesicles
.

Research methods

The researchers constructed a dual-peptide-modified functional small extracellular vesicle Ang/TAT-sEVs drug loading system, and verified the targeting, BBB penetration, glioma tissue penetration, anti-tumor activity and related mechanisms of Ang/TAT-sEVs-DOX through in vitro and in vivo cell uptake experiments, BBB models and animal models
.

Study results

The research process and results are as follows:

1.
Preparation of Ang/TAT-sEVs and verification of their characterization
.

The authors fused the targeted peptide Ang and the cell penetrating peptide TAT with extracellular vesicles to generate the corresponding plasmids, and then infected HEK293T cells by lentiviral particles, and isolated the transfected Ang/TAT-sEVs
from the transfected cell culture supernatant by ultracentrifugation.

Western blotting (WB), transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) showed that the isolated extracellular vesicles were sEVs, and peptide modification had no effect
on the morphology and size distribution of sEVs.

2.
To test the targeting effect, cell uptake and BBB penetration ability
of Ang/TAT-sEVs.

The results of flow cytometry and confocal microscopy showed that Ang peptide modification could improve the uptake effect of sEVs and TAT-sEVs in LRP1-positive cells (U87MG, bEnd.
3), indicating that Ang/TAT-sEVs could mediate high levels of cell targeting and cell uptake
.

The authors constructed an in vitro BBB model and used quantitative analysis of fluorescence and confocal microscopy imaging to find that the BBB penetration effect of Ang/TAT-sEVs and the ability to target U87MG cells were significantly increased
.

3.
To examine the antitumor effects
of Ang/TAT-sEVs in vitro.

In vitro experiments, fluorescence imaging showed that ANG/TAT-sEVs could effectively deliver doxorubicin (DOX) to LRP1-positive U87MG, and the experiments showed that free DOX was comparable
to Ang/TAT-sEVs-DOX cell viability inhibition.

The authors also validated the high targeting and DOX delivery effects
of Ang/TAT-sEVs with other LRP1-positive cell lines (human hepatoma cells).

The results showed that the binding of the receptor to the target cell can effectively improve the delivery
of the drug.

Neither the blank group nor the Ang/TAT-sEVs group significantly inhibited cell growth, indicating that sEVs were free or had low
cytotoxicity.

4.
Verify the effect
on in vivo targeting, BBB penetration and glioma penetration.

The authors constructed a mouse model of U87MG subcutaneous glioma; The fluorescence signal analysis and quantitative results after treatment with Ang/TAT-sEVs showed that Ang/TAT-sEVs had the best
targeting for glioma.

In vivo fluorescence imaging and quantification showed that Ang/TAT-sEVs had the strongest
fluorescence signal in tumor tissues.

The above results show that Ang/TAT-sEVs can effectively target tumor tissues
in subcutaneous glioma models.

The authors further used the in situ U87MG glioma model to evaluate the ability of Ang/TAT-sEVs to cross the BBB and penetrate gliomas.
The results of fluorescence analysis and quantification showed that the cumulative amount of Ang/TAT-sEVs in brain tissue was the highest, indicating that Ang/TAT-sEVs could penetrate well into BBBs and target gliomas
in situ.

Finally, the authors used immunohistochemistry and confocal imaging to find that the accumulation of Ang/TAT-sEVs in gliomas increased significantly, indicating that Ang/TAT-sEVs have good ability
to penetrate gliomas.

Therefore, Ang/TAT-sEVs have good targeting of BBB and glioma, and can effectively penetrate into BBB and glioma tissues
.