Engineered Exosomes Containing microRNA-29b-2 and Targeting the Somatostatin Receptor Reduce Presenilin 1 Expression and Decrease the β-Amyloid Accumulation in the Brains of Mice with Alzheimer's Disease

Abstract

Purpose: Exosomes are membrane vesicles secreted by various cells and play a crucial role in intercellular communication. They can be excellent delivery vehicles for oligonucleotide drugs, such as microRNAs, due to their high biocompatibility. MicroRNAs have been shown to be more stable when incorporated into exosomes; however, the lack of targeting and immune evasion is still the obstacle to the use of these microRNA-containing nanocarriers in clinical settings. Our goal was to produce functional exosomes loaded with target ligands, immune evasion ligand, and oligonucleotide drug through genetic engineering in order to achieve more precise medical effects.

Methods: To address the problem, we designed engineered exosomes with exogenous cholecystokinin (CCK) or somatostatin (SST) as the targeting ligand to direct the exosomes to the brain, as well as transduced CD47 proteins to reduce the elimination or phagocytosis of the targeted exosomes. MicroRNA-29b-2 was the tested oligonucleotide drug for delivery because our previous research showed that this type of microRNA was capable of reducing presenilin 1 (PSEN1) gene expression and decreasing the β-amyloid accumulation for Alzheimer's disease (AD) in vitro and in vivo.

Results: The engineered exosomes, containing miR29b-2 and expressing SST and CD47, were produced by gene-modified dendritic cells and used in the subsequent experiments. In comparison with CD47-CCK exosomes, CD47-SST exosomes showed a more significant increase in delivery efficiency. In addition, CD47-SST exosomes led to a higher delivery level of exosomes to the brains of nude mice when administered intravenously. Moreover, it was found that the miR29b-2-loaded CD47-SST exosomes could effectively reduce PSEN1 in translational levels, which resulted in an inhibition of beta-amyloid oligomers production both in the cell model and in the 3xTg-AD animal model.

Conclusion: Our results demonstrated the feasibility of the designed engineered exosomes. The application of this exosomal nanocarrier platform can be extended to the delivery of other oligonucleotide drugs to specific tissues for the treatment of diseases while evading the immune system.

Keywords: 3xTG-AD; SH-SY5Y; functional nanocarriers; hippocampus; oligonucleotide drug.

Figure 1

Dendritic cells transfected with miR-29b-2 and CD47-SST lentiviral vectors. (A) Construction design for CD47-CCK and CD47-SST plasmids. (B) Designs for two different constructs. (C) pMIRNA1 lenti-vector (green fluorescent) carrying miR29b-2 transfected dendritic cells. (Scale bar represents 50 µm) (D) pLVX-IRES-tdTomato lenti-vectors (red fluorescence) were used to transfect the miR29b-2-DC with target gene CCK or SST, and immune immunity gene CD47. (Scale bar represents 50 µm) (E) The sorting of cells. DC cell sorting with a double positive ratio. (F) Detection of target gene (CCK or SST) and immune immunity gene CD47 on Western blots.

Figure 2

An analysis of exosomes released by dendritic cells transfected with lentiviral vectors containing miR-29b-2 and CD47-CCK /CD47-SST. (A) Particle size distribution range. (B) Protein expression in engineered exosomes. (C) Transmission electron microscopy images of engineered exosomes. (Scale bar represents 100 nm).

Figure 3

Analysis of engineered exosomes uptake by SH-SY5Y cell line and analysis of engineered exosomes inhibition of disease protein expression. (A) DiO-labeled exosomes (green fluorescence) and nuclei of entering cells (blue fluorescence). (Scale bar represents 50 µm). (B) Quantification of exosomes entering SHSY5Y cell line. (C) The expression of receptor proteins in SH-SY5Y cell line. (D) A Western blot analysis of cells treated with different exosome groups in the disease model SH-SY5Y. (E) Quantification of disease protein levels in SH-SY5Y cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Engineered exosomes inhibit disease proteins in 3xTg-AD mice. (A–C) The Western blot and quantitative results of the brain tissue from 3xTg-AD mice were analyzed after three days of treatment. * p < 0.05 *** p < 0.001. (D) The Aβ 1–42 oligomers protein content of the 3xTg-AD mouse hippocampus was examined with IHC. (Scale bar represents 200 µm).

Figure 5

Engineered exosomes delivered intravenously to mice’s brains. (A) Nude mice were injected intravenously with different groups of exosomes for 24 hours and their IVIS results were analyzed in vivo. (B) An analysis of the quantitative results of IVIS experiments on brain brightness. (C) The exosomal protein CD63 with CD47 and SST in the hippocampal region. (Scale bar represents 200 µm).

Figure 6

The uptake of SST target exosomes in knockdown SST receptor cells. (A) Silence RNA inhibition of SST receptor 3 in SH-SY5Y cells. (B) An analysis of the quantitative results of silence RNA inhibition of the SST receptor 3 in SH-SY5Y cells. (C) Quantitative analysis of the entrance of exosomes into SH-SY5Y cells (***p < 0.001). (D) As the SST receptor 3 was inhibited by silence RNA, the number of CD47-SST exosomes entering SH-SY5Y cells decreased. (Scale bar represents 20 µm).