Identification of tPep exosomes and the packaging of Bcl-2 siRNA into tPep exosomes.

To target white adipocytes specifically to deliver Bcl-2 siRNAs, exosomes were modified to express CKGGRAKDC peptides (tPep) on surfaces of bilayer membranes. We cloned tPep into Lamp2b, a stable protein expressed on the membrane surface of exosomes. HEK 293T cells were transfected with a plasmid coding tPep-Lamb2b and Bcl-2 siRNA. 48 hours later, exosomes were harvested to be characterized using TEM and Nanosight. Captured images by TEM illustrated that the average size of exosomes with lipid bilayer membranes was around 100nm (Fig. 1A). In agreement with TEM images, the Nanosight nanoparticle tracking analysis showed size distribution of exosomes and the diameter of majority of particles was 172nm (Fig. 1B). Therefore,the identity of exosomes isolated from HEK 293T cells was confirmed.

Fig 1. Identification of tPep exosomes morphologically. (A) TEM micrographs of tPep exosomes secreted by HEK 293T cells. (B) Nanosight NS 300 system was employed to measured exosomes from HEK 293T cells. The histogram represents particle size distribution.

tPep exosomes can specifically deliver siRNA into 3T3L1 cells and downregulate Bcl-2 expression levels.

To certify that tPep exosomes can deliver siRNAs into adipocytes specifically, the mouse white fat cell line 3T3L1 and mouse skeletal muscle cell line C2C12 were employed to be incubated with exosomes. We transfected Alexa Fluor 555 labelled oligonucleotide(red fluorescence) together with plasmid coding tPep into HEK 293T cells and harvested exosomes after 48 hours. Under fluorescence confocal microscope, the red fluorescence only exhibited in 3T3L1 cells incubated with tPep exosomes loaded with labelled oligonucleotide. However, we cannot observed red fluorescent signals in 3T3L1 cells treated with non-tPep exosomes. What’s more, red fluorescent signals did not present in C2C12 cells incubated with non-tPep exosomes or tPep exosomes loaded with oligonucleotide (Fig. 2A). Subsequently, Bcl-2 protein levels in 3T3L1 cells was significantly reduced. We observed that Bcl-2 siRNA-2 showed the most significant function amongst the four candidates, so we selected it for further study subsequently (Fig. 2B). Hence, Bcl-2 siRNA can be delivered by exosomes with tPep on the surfaces of membranes into 3T3L1 cells to repress Bcl-2 expression levels.

Fig 2. tPep exosomes deliver small RNAs into white adipose cell line 3T3L1 specifically and Bcl-2 siRNAs delivered by tPep exosomes can reduce Bcl-2 protein level. (A) Confocal microscopy images of fluorescently labelled oligonucleotides(red colors) in 3T3L1 cells and C2C12 cells incubated with or without tPep exosomes. (B) Western blotting analysis of Bcl-2 protein levels of 3T3L1 cells transfected with 4 Bcl-2 siRNAs.

tPep exosomes can transfer Bcl-2 siRNAs to white fat tissue specifically and reduce Bcl-2 expression levels.

Experiments in vivo were performed to study whether tPep exosomes can transport Bcl-2 siRNAs to target white fat tissue specifically and mediate Bcl-2 expression levels. tPep or non-tPep exosomes containing Bcl-2 siRNAs were injected into mice through caudal veins. After 24 hours, the Bcl-2 siRNA levels in white fat and liver tissues were detected. Concentration of Bcl-2 siRNA delivered by tPep exosomes was higher in white fat tissue compared with that delivered by non-tPep exosomes. Besides, lower level of Bcl-2 siRNA was observed in liver tissue, suggesting that tPep exosomes can transfer Bcl-2 siRNAs to specific white fat tissue other than liver tissue (Fig. 3A). Consistently, Bcl-2 mRNA levels were reduced obviously in white fat tissue after injected with tPep exosomes containing Bcl-2 siRNAs (Fig. 3B). Taken together, our study in vivo demonstrated that tPep exosomes have the ability to deliver Bcl-2 siRNAs to white fat tissue, but not liver tissue, to negatively regulate gene expression levels.

Fig 3. tPep exosome-mediated Bcl-2 siRNA can target fat tissue specically and reduce Bcl-2 expression levels in vivo. (A) Bcl-2 siRNA enriched in adipose tissue other than other tissues such as liver, kidney and heart tissues from mice. (B) qRT-PCR analysis of Bcl-2 mRNA levels in the livers of mice treatment with PBS or tPep exosomes. (C) qRT-PCR assay of levels of Bcl-2 mRNA in fat tissues of mice injected with PBS or tPep exosomes. n=15.

Bcl-2 siRNA delivered by tPep exosomes can induce targeted apoptosis of adipocytes and curb effects of high fat diets (HFD) on mice.

After verifying that Bcl-2 siRNA delivered by exosomes modified with tPep can downregulate Bcl-2 levels, we study the functional effects on mice. It is known that Bcl-2 is an integral outer mitochondrial membrane protein that blocks the apoptotic death of some cells such as lymphocytes and downregulation of Bcl-2 can trigger apoptosis. Fig. 4A depicts the experiment we performed. Mice fed with high fat diets (HFD) throughout the experiment were injected with tPep exosomes or PBS on days 1, 3, 5, 7, 9, 11, 13. On day 14, mice were dissected for serum, cervicodorsal and inguinal adipose tissues. Total weights of cervicodorsal and inguinal adipose tissues and serum cholesterol levels were quantified. The mice injected with tPep exosomes showed lower cervicodorsal and inguinal adipose weights, suggesting the positive psychological effects caused by delivery of Bcl-2 siRNAs with tPep exosomes (Fig. 4B). Further investigation of the levels of serum cholesterol also confirmed that the treatment of tPep exosomes can positively improve the psychological condition of mice. Mice treated with tPep exosomes displayed a progressive decline in the serum level cholesterol compared with the level in mice treated with PBS (Fig. 4C). Histological analysis of tissues from mice after 2 weeks of treatment demonstrated that mice injected with PBS had more fat deposits in the white adipose tissues, whereas those injected with tPep exosomes regained glossily normal histological patterns. Notably, control tissues like liver tissues from mice treated with exosomes or PBS remained microanatomically stable in contrast. Consequently, Bcl-2 siRNAs deliver by tPep exosomes can suppress the negative influence of high fat diets (HFD) on mice by inducing apoptosis of white adipocytes.

Fig 4. The effects of Bcl-2 siRNA- tPep exosomes on mice fed with high fat diet (HFD). (A) The experimental procedure is described by the flow diagram. (B) Measurement of weights of adipose tissues from mice injected with PBS or tPep exosomes. (C) Level of cholesterol from mice serum treated with tPep exosome was decreased. (D) Paraffin sections of adipose tissues and liver tissues from mice stained with hematoxylin and eosin showed morphological changes of cells, suggesting apoptosis of adipocytes in mice treated with tPep exosomes while preservation of liver tissues was observed. n=15, *P < 0.05; **P < 0.01; ***P< 0.001.