Nanotechnology

Engineered extracellular vesicles for ischemic stroke: a systematic review and meta-analysis of preclinical studies | Journal of Nanobiotechnology


Study characteristics

We identified 2793 studies from the databases, which we then screened based on our inclusion and exclusion criteria. As shown in Fig. 1 and 28 studies [17, 21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47] ultimately met our criteria and were included in this review. Details of these studies are presented in Table 1. All studies were conducted using rats (n = 19) and mice (n = 9). Apart from two studies that utilized photochemistry and electrocoagulation techniques, the prevalent approach was the suture method of middle cerebral artery occlusion (MCAO) (n = 26). Mesenchymal stem cells (MSC) were the primary source of EVs in most studies (n = 15), with other sources including neural stem cells (NSC) (n = 5), blood (n = 5), and soma (n = 3). The predominant method of engineering EVs was through lentiviral transfection (n = 16), followed by coculture (n = 7), ultrasonic techniques (n = 3), electroporation (n = 1), and surface modification (n = 1). The preferred route of EEVs administration was intravenous injection (n = 21), though some studies opted for intracerebral injection (n = 5) or nasal administration (n = 2). Administration timing varied, spanning from a day before IS (n = 2) to 14 days post-IS (n = 26), with select studies administering EVs on multiple occasions (n = 5). Notably, a significant portion of studies engineered the parent cells (n = 19), as opposed to directly engineering the EVs (n = 9).

Fig. 1
figure 1

PRISMA flow diagram. Summary of the number of studies identified, screened, and ultimately included in the systematic review and meta-analysis

Table 1 Summary of studies included in the systematic review

Outcomes

EEVs reduce infarct volume and improve neurological scores after IS

The effects of EEVs therapy on infarct volume and neurological scores were shown in Fig. 2a-d. A total of 321 animals in 25 studies reported changes in infarct volume after treatment with EEVs, of which 21 studies reported the percentage of infarct volume (Fig. 2a) and 4 studies reported the size of infarct volume (Fig. 2b). The results showed that the EEVs reduced the percentage of infarct volume (SMD = -2.33, 95% CI: -2.92, -1.73, p < 0.00001, Tau2 = 0.75, I2 = 50%) and the size of infarct volume (SMD = -2.36, 95% CI: -4.09, -0.63, p = 0.008, Tau2 = 2.55, I2 = 85%) compared to natural EVs therapy.

Fig. 2
figure 2

Forest plots show the effect of EEVs therapy on infarct volume and neurological scores in IS. (a) The percentage of infarct volume. (b) The size of infarct volume. (c) MNSS. (d) Zea-Longa score

Furthermore, we examined the effect of EEVs therapy on neurological scores after IS. In 8 studies, 126 animals were assessed using the modified neurological severity score (mNSS) (Fig. 2c), and 44 animals in 5 studies used the Zea-Longa score (Fig. 2d). The results showed that treatment with EEVs significantly improved mNSS after IS (SMD = -1.78, 95% CI: -2.39, -1.17, p < 0.00001, Tau2 = 0.34, I2 = 46%). Similarly, the Zea-Longa score demonstrated comparable results (SMD = -2.75, 95% CI: -3.79, -1.71, p < 0.00001, I2 = 0%).

EEVs promote behavioral recovery after IS

Behavioral tests were conducted on a total of 274 animals across 11 studies as shown in Fig. 3a-d. For motor and coordination function, 5 studies performed the rotarod test (SMD = 2.50, 95% CI: 1.81, 3.18, p < 0.00001, I2 = 41%) as shown in Fig. 3a, while 4 studies performed the grid-walking test (SMD = -3.45, 95% CI: -5.15, -1.75, p < 0.0001, Tau2 = 2.28, I2 = 76%) as shown in Fig. 3b. For motor and sensory function, 4 studies performed adhesive removal test (SMD = -2.60, 95% CI: -4.27, -0.93, p = 0.002, Tau2 = 2.44, I2 = 87%) as shown in Fig. 3c. For learning and memory function, 3 studies performed the morris water maze test (SMD = -3.91, 95% CI: -7.03, -0.79, p = 0.01, Tau2 = 6.44, I2 = 86%) as shown in Fig. 3d. In summary, all these tests suggest that treatment with EEVs improves behavioral recovery after IS.

Fig. 3
figure 3

The forest plot of the effect of EEVs treatment on IS behavior is shown. (a) Rotarod test. (b) Grid-walking test. (c) Adhesive removal test. (d) Morris water maze test

EEVs reduce the release of pro-inflammatory factors after IS

9 studies involving 190 animals reported the release of pro-inflammatory factors after IS as shown in Fig. 4a-c. 4 studies reported that EEVs can reduce IL-1β (SMD = -2.02, 95% CI: -2.77, -1.27, p < 0.00001, I2 = 0%) as shown in Fig. 4a. 6 studies reported that EEVs can reduce the release of IL-6 (SMD = -3.01, 95% CI: -4.47, -1.55, p < 0.0001, Tau2 = 1.83, I2 = 61%) as shown in Fig. 4b. 7 studies reported that EEVs can also reduce the release of TNF-α (SMD = -2.72, 95% CI: -4.30, -1.13, p = 0.0008, Tau2 = 2.55, I2 = 72%) as shown in Fig. 4c. In summary, these studies all demonstrate that treatment with EEVs can reduce the release of pro-inflammatory factors after IS.

Fig. 4
figure 4

Forest plot of the effect of EEVs treatment on pro-inflammatory factor release after IS. (a) IL-1β. (b) IL-6. (c) TNF-α.

EEVs reduce apoptosis rate and increase the number of neurons after IS

11 studies involving 158 animals reported on the apoptosis rate and the number of neurons after IS, as shown in Fig. 5a-b. 9 studies reported that treatment with EEVs reduce apoptosis rate (SMD = -2.24, 95% CI: -3.32, -1.16, p < 0.0001, Tau2 = 1.61, I2 = 72%) as shown in Fig. 5a. 4 studies reported that treatment with EEVs significantly increase neuron numbers after IS (SMD = 3.70, 95% CI: 2.44, 4.96, p < 0.00001, I2 = 38%) as shown in Fig. 5b.

Fig. 5
figure 5

Forest plot of the effect of EEVs treatment on apoptotic rate and the number of neurons after IS. (a) Apoptotic rate. (b) The number of neurons

Subgroup and sensitivity analyses

We conducted a subgroup analysis to explore the source of heterogeneity. As shown in Table 2, we did not observe significant sources of heterogeneity in the outcome of infarct volume among subgroups of randomization, blinding, animal species, source of EVs, methods of engineering, engineering targets, route of administration, and the timepoint of administration. We also conducted a sensitivity analysis to ensure the robustness of determining the overall effect size of the observed outcome measurements. We removed one study at a time and recalculated the pooled effect size for the remaining studies. The results showed that for all outcomes, there was no significant improvement in heterogeneity between studies, indicating that no study had driven the source of heterogeneity.

Table 2 Subgroup analysis of infarct volume

Research quality and bias risk

As shown in Table 3, the median quality assessment score for the studies was 7 points (IQR: 6–9). However, most studies employed the principle of random allocation and only a few reported concealment of allocation. Half of the studies used a blinding to evaluate the results. Only one study provided information on sample size calculation, which received a risk of bias score of 10 points, as shown in Table 4.

Table 3 CAMARADES Checklist Assessment Bias Risk
Table 4 Extended risk of bias checklist data

Publication bias

We also conducted a publication bias test and generated funnel plots for outcome measures that included more than ten studies. The results indicated publication bias for both of our outcome measures. The funnel plots for infarct volume and neurological scores appeared asymmetrical, as illustrated in Fig. 6, with a majority of the studies indicating more positive effects of EEVs.

Fig. 6
figure 6

Publication bias funnel plots for infarct volume and neurological scores. (a) Infarct volume. (b) Neurological scores