Preparation and characterization of MSN based miR-26a-5p targeted delivery systems

In contrast to other materials widely used in drug delivery such as liposomes and polymeric nanoparticles [27,28,29], MSNs are resistant and allow sustained release over time due to their well stability and controllable-release capacity [30, 31]. Thus, MSNs are potentially good choices among various nanocarrier for solving the problems of sustained drug release and targeted delivery [32, 33]. For better delivery of miR-26a-5p to microglia, as shown in Figs. 1A and 2A, herein we prepared four different types of MSN: (i) bare ones, with silanol groups on the surface and negative ζ potentials, denoted as MSN, (ii) surface amino-functionalized mesoporous silica nanoparticles A-MSN, obtained by co-condensation between tetraethylorthosilicate (TEOS) and (3-aminopropyl) triethoxysilane (APTES), which present amino groups and possess positive ζ potentials for better anchoring of therapeutic miRNAs, (iii) miR@A-MSN, prepared by loading miR-26a-5p into A-MSN via multiple non-covalent interactions, including electrostatic and macromolecular entanglement (iv) miR@A-MSN-MG1, obtained by grafted targeted peptides on the surface of miR@A-MSN. To investigate the physical properties of the nanoparticles, we performed the following experiments by transmission electron microscopy (TEM), and dynamic light scattering (DLS) to characterize the various nanoparticle systems. The results showed that all four types of nanoparticles exhibit similar pore arrangements and textural properties with concentrated particle sizes ranging between 100 and 200 nm (Fig. 2B, D). The mean hydrodynamic sizes, as determined by DLS, of A-MSN, miR@A-MSN, miR@A-MSN-MG1, were found to be increased to varying extents compared to bare MSN, ranging from about 120 nm to 150 nm, also suggesting the successful surface modification (Fig. 2D). Moreover, the results of the zeta potential test showed that after the surface of MSN was modified with amination, the potential increased from approximately − 10 mV to + 43 mV, indicating successful grafting of a large number of amino groups onto the surface of MSNs, and the dispersion stability of the nanoparticles in solution was improved. When the surface of A-MSN was modified with the targeting peptide MG1, its zeta potential started to decrease, indicating that part of the NH2 reacted with MG1, while the potential of the nanoparticles further decreased after loading miR-26a-5p, also indicating the successful loading of miR-26a-5p and its potential was around + 20 mV, which still had good dispersibility (Fig. 2C).

Fig. 1

Schematic of miR-26a-5p nanoparticle targeted delivery system and long-lasting analgesia through anti-inflammation. A Mesoporous silica nanoparticles modified with MG1-targeting peptide and loaded with miR-26a-5p which targeted Wnt5a. B miR-26a-5p nanoparticle targeted delivery system provides long-lasting analgesia. C MG1 modification increases nanoparticle enrichment in microglia and efficiently regulate neuroinflammation. (A: Ethanol APTES, MSN: Mesoporous silica nanoparticle)

Fig. 2

Preparation of nanoparticles and their particle size and potential characterization. A Schematic diagram of the preparation of mesoporous silica nanoparticles (MSN), aminated mesoporous silica nanoparticles (A-MSN), miR-loaded mesoporous silica nanoparticles (miR@A-MSN), and miR-loaded mesoporous silica nanoparticles with modified targeting peptides (miR@A-MSN-MG1). B Transmission electron microscope (TEM) images of MSN, A-MSN, A-MSN-MG1 and miR@A-MSN-MG1. C Zeta potential of MSN, A-MSN, A-MSN-MG1 and miR@A-MSN-MG1. D Particle size distribution of MSN, A-MSN, A-MSN-MG1 and miR@A-MSN-MG1

To confirm and determine the successful drug loading and targeting peptide grafting, we used an ultramicro ultraviolet spectrophotometer to check the miR-26a-5p loading rate and MG1 grafting rate in MSN based miR-26a-5p targeted delivery systems. As shown in Fig. 3A, C, the absorbance peak values of both MG1 and miR-26a-5p solutions were found to be linear with respect to their concentrations, so we further determined the response rate of MG1 and the loading rate of miR-26a-5p by the standard curve of the MG1 and miR-26a-5p. As shown in Fig. 3A, B, the absorbance of MG1 in the solution decreased from 11.04 to 4.64 at the end of the reaction, with a response rate of 57.97%. Similarly, from Fig. 3C, D, it can be seen that the concentration of miR-26a-5p in the solution changed significantly before and after the reaction, and the loading rate was calculated to be 78.54%. The results of in vitro miR-26a-5p release assay also fully demonstrated the slow and sustained release behaves of the nano-delivery system, which showed a linear release within the first 48 h, followed by a faster release, and then a slower release between 48 and 96 h, and the final release rate could reach nearly 92%. In addition, considering the formulated forms of miRNAs (including miR-26a-5p) are easily degraded. Therefore, we performed a stability study. We measured the miRNA content in nanoparticles stored for 9 and 12 months (Additional file 1: Figure S1A). The results showed that the miRNA content in the nanoparticles remained almost unchanged between 9 and 12 months compared to freshly prepared nanoparticles, reflecting the good stability of our system. In addition, we also used artificial cerebrospinal fluid (ACSF) to simulate the in vivo environment and performed release kinetics experiments on nanomaterials stored for 12 months. The results showed that the release rates of nanoparticles in ACSF were almost the same for 0 and 12 months (Additional file 1: Fig. S1B). Then the thermogravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FTIR) were performed to verify the successful surface modifications of nano-systems (Fig. 3F and 3G). From the TGA results in Fig. 3F, it is indicated that the weight reduction of MSN-NH2 was about 74% of that at room temperature when the temperature was increased to 800 ℃, indicating that the amino siloxane group was successfully modified to MSN. Meanwhile, the thermogravimetric curves of A-MSN-MG1 showed that the mass of the nanoparticles modified with MG1 decreased more with the increase of temperature (the final dry weight percentage was 57%), and the two curves remained approximate at the later stage, which also fully indicated that MG1 was successfully modified into A-MSN nanoparticles.

Fig. 3

Physicochemical characterization and cytocompatibility testing of nanoparticles. A Standard curve for targeting peptide MG1 (absorbance—concentration). B Variation of targeting peptide MG1 concentration in solution with time of chemical grafting reaction. C Standard curve of miR (absorbance-concentration). D Concentration changes during the loading of miR into nanoparticles in solution. E In vitro simulation of miR release profile (release ratio–time). F Thermogravimetric analysis of A-MSN, A-MSN-MG1. G Fourier transform infrared (FTUR) spectra of MSN, A-MSN and A-MSN-MG1. H Toxicity test for cell proliferation viability of MSN, A-MSN, A-MSN-MG1 and miR@ A-MSN-MG1 (CCK-8). I Cytotoxicity experiments of MSN, A-MSN, A-MSN-MG1 and miR@ A-MSN-MG1 (live-dead staining)

As observed in Fig. 3G, the characteristic peak of the hydroxyl group on the surface of MSNs (σ = 1081 ppm) was significantly reduced after chemical modification, indicating that the reaction with APTES occurred and consumed part of the hydroxyl group. New characteristic absorption peaks (σ = 2350 ppm) appeared in the FTIR spectra of A-MSN and A-MSN-MG1, which likely represent the characteristic absorption peaks of amino groups [31]. Also a new absorption peak appeared at σ = 1490 ppm, which is the characteristic absorption peak of the amide group, indicating the successful modification of the targeting peptide onto the MSN-NH2 nanoparticles. In accordance with the results of TEM and DLS measurements, the above results of thermogravimetric analysis and infrared spectroscopy further confirmed the effective chemical modification with amino groups and MG1 peptides and of the nanoparticles. The design and establishment of targeted nano-delivery system are expected to not only achieve targeting, but also endow the potential for prolonged release and therapeutic performance.

In vitro biocompatibility of nano-delivery systems

Besides the above physicochemical properties, we further checked the in vitro cytocompatibility of nanoparticles delivery systems via cell proliferation toxicity assay and cell live-dead staining assay (Fig. 3H, I). We incubated human umbilical vein endothelial cells (HUVECs) with bare MSNs, A-MSN, miR@A-MSN, miR@A-MSN-MG1 at the same concentrations in the culture medium until examination, alongside control groups receiving no treatment. As expected, from the results of the CCK-8 assay, the results showed that the absorbance of HUVECs cultured in nanoparticle-containing medium was not significantly different from that of the control group at 450 nm, indicating that the four types of nanoparticles no noticeable toxicity to cell proliferation. In addition, as seen in Fig. 3I, the live-dead staining results revealed robust cell growth with almost no dead cells observed. Also the cell density and morphology were not significantly different from the control group after cultured by the medium containing nanoparticles, with a significantly higher number of live cells compared to dead cells, suggesting the good cytocompatibility of nanoparticles. The results of in vitro biocompatibility of nano-delivery systems fully show that the surface modification with amino- functionality and MG1 graft did not decrease the inherent advantages of well biocompatibility for MSNs systems, while also imparting the capability to target microglia, indicating a great potential for long-lasting targeted release system [34, 35]. Furthermore, HE staining of the liver and kidneys from mice in different treatment groups revealed no apparent alterations or signs of inflammation following nanoparticle administration (Additional file 1: Fig. S2A). Additionally, we evaluated liver function markers (AST and ALT) and kidney function markers (BUN and Cr) (Additional file 1: Fig. S2B), and the results showed no significant changes, further supporting the safety profile of our nanoparticles.

miR-26a-5p@A-MSN-MG1 delivery system produce long-lasting analgesia duration

Although increasing specific miRNAs has been accepted as a promising therapeutic strategy in chronic pain, current miRNA mimics cannot provide long-lasting or sustainable analgesia which may be partly due to the nature of miRNAs, e.g. their negative charge [39], short half-life in vivo [36, 37], and rapid degradation and inactivation by endogenous nucleases [38, 39]. Apart from that, the non-specificity of miRNA on different cell types also attenuates the effect in pain conditions [12].

To address these challenges, we employed mesoporous silica nanoparticles (MSN) for miR-26a-5p mimic delivery and further modified the surface of MSN with a microglia-targeting peptide, MG1, to improve its duration and targetability in vivo. Subsequently, we established a spared nerve injury (SNI) mouse model to evaluate the pain-relieving effect adopting different delivery strategies. Our behavioral tests showed that miR-26a-5p@A-MSN-MG1 could provide a 42-days analgesia period after single administration, whereas miR-26a-5p@A-MSN or miR-26a-5p mimic only provide analgesic periods of 21 days and 5 days, respectively (Fig. 4B–E). These results suggested that mesoporous silica nanoparticles (MSN) could efficiently elongate the effect of miRNA mimic, which may be attributed to its notable stability and controllable-release capacity [30, 31]. More importantly, MG1-targeted modification further extend the analgesic duration to 42 days (Fig. 4B–E), which, to the best of our knowledge, is longer than any previously reported analgesic duration using nanoparticles or exosomes to deliver miRNA or small molecule (Table 1) [32, 40, 41]. Data of mechanical pain for D8, 10, 28, 35, 42 and 49 are shown in Additional file 1: Fig.S3.

Fig. 4

mir-26a-5p@A-MSN-MG1 delivery system produce long-lasting analgesia duration. A Experimental schematic plot for the establishment of the mouse model of spared nerve injury (SNI). B 50% paw withdraw threshold (PWT) of left hind paw of different treatment groups mice. At the day of 14, 21, 28, 35, 42 and 49, the paw withdrawal thresholds (WTs) of SNI + miR@A-MSN-MG1 group was significantly higher than those of SNI + miR group after single intrathecal injection at the 7th day (*p < 0.05, **p < 0.01, ***p < 0.001 compared with SNI + miR group by Two-way ANOVA followed by Tukey post hoc test, n = 8 in each group). C-E 50% paw withdraw threshold (PWT) of left hind paw of different treatment groups mice at day 12, 14 and 21. F Immunofluorescent study revealed that the enrichment of nanoparticles (Cy5 red) in microglia (IBA1 green) in the L4–5 spinal dorsal horn of SNI mice was significantly increased after MG1 targeting peptide modification but there was no significant change in neurons (MAP2 green). The blue spots are DAPI nuclear staining (Scale bar: 50 μm)

Table 1 Analgesic efficacy of different delivery systems

We have also conducted open-field and rotarod experiments at corresponding time points (Additional file 1: Fig. S4). The results indicate no significant differences in the mice's motor function, excluded that pain-like behavior obtained are due to compounding factors.

To further observe the targeting effect of MG1 modification in vivo, we labeled the nanoparticles with Cy5 and conducted immunofluorescence staining on the L4-5 segments of the mouse spinal cord. We observed that following intrathecal injection of miR-26a-5p@A-MSN, red fluorescence co-localization was noted in both microglia (IBA1+) and neurons (MAP2+) (Fig. 4F), but almost no co-localization in astrocytes (GFAP+) (Additional file 1: Fig. S2A). After modification with the targeting peptide MG1, an increased presence of Cy5-labeled nanoparticles was observed in microglia, with a greater number of microglia colocalizing with these nanoparticles (Fig. 4F, Additional file 1: Fig. S2B). We have also captured images of the contralateral side, revealing that there is more Cy5 red fluorescence on the ipsilateral side, indicating a higher concentration of miR@A-MSN-MG1 (Additional file 1: Fig. S5). This may be attributed to the greater activation of M1-type microglial cells on the ipsilateral side compared to the contralateral side, as clearly observed in the figure. Compared with microglia, MG1 modification did not changed the colocalization of nanoparticles with neurons and astrocytes (Additional file 1: Fig. S6A). These results indicated that MG1 modification could significantly facilitate more nanoparticles targeting microglia, and thus result in the extended analgesic duration after modification. Regrettably, the nanoparticles could still be observed in neuron after MG1 modification, suggesting a need for further efforts to improve the specificity and exclusivity of the nano-delivery system for microglia. MG1 peptide, a homing peptide that specifically recognizes M1 microglia, was identified and isolated by phage display technology [42]. Previous studies showed that BV2-M1 cells showed significantly higher uptake of MG1-modified nanoparticles than mouse brain vascular endothelial cells (bEnd.3), neuron-like PC12 cells, and rat brain astrocytes (CTX) [26]. Although miR@A-MSN-MG1 could not avoid uptake by neurons or other cells, it significantly enhanced enrichment in microglia, and the MG1 peptide still exhibited excellent targeting effects on microglia under inflammatory conditions [26].

We also confirmed the presence of nanoparticle accumulation in the DRG (Additional file 1: Fig. S7). However, our primary therapeutic target is the abundant activation of microglial cells in the spinal cord following neuropathic pain. The MG1 targeting peptide significantly increases the enrichment of nanoparticles in spinal microglial cells, enhancing our therapeutic strategy's specificity.

Moreover, in the current study, although nanoparticles have the ability to cross the blood–brain barrier [43], we adopted intrathecal injection as administration route instead of oral, intravenous or intraperitoneal route. The main reason is that intrathecal injection enables the miRNA mimic to act directly on spinal cord by delivering the nanoparticles to the subarachnoid space. Furthermore, intrathecal injection is a common method to reduce medicine consumption and minimize systemic medicine effects in pain management [44]. Therefore, in the current study, we utilized intrathecal injection to achieve optimal therapeutic effects with a lower dosage and minimal side effects [45].

miR-26a-5p@A-MSN-MG1 delivery system contribute to the prolonged suppression of microglia activation and neuroinflammation

The role of microglia in the onset, progression, and maintenance in chronic pain is well established [23, 46]. A core feature of microglia adaptation to peripheral nerve injury is their activation, leading to persistent neuroinflammation [47]. Therefore, modulating microglia-associated inflammation has been considered as an effective strategy for pain management [22]. In current study, we further evaluated the effect of different delivery strategies on microglia activation and its associated inflammation.

We firstly evaluated the anti-inflammatory effect of different delivery strategies in the L4-5 segment of spinal cord at POD 12, 14 and 21 after SNI. All three strategies to deliver miR-26a-5p mimic significantly attenuated the level of IL-1β and IL-6 (Fig. 5A, G). Consistent with behavioral results of mechanical allodynia, miR@A-MSN and miR@A-MSN-MG1 exhibited a prolonged anti-inflammatory effect untill POD 14 and 21, respectively (Fig. 5A–I). Next, we observed the long-term effect of MG1 modification on microglia activation in the dorsal horn of spinal cord, the L4-5 segment of mice spinal cord were sampled at POD 21, a timepoint that clearly distinguishes the analgesic and anti-inflammatory effects of A-MSN and A-MSN-MG1 delivery strategies. The immunofluorescent staining results showed that the effect of miR-26a-5p in inhibiting microglial activation showed no statistical difference compared to the NC group at POD 21 (Fig. 5J and Additional file 1: Fig. S3). Compared with other group, the number of activated microglia in the miR@A-MSN-MG1 group is the lowest (Additional file 1: Fig. S8). These results indicated that the longer analgesic duration of A-MSN or A-MSN-MG1 delivery system is mainly due to the sustained effect on microglia activation and anti-inflammation. We also noted that the decreases in IL-1β, TNF-α, and IL-6 levels did not occur in parallel at each timepoint, which may be related to the distinct roles of these inflammatory cytokines in the progression of chronic pain [48].

Fig. 5

miR-26a-5p@A-MSN-MG1 delivery system contribute to the prolonged suppression of microglia activation and neuroinflammation. A–I Protein levels of IL-1β (A–C), TNF-α (D–F), and IL-6 (G–I), in the L4–L5 dorsal spinal cord of mice were tested by ELISA at postoperative day 12, 14 and 21. N = 6 mice for each group. Data are represented as mean ± sem. J Immunofluorescent study revealed IBA1-positive microglia (green) in the spinal dorsal horn of different treatment groups mice at day 21 (Scale bar: 100 μm). The blue spots are DAPI nuclear staining. K-N The protein levels of IL-1β (K), TNF-α (L), IL-6 (M), IL-10 (N) in BV2 cell culture supernatant were tested by ELISA

Subsequently, we further evaluated the anti-inflammatory effect of MG1 modification in vitro. BV2 cells, cultured and stimulated with LPS (1 μg/mL) for 24 h. Meanwhile, the miR-26a-5p@A-MSN or miR-26a-5p@A-MSN-MG1 were added to the culture medium to reach the 100 nM concentration. Compared with other groups stimulated with LPS, miR-26a-5p@A-MSN or miR-26a-5p@A-MSN-MG1 both significantly decreased the level of IL-1β, TNF-α and IL-6, and increased the level of IL-10 in culture supernatant (Fig. 5K–N). These in vitro results suggest that both MSN and MG1 modified MSN strategies could exert excellent anti-inflammatory effects. Of note, miR-26a-5p@A-MSN-MG1 exhibits a more evident effect in TNF-α and IL-10 level (Fig. 5L, N) compared with miR@A-MSN in vitro, this further indicates that targeting microglia is a better approach to combat inflammation.

In past decades, accumulating studies have provided evidence for the important role of microglia in allodynia and central sensitization of chronic pain and have shed light on the development of therapeutic strategy [22]. Previous studies adopted minocycline (an inhibitor of microglia activation) and chemogenetic manipulation of microglia are both effective targeting strategies in rodent chronic pain model through inhibiting microglia activation and inflammation [48]. These studies and our targeting strategy demonstrated that target microglia to regulate inflammation is a potent approach for chronic pain management. Moreover, MG1 modified strategies are unlikely to cause antibiotic-related adverse events resulting from minocycline and are more feasible to perform compared with chemogenetic manipulation.

miR-26a-5p@A-MSN-MG1 delivery system maintains a prolonged regulation on Wnt5a signaling pathway

Wnt5a, a member of non-classical Wnt signaling pathway, is a promising target for cancers, inflammatory diseases, and chronic pain [49,50,51]. Our previous research found that miR-26a-5p targets Wnt5a to produce analgesia through regulating neuroinflammation. On this basis, we observed the expression level of Wnt5a signaling molecules at different timepoints after SNI. We found that Wnt5a and its membrane receptor ROR2 were significantly elevated at POD 3,7,14,21, with the highest expression at POD 14(Fig. 6A–C, similar expression pattern could be found in p-JNK and the downstream transcription factor NFAT (Fig. 6A, D, E). These results demonstrated that Wnt5a signaling molecules, including Wnt5a, Ror2, p-JNK and NFAT, continue to elevate after SNI till POD 21. This also suggests that Wnt5a/Ror2-mediated non-classical Wnt signaling is an important signaling pathway in the progression of SNI-induced neuropathic pain towards chronicity, which continues to be activated and plays an important role for a longer period of time after the onset of pain [52], and thus Wnt5a/Ror2 signaling may be an important therapeutic target for chronic pain.

Fig. 6

miR-26a-5p@A-MSN-MG1 delivery system maintains a prolonged regulation on Wnt5a signaling pathway. A–E Western blot was used to assess the level of Wnt5a (B), Ror2 (C), p-JNK (D) and NFAT (E) at day 3, 7, 14, and 21 after SNI modeling. Data are expressed as fold change compared to the sham group. F–L Western blot was used to assess the level of Wnt5a (G), Ror2 (H), p-JNK (I) and NFAT (J), iNOS (K) and CD206 (L) in different treatment groups. Representative immunoblots and quantification showing Foxy-5 reversed the effect of miR@A-MSN-MG1. Data are expressed as fold change compared to the sham group. M–P Protein levels of IL-1β (M), TNF-α (N), IL-6 (O) and IL-10 (P), in the L4–L5 dorsal spinal cord of mice after Foxy-5 reverse were tested by ELISA at day 21. N = 6 mice for each group. Data are represented as mean ± sem. *p < 0.05, **p < 0.01, ***p < 0.001

Next, we examined the expression of Wnt5a, ROR2, p-JNK and NFAT at POD 21 after SNI, employing different miR-26a-5p delivery strategies through intrathecal administration. Consistent with above results, all three miR-26a-5p delivery strategies could significantly decrease the protein expression of Wnt5a, ROR2, p-JNK and NFAT. miR-26a-5p@A-MSN-MG1 is the most effective deliver strategy in regulating molecules of Wnt5a signaling pathway (Fig. 6G–I). We also observed that miR-26a-5p decreased iNOS and increased CD206 (hallmark molecules of M1 and M2 microglia respectively) expression respectively which indicated that miR-26a-5p could influence the equilibrium of M1/M2 microglia and thus regulate microglia activation and inflammatory cytokines releasing. Furthermore, we also demonstrated the effect of miR-26a-5p on Wnt5a signaling pathway and microglia function could be reversed by Foxy5, a Wnt5a mimetic peptide, in miR-26a-5p@A-MSN-MG1 deliver system (Fig. 6G–L). These results further confirmed that A-MSN-MG1 could prolong the effect of miR-26a-5p on Wnt5a signaling pathway to regulate microglia function at least to POD 21 after SNI.

Wnt5a is a critical signaling molecule in physiological and pathological conditions, including embryonic development, tumors, inflammatory diseases, and neurodegeneration [53,54,55]. Previous research found that Wnt5a expression is responsible for sustained inflammation and macrophage activation through autocrine and paracrine signaling [56]. Our results also found that Wnt5a maintains an evident increasing expression pattern after SNI till POD 21 (Fig. 6A, B). Corresponding to Wnt5a expression level, the activation of microglia remained significantly elevated, accompanied by a marked upregulation of inflammatory cytokine expression (Fig. 5A–J). ROR2 and NFAT are both key molecules in Wnt5a signaling pathway to regulate inflammation and microglia function [54, 57]. Furthermore, recent studies showed that NFAT (Nuclear Factor of Activated T-cells) not just mediated T cells function, but contribute to innate immunity, including employed by myeloid cells during early immune response to pathogens and/or tissue lesion, and promote inflammation [58, 59]. In a mouse model of Alzheimer's disease, it has been demonstrated that inhibiting NFAT can attenuate microglial activation and subsequent inflammation [60]. Additionally, triggered NFAT-mediated transcription could amplify the pro-inflammatory responses of microglia [61]. Our results demonstrated that targeting microglia Wnt5a is a feasible strategy to regulate the ROR2 and NFAT expression, which are important molecules in Wnt5a signaling pathway to induce neuroinflammation and microglia activation. What’s more, our strategy to deliver miR-26a-5p provides a sustainable miRNA release and improve the cell-specificity, which contributes to the long-term inhibition on Wnt5a signaling induced neuroinflammation and microglia activation.

miR-26a-5p@A-MSN-MG1 delivery system provides sustained relief from inflammatory pain and chemotherapy-induced peripheral neuropathic (CIPN) pain

Accumulating evidence suggests that Wnt5a is a crucial target in various types of chronic pain. Its involvement has been observed in EAE-induced chronic pain [62], gp120-induced mechanical abnormal pain [