Hongsen Tian,Chenhui Gu,Wenshuai Li,Tong Tong,Yunsheng Wang,Yang Yang,Haoli Wang,Zhanqiu Dai,Pengfei Chen,Feng Wang,Xianfeng Lin,Liqing Shangguan,Linfeng Wang
Neutralization of Intracellular pH Homeostasis to Inhibit Osteoclasts Based on a Spatiotemporally Selective Delivery System
nanovesicles,osteoclasts,intracellular pH,membrane hybridization,osteoporosis
Osteoporosis is a global disease caused by abnormal overactivation of osteoclasts. The acidic environment in sealing zone of osteoclasts with H+ pumped from cytoplasm is critical to the maturation of osteoclasts. Therefore, reducing the intracellular H+ concentration can reduce the H+ secretion of osteoclasts from the source. In our study, we developed a novel nanovesicle which encapsulates Na2HPO4 with a liposome hybridizes with preosteoclast membrane (Na2HPO4@Lipo-pOCm). These nanovesicles release Na2HPO4 into the preosteoclast by targeting preosteoclasts and membrane fusion, reducing the intracellular H+ concentration, and achieve biological cascade regulation of osteoclasts through simple pH regulation. In vitro and in vivo experiments confirmed that these nanovesicles reduce mitochondrial membrane potential by decreasing intracellular H+ concentration, thereby reducing the ROS in osteoclasts as well as the expression of the upstream transcription factor FOXM1 of Acp5. In short, this nanovesicle can significantly inhibit the osteoclasts and ameliorate osteoporosis caused by OVX.
Neutralization of Intracellular pH Homeostasis to Inhibit Osteoclasts Based on a Spatiotemporally Selective Delivery System

Osteoporosis is a global disease caused by abnormal overactivation of osteoclasts. The acidic environment in sealing zone of osteoclasts with H+ pumped from cytoplasm is critical to the maturation of osteoclasts. Therefore, reducing the intracellular H+ concentration can reduce the H+ secretion of osteoclasts from the source. In our study, we developed a novel nanovesicle which encapsulates Na2HPO4 with a liposome hybridizes with preosteoclast membrane (Na2HPO4@Lipo-pOCm). These nanovesicles release Na2HPO4 into the preosteoclast by targeting preosteoclasts and membrane fusion, reducing the intracellular H+ concentration, and achieve biological cascade regulation of osteoclasts through simple pH regulation. In vitro and in vivo experiments confirmed that these nanovesicles reduce mitochondrial membrane potential by decreasing intracellular H+ concentration, thereby reducing the ROS in osteoclasts as well as the expression of the upstream transcription factor FOXM1 of Acp5. In short, this nanovesicle can significantly inhibit the osteoclasts and ameliorate osteoporosis caused by OVX.

Osteoporosis is a globalized disease, which increase the propensity for fragility fracture. Abnormal overactivation of osteoclasts leads to increased bone resorption capacity and bone loss, which in turn lead to the occurrence of osteoporosis. Many conventional drugs (e.g., risedronate or bisphosphonate) have been used for the treatment of osteoporosis in the elderly; however, traditional drugs cannot precisely target osteoclasts and can damage normal cells such as osteoblasts and normal bone tissue, leading to undesirable off-target effects and potential side effects at nonbone sites. Therefore, it is urgent to develop a drug that can precisely target osteoclasts to treat osteoporosis. In the process of bone resorption by osteoclasts, the osteoclasts secrete intracellular H+ into the sealing zones to form an acidic environment. In the acidic environment, cathepsin K (CTSK) is activated to degrade bone. At the same time, the secreted H+ first dissolves the inorganic components of the bone, exposing the organic components, and then CTSK degrades the organic components. Therefore, the acidification of the sealing zones is a prerequisite for bone resorption. There is a specific acidic environment within the vigorous osteoclast cytoplasm. At the same time, when the intracellular pH increases, the formation of the podosome belt is also reduced. In the process of osteoclast maturation, intracellular pH is progressively acidified. Thus, increasing the intracellular pH at the preosteoclast stage can inhibit osteoclast H+ secretion at the source. Therefore, increasing the pH in osteoclasts is a feasible method to inhibit osteoclasts. However, it is not easy to increase pH in osteoclasts. Cell membrane coating can be used to prolong systemic circulation of drugs and carry out cell-specific targeting. Therefore, cell membrane coating technology as a tool for drug delivery is a possible disease treatment strategy. During the maturation of osteoclasts, preosteoclasts form mature osteoclasts, mediated by fusion-related proteins through membrane fusion. There are proteins involved in recognition, adhesion, and fusion on the osteoclast membrane, such as DC-STAMP and CD-47. In our previous study, the precursor osteoclast membrane was used as a spatiotemporally targeted drug delivery system. As a carrier for drug delivery, the precursor osteoclast membrane can be used to recognize the precursor osteoclast through membrane fusion and enter osteoclasts to function in cells. Therefore, the precursor osteoclast membrane is a particularly suitable carrier for drug delivery. Na2HPO4 is a weak alkaline substance that widely exists in the internal environment and cells. It regulates the acid–base balance and has good biological safety. It is widely used to inhibit acidosis, compared with other PH regulators, Na2HPO4 has higher biological safety, and phosphate is also the main component of bone. Based on previous studies, we hybridized preosteoclast membranes to liposomes entrapped with an alkaline solution of Na2HPO4 (Na2HPO4@Lipo-pOCm). The nanovesicles can precisely target preosteoclasts and release their encapsulated Na2HPO4 to reduce the intracellular H+ concentration, solving the problem of bone resorption caused by the secretion of H+ by osteoclasts at the source and through simple acid-based chemical neutralization reaction, biological cascade regulation of osteoclasts is realized to inhibit osteoclast differentiation. In vitro experiments show that Na2HPO4@Lipo-pOCm can inhibit osteoclast formation and the bone resorption function of osteoclasts. In an osteoporosis model, Na2HPO4@Lipo-pOCm could significantly inhibit osteoporosis induced by ovariectomy. In conclusion, the Na2HPO4@Lipo-pOCm we designed can precisely target preosteoclasts and release Na2HPO4 through membrane fusion to reduce the H+ concentration in osteoclasts, thereby inhibiting their bone resorption function.

The morphology of the extracted cell membrane was observed by confocal microscopy (FigureS1A). Compared with whole cells, the extracted cell membrane is irregular in shape and does not contain nucleus. Membrane protein retention on the precursor osteoclast membrane (pOCm) is determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Some proteins in whole cells were retained in the extracted cell membrane (Figure S1B). Western blots showed that only Na+/K+-ATPase was contained in our extracted cell membranes, and the results showed that the extracted cell membranes only contained cell membrane components and did not contain other intracellular components (Figure 1A). The above results indicate that the cell membrane extracted in this study contains no other cellular structure. As a drug delivery carrier, the purity of preosteoclasts is very important, so we have analyzed the proportion of preosteoclasts in the cells we use. The result shows that 81.1% of the cells we use are preosteoclasts (Figure S1E). Na2HPO4-Lipo were synthesized according to the method reported in the literature. The hybridization of the cell membrane and liposomes was observed under a confocal microscope (Figure S2A, B). Compared with the preosteoclast membrane, the hybridized nanovesicles had all the proteins on the preosteoclast membrane (Figure S1D). Fluorescein isothiocyanate (FITC) has a significantly stronger green fluorescence signal in alkaline solutions than in neutral or acidic solutions, so it can be used for pH detection. Therefore, we simultaneously prepared vesicles encapsulating NaCl-FITC and Na2HPO4-FITC (NaCl-FITC@Lipo-pOCm, Na2HPO4-FITC@Lipo-pOCm). Na2HPO4-FITC@Lipo-pOCm showed stronger fluorescence intensity than NaCl-FITC@Lipo-pOCm, proving the alkaline environment in liposomes (Figure S1C). The concentration of Na2HPO4@Lipo-pOCm is 6.84 × 1012 ± 1.36 × 1012 particles/mL. The peak particle sizes of Na2HPO4@Lipo, pOCm, and Na2HPO4@Lipo-pOCm were 92.6, 101.367, and 97.3 nm, respectively, and the zeta-potentials were −20.4333, −28.3, and −25.9 mV, respectively (Figure 1B). Cryo-transmission electron microscopy showed Lipo-pOCm to be uniformLy spherical (Figure S1F). Western blot analysis showed that the hybridized nanovesicles retained DC-STAMP, CD-47 and other proteins related to membrane fusion on the precursor osteoclast membrane (Figure 1C). This provides a prerequisite for the targeting of the nanovesicles to preosteoclasts and membrane fusion to release Na2HPO4 into the cytoplasm. At the same time, the particle size of Lipo-pOCm in serum can be stable, while the particle size of pOCm vesicles is constantly changing in serum. (Figure S1G). In vitro drug release experiments showed that the release rate of Na2HPO4 of Lipo-pOCm was significantly slower than that of pOCm. (Figure S1H) To prove that Na2HPO4 enters the cell by means of membrane fusion in Na2HPO4@Lipo-pOCm, we used a endocytosis inhibition assay. Osteoclast precursor cells were treated with four inhibitors: chlorpromazine (a clathrin-mediated endocytosis inhibitor), filipin III (a caveolae-mediated endocytosis inhibitor), cytochalasin D (an actin polymerization inhibitor), and wortmannin (a phosphatidylinositol 3-kinase-mediated micropinocytosis inhibitor), which inhibit the endocytosis of the cells. Detection was performed using flow cytometry. The amount of Na2HPO4@Lipo-pOCm entering the cells was significantly reduced at 4 °C. The four inhibitors had no inhibitory effect on the entry of Lipo-pOCm into the cells (Figures 1D and S2C). At the same time, we conducted the endocytosis inhibition experiment with pOCm (Figure S2D,E), and the results showed that the membrane fusion effect of Lipo-pOCm was mediated by the pOCm, and the addition of liposomes would not affect the efficiency of membrane fusion. In order to prove that DC-STAMP and CD-47 are the main functional proteins in the fusion process, we knock down DC-STAMP and CD-47, respectively (Figure S2F,G), and the efficiency of membrane fusion significantly decreases after knock down (Figure 1E), which proved that Na2HPO4@Lipo-pOCm entered the cells by membrane fusion. Confocal microscopy more clearly showed the process of membrane fusion (Figure S3I,J). To further investigate the role of membrane fusion in lysosomal escape, we synthesized TRITC-labeled fluorescent nanoparticle(NPsTRITC) according to the literature reports and packaged them by Lipo or Lipo-pOCm, respectively. Confocal microscopy imaging was performed after the preosteoclasts were treated with NPsTRITC@Lipo or NPsTRITC@Lipo-pOCm (Figures 1F and S2K). The NPsTRITC@Lipo colocalized with lysosomes, indicating that the nanoparticles all entered the lysosomes, while the NPsTRITC@Lipo-pOCm had no colocalization with lysosomes, a result that suggests that Lipo-pOCm can escape the phagocytosis of lysosomes. To verify that Na2HPO4@Lipo-pOCm has a bone-targeting effect in vivo, we synthesized ICG-encapsulated liposomes (ICG@Lipo) and ICG-encapsulated liposomes and preosteoclast membrane hybrid vesicles (ICG@Lipo-pOCm) using the same method. Osteoporosis mice were injected with ICG@Lipo and ICG@Lipo-pOCm into the tail vein, and in vivo imaging was performed 0.5, 3, and 6 h after injection. The results showed that the proportion of spine and limbs targeted in the ICG@Lipo-pOCm group reached 18% and 14%, respectively, significantly higher than 8% and 5% in the ICG@Lipo group (Figure. 1G). In order to further verify the targeting at the cell level, we injected Lipo-pOCm labeled with DiI (Lipo-pOCmDiI) and took the femur for fluorescent staining (Figure 1H). The results showed that Lipo-pOCm could specifically target the preosteoclasts in vivo. The above experimental results show that the Lipo-pOCm have a good bone targeting effect.

In the process of osteoclast differentiation, the cytoplasmic pH gradually decreased, and the intracellular pH reached its lowest value after 3 days of RANKL stimulation (FigureS3). The cytotoxicity (Figure S4) of Na2HPO4@Lipo-pOCm was determined by the CCK-8 method, which showed no significant inhibition of bone marrow stromal cells (BMMs) at a concentration of 1.71 × 1011 particles/mL. To determine the effect of Na2HPO4@Lipo-pOCm on the generation of osteoclasts, BMMs were cultured in 96-well plates, and 1.71 × 1011 particles/mL of Na2HPO4@Lipo-pOCm or NaCl@Lipo-pOCm was added 3 days after RANKL stimulation. In the RANKL-treated control and NaCl@Lipo-pOCm treatment groups, a large number of TRACP-positive multinucleated osteoclasts were formed, while the number of TRACP-positive multinucleated osteoclasts was significantly reduced under Na2HPO4@Lipo-pOCm treatment (Figure 2A,B). This result indicates that Na2HPO4@Lipo-pOCm treatment significantly inhibited the formation of osteoclasts. Bone resorption assay shows a significant reduction in the number and area of bone resorption observed in the Na2HPO4@Lipo-pOCm group under scanning electron microscopy. In contrast, the OCs and NaCl@Lipo-pOCm groups showed more acid-etching bone defects and severe inorganic mineral loss (Figure 2D,E). Western blot analysis and real-time quantitative polymerase chain reaction (RT-qPCR) assays showed that Na2HPO4@Lipo-pOCm had an inhibitory effect on osteoclasts. After Na2HPO4@Lipo-pOCm treatment, osteoclast function-related genes (c-fos, Atp6v0d2, Acp5, Ctsk, and Nfatc1) (Figure 2C) and the expression of the corresponding proteins (CTSK and NFATc1) decreased (Figure 2F,G). The above results indicate that Na2HPO4@Lipo-pOCm could inhibit the bone resorption function of osteoclasts.

To explore the changes in the transcription level inside the cells after Na2HPO4@Lipo-pOCm was added. We analyzed BMMs, OCs, and Na2HPO4@Lipo-pOCm-treated OCs cells by transcriptome sequencing. We performed a Venn diagram analysis of the sequencing results, and identified 974, 654, and 330 specific genes in the gene expression of BMMs, OCs, and OCs+Na2HPO4@Lipo-pOCm, respectively (Figure 3A). Principal component analysis (PCA) revealed that the repeats were tightly clustered and that the three cells constituted distinct, repeatable subsets (Figure 3B). Based on the top 300 upregulated genes, we found that the expression levels of osteoclast-related genes such as Ctsk, Acp5, Nfatc1, and Atp6v0d2 were decreased in OCs treated with Na2HPO4@Lipo-pOCm, which is consistent with our previous study (Figure 3D). Gene set enrichment analysis (GSEA) revealed decreased oxidative phosphorylation levels in the Na2HPO4@Lipo-pOCm group compared to the OCs group (Figure 3C). To identify the key transcription factors that cause osteoclast differentiation and functional changes after the addition of Na2HPO4@Lipo-pOCm, we selected the top 300 genes with the largest changes, constructed the mRNA expression profiles of transcription factors, and used CHEA3’s original pathway analysis to predict upstream regulators. The predicted transcription factor with the highest Z score was FOXM1 (Figure 3E), with a 10.09-fold change. We further verified the changes in FOXM1 expression in cells. The RT-qPCR results show decreased FOXM1 transcript levels in the Na2HPO4@Lipo-pOCm group (Figure 3F). Western blot results further demonstrate that the FOXM1 protein expression level decreased after the addition of Na2HPO4@Lipo-pOCm (Figure 3G,H).

During the process of osteoclast maturation, the level of ROS was significantly increased. In mitochondria, the free energy provided by the flow of electrons from NADH or flavin nucleotides to O2 is conserved as a transmembrane electrochemical proton gradient (proton motive force, PMF), which is then exploited to generate ATP from ADP and Pi. When the mitochondrial electron transport chain (ETC) is inhibited, the mtROS generated by mitochondria will increase. In the electron transport chain, as electrons are transported along the chain, H+ is pumped from the mitochondrial matrix into the intermembrane space, resulting in the formation of negative and positive transmembrane electrochemical proton gradients (PMF) inside and outside the mitochondrial inner membrane. When the extramitochondrial H+ concentration decreases, the proton gradient pressure on the ETC decreases, and the inhibition of PMF during electron chain transfer decreases, resulting in a decrease in the production of ROS. We selected BCECF-AM stained cells for flow cytometry and confocal microscopy analysis. Flow cytometry and confocal microscope showed that in the Na2HPO4@Lipo-pOCm group, the pH of osteoclasts increased significantly compared with that in the OCs group (Figures 4A,B and S5A,B). We used flow cytometry to further determine the changes in intracellular reactive oxygen species levels (Figures 4C and S5C). The level of ROS increased during the differentiation of BMMs into OCs, while that in the Na2HPO4@Lipo-pOCm group was significantly lower than the OCs group. To explore whether the decrease in ROS levels was caused by the decrease in mitochondrial PMF, we stained cells with JC-10 stain and observed them under a confocal microscope. The results show that the mitochondrial membrane potential in osteoclasts decreased significantly after the addition of Na2HPO4@Lipo-pOCm (Figures 4D and S5D). Based on the above increases in pH, it can be determined that the changes in mitochondrial membrane potential were caused by increases in cytoplasmic pH. In addition, by seeding osteoclasts on FITC labeled bone slices, we demonstrated that Na2HPO4@Lipo-pOCm reduces the acidic environment of the sealing zones (Figures 4E and S5E). Compared with normal osteoclasts, the fluorescence intensity of FITC in the sealing zones increased after 1 day of coincubation with Na2HPO4@Lipo-pOCm. Among the predicted appellate transcription factors, FOXM1 was the transcription factor with the highest score. Forkhead Box M1 (FOXM1), a member of the Forkhead box (Fox) transcription factor family, is a multifaceted transcription factor with multiple cell-type-specific functions in vivo, and ACP5 is a direct transcriptional and functional target of FOXM1. ROS is an influential factor on FOXM1. ROS increased after stimulation of BMMs with H2O2 for 24 h (Figures 4F and S5F). The RT-qPCR results show that the expression of FOXM1 increased with increasing ROS (Figure 4G). Then we transfected siRNAFOXM1 to knock down the expression of the FOXM1 gene (Figure S5G), and the RT-qPCR results show that after transfection of siRNAFOXM1 to knock down the FOXM1 gene, osteoclast differentiation and function were reduced (Figure S5G). TRAP staining also showed decreased generation of osteoclasts (Figure 4H). A natural product, thiostrepton, directly interacts with FOXM1 and inhibits its binding to genomic targets. Thiostrepton also prevents FOXM1 from binding and activating its own promoter. The expression of FOXM1 was also significantly attenuated after thiostrepton treatment of osteoclasts (Figure S5G), without inducing apoptosis or inhibiting cell proliferation at ≤1 μM concentrations (Figure S5J). The RT-qPCR results indicate that osteoclast differentiation was inhibited (Figure S5H), and TRAP staining also indicated a decrease in the generation of osteoclasts (Figure 4H). The above results indicate that FOXM1 is a key transcription factor in hydrogen ion mediated osteoclast inhibition.

To further verify the effect of Na2HPO4@Lipo-pOCm in vivo, a common OVX model was used in this study (Figure 5A). No adverse events or deaths were reported in any of the groups, there was no impairment of liver and kidney function or vital organs in mice injected with NaCl@Lipo-pOCm solution or Na2HPO4@Lipo-pOCm solution compared with the OVX and control groups (Figure S6A,B). The mice were sacrificed 6 weeks after surgery, and the femur and tibia were taken for micro-CT scanning. The scans show that Na2HPO4@Lipo-pOCm prevented extensive bone loss in several parts of the mouse body after ovariectomy (Figure 5C). Quantitative analysis of bone parameters also confirmed increased BMD, BV/TV, Tb.Th, Tb.N, and Tb.Sp in Na2HPO4@Lipo-pOCm treated group relative to the OVX mice without Na2HPO4@Lipo-pOCm treatment (Figure 5D). Meanwhile, there was no significant improvement in whole-body bone loss in the NaCl@Lipo-pOCm and Na2HPO4@Lipo group compared to the OVX group (Figure S7). Since the drug is administered intravenously, we have carried out pharmacokinetic analysis of the Lipo-pOCm (compare with Lipo). The results showed that Lipo-pOCm revealed remarkably longer blood circulation time (t1/2 = 21 h) than Lipo (t1/2 = 8 h), which may be caused by the presence of CD47 protein on the cell membrane. Histological examination (HE staining) showed that both the bone volume and bone surface were well maintained in the Na2HPO4@Lipo-pOCm group compared to the OVX group (Figure 5E,F). However, there was no difference between the NaCl@Lipo-pOCm group and the OVX group (Figure S7B). TRAP staining showed that OVX led to an increase in the number of osteoclasts (TRAP-positive cells), while Na2HPO4@Lipo-pOCm treatment significantly decreased the number of osteoclasts (Figure 5E,F). However, there was no difference between the NaCl@Lipo-pOCm group and the OVX group (Figure S7B). It suggests that Na2HPO4@Lipo-pOCm can significantly improve the osteoporosis caused by devitalization through the inhibition of osteoclasts. In this study, we successfully constructed Na2HPO4@Lipo-pOCm formed by the hybridization of liposomes encapsulating a mild alkaline solution with the membrane of precursor osteoclast. The targeting of the precursor osteoclast membrane enables the nanovesicles to target the precursor osteoclasts and release the mild alkaline solution into the cell through membrane fusion, changing the intracellular pH, which in turn inhibits the function of osteoclasts and ameliorates osteoporosis. In conclusion, through the combination of natural cell membranes and liposomes, these novel nanovesicles provide a potential strategy for the treatment of osteoporosis.