Our study initially synthesized of oridonin-loaded liposome, incorporating DSPE-PEG2000-maleimide as needed. With an established standard curve via HPLC (Figure S1B) of oridonin, we quantified oridonin content in these liposomes at 238 nm, as depicted in Fig. 1E and S1A. To ascertain the loading efficacy of oridonin in TLR2 pep-orid-liposome, batches of products were synthesized, subsequently analyzing the oridonin concentration post-encapsulation through HPLC. The calculated loading efficiency was 9.83 ± 0.15%, indicating successful drug incorporation. Furthermore, we synthesized the TLR2 targeting peptide with an additional Cys residue at the C-terminal. The purity and molecular weight of this TLR2 pep-Cys were verified using HPLC and MS, as shown in Figure S2A and S2B. This peptide was then conjugated to the maleimide-doped orid-liposome at two distinct molar ratios: 1:1 and 5:1. HPLC analysis, employing UV absorbance at 214 nm, was used to characterize TLR2 peptide loading, with volumes set at 100 µl and 20 µl for the 1:1 and 5:1 molar ratio to orid-liposome-MAL, respectively.
As detailed in Figure S3A to C and 1F, more studies demonstrated that TLR2 peptide release from both TLR2 pep-orid-liposome 1:1 and 5:1 was induced in the presence of 50 mg/ml dithiothreitol (DTT), peaking at 10 minutes. In the absence of DTT, no free TLR2 peptide was detectable. These results indicate successful loading of TLR2 peptide onto the orid-liposome-MAL via covalent conjugation (Michael addition) between the maleimide group of the liposome and the thiol group of the peptide. Unless otherwise specified, the TLR2 pep-orid-liposome at the 5:1 molar ratio is referred to as the standard TLR2 pep-orid-liposome in subsequent documentation.
The morphological characteristics of TLR2 pep-conjugated oridonin-loaded liposomes (TLR2 pep-orid-liposome) were investigated using high-resolution electron transmission microscopy (TEM). The image (Fig. 1G) revealed that TLR2 pep-orid-liposome at a 5:1 ratio exhibited a uniform, monodispersed nano-spheric morphology with an average diameter of 78.13 nm, determined from 10 randomly selected particles. Dynamic light scattering in PBS yielded hydrodynamic diameter of 108.2 nm, 81.58 nm, 78.49 nm and 72.71 nm for orid-liposome, orid-liposome-MAL, TLR2 pep-orid-liposome 1:1, and TLR2 pep-orid-liposome 5:1, respectively (Figure S4A to C and 1H). Additionally, zeta potential values were determined to be -49.7 mV, -46.9 mV, -43.7 mV and − 47.5 mV for the respective formulations (Figure S5A to C and 1I).
Moreover, stability of TLR2 pep-orid-liposome was assessed under varying conditions. Notably, TLR2 pep-orid-liposome was observed instability exclusively when exposed to alkaline conditions (NaOH). Conversely, 24-hour incubation in 50% FBS maintained approximately 70% stability of these nanoparticles (Figure S6A to B). Release profiles were examined following similar procedures as previous experiments, with an additional step of post-centrifugation precipitate evaluation (Figure S6C). The results indicated little precipitation from nanoparticles, with the majority of active ingredients being retained while only a small fraction of the drug was released into the supernatant. These findings align with the notion of physiological stability.
TLR2 pep conjugation, together with maleimide, was expected to improve the AML cell killing ability of oridonin. To determine the most effective maleimide to TLR2 peptide ratio, AML cell lines (HL60, U937 and Molm13) and CML cell line (K562) as control were treated with various liposomal formulations, including orid-liposome, orid-liposome-MAL, TLR2 pep-orid-liposome 1:1, and TLR2 pep-orid-liposome 5:1 over 24 or 48 hours. As depicted in Fig. 2A and S7, a concentration- and time- dependent inhibition of cell viability was observed in all AML cell lines and K562. Whereas TLR2 pep alone displayed no inhibitory effect at concentration up to 64 µM (Figure S8). Besides, negative control groups with non-oridonin-loaded liposomes indicated that neither liposome nor liposome-MAL formulations exhibited significant anti-leukemic activity (Figure S9 and S10). Comparative analysis revealed that all oridonin formulations improved the drug's efficacy to varying extents. Notably, orid-liposome-MAL and TLR2 pep-orid-liposome 5:1, which containing higher free maleimide levels than TLR2 pep-orid-liposome 1:1, demonstrated superior efficacy (Table S1). Considering both target-specific delivery and AML cell killing capacity, TLR2 pep-orid-liposome 5:1 showed to be optimal among different forms of nanoparticles.
To further ascertain the enhanced apoptotic induction by TLR2 pep-orid-liposome in AML cells, we employed Annexin V/PI-based apoptosis analysis. This study encompassed HL60, U937, Molm13 and K562 cells, which treated by liposome-MAL, oridonin, orid-liposome, orid-liposome-MAL or TLR2 pep-orid-liposome at a concentration of 4 µM (half maximal inhibitory concentration, IC50) for 24 hours. As anticipated, the results demonstrated that TLR2 pep-orid-liposome notably augmented oridonin’s ability to induce apoptosis in AML cells (Fig. 2B, S11 and S12).
To investigate the intracellular mechanisms of TLR2 pep-orid-liposome, we analyzed the expression levels of total and cleaved caspase 3 in HL60, U937, and Molm13 cells. These cell lines were treated with 4 µM of various formulations for 12 hours. As shown in Fig. 2C, western-blotting results revealed that TLR2 pep-orid-liposome significantly induced caspase 3 cleavage. Notably, the ratio of cleaved to total caspase 3 is much higher in the TLR2 pep-orid-liposome group compared to others. This indicates a more rapid cellular penetration of TLR2 pep-orid-liposome and an enhanced ability to induce apoptosis in AML cells via a caspase 3-dependent pathway.
Validation of TLR2 targeting peptide uptake by AML cells
To investigate how TLR2 pep-orid-liposome improved AML killing ability of oridonin, cellular uptake assays and GSH quantification tests were conducted at the cellular and molecular level. Firstly, to ensure the modified TLR2-binding peptide with an extra Cys-residue maintained the TLR2-binding ability, TLR2 pep-Cys was labeled with rhodamine at N-terminus. This labeled peptide was then incubated at a concentration of 2 µM for 2 hours with cell lines (K562, HL60, U937 and Molm13) and PBMCs as a control. Subsequent flow cytometry analysis was used to measure cellular fluorescence. As illustrated in Fig. 3A, the modified TLR2 pep-Cys effectively recognized the high TLR2-expressing cell lines HL60, U937, Molm13, and to a lesser extent, the moderately expressing K562 cell line, while showing minimal interaction with TLR2 negative PBMCs. These findings indicate that TLR2 pep-Cys successfully retained its TLR2-specific binding capacity.
To visually track the internalization process of TLR2 pep-guided delivery, TLR2 pep-orid-liposome was prepared using rhodamine-labeled TLR2 pep-Cys to accomplish cellular up-take assays. As shown in Fig. 3B, confocal microscopic images demonstrated rapid labelling of AML cells (HL60, U937, and Molm13) by TLR2 pep-orid-liposome within 5 minutes of incubation. These liposomes demonstrated efficient cytosolic internalization in a time-dependent manner. Conversely, minimal internalization of TLR2 pep-orid-liposome was observed in K562 cells. Together with the flow cytometry results, these confocal images indicate TLR2 pep-orid-liposome could efficiently facilitate drug delivery into AML cells, with the efficiency being dependent on the level of TLR2 expression.
To further verify whether TLR2 targeting mediates endocytosis, endocytosis inhibitor amiloride (3 mM) was used to treat AML cells 12 hours in advance, and then rhodamine-labeled TLR2 pep-orid-liposome was co-incubated for 2 hours. The results of flow cytometer detection indicate that amiloride could effectively inhibit TLR2 pep-orid-liposome uptake by HL60, U937 and Molm13 cells, suggesting that TLR2 pep-orid-liposome penetrates TLR2 + cells through receptor-mediated endocytosis (Figure S13).
Maleimide doped liposome further triggered ROS imbalance in AML cells and ameliorated the killing ability of oridonin
TLR2 pep-orid-liposome was engineered to trigger GSH exhaustion via maleimide, thereby intensifying redox imbalance and enhancing the cytotoxicity against AML cells. To verify the ROS augmentation triggered by this nano-machine, ROS assays were employed using a fluorescent probe in AML cells HL60, U937 and Molm13. These experiments were repeated three times, and statistical analysis confirmed that oridonin triggered ROS augmentation in AML cells. Interestingly, empty liposome-MAL unexpectedly demonstrated superior ROS enhancement than oridonin alone. TLR2 pep-orid-liposome 5:1 showed greater efficacy in inducing ROS than oridonin, orid-liposome, orid-liposome-MAL, or TLR2 pep-orid-liposome 1:1. This augmented efficiency of oridonin in orid-liposome-MAL can be attributed not only to its liposomal nature but also to the presence of maleimide, as indicated in Fig. 3C and S14. The lesser effect observed with TLR2 pep-orid-liposome 1:1, compared to the 5:1 ratio, and is likely due to a higher occupancy of DSPE-PEG2000-MAL sites by TLR2 pep.
To delve deeper into the mechanism, we performed reduced GSH quantification assays in AML cells, following treatment by liposome-MAL, oridonin, orid-liposome, orid-liposome-MAL, TLR2 pep-orid-liposome 1:1 and TLR2 pep-orid-liposome 5:1. All treatments were administered at an oridonin-equivalent concentration of 4 µM, with PBS serving as the control. This biochemical reaction results in the production of a yellow compound, 2-nitro-5-mercaptobenzoic acid, identifiable at a wavelength of 412 nm. As demonstrated in Fig. 3D, the treatments with orid-liposome, orid-liposome-MAL, and TLR2 pep-orid-liposome 1:1 and TLR2 pep-orid-liposome 5:1 notably depleted reductive GSH within 4 hours. This effect was significantly more pronounced compared to the control, oridonin, and liposome-MAL. Among these, orid-liposome-MAL and TLR2 pep-orid-liposome 5:1 exhibited superior efficacy, attributable to the incorporation of maleimide.
At the molecular level, GSH depletion assay was also conducted by co-incubating 1 mM GSH with various compounds: oridonin, liposome-MAL, orid-liposome, orid-liposome-MAL, and orid-liposome-TLR2 pep 1:1 or 5:1, each at a concentration of 1 mM for 4 hours. Figure 3E demonstrated that all treatment groups effectively depleted GSH. Notably, orid-liposome-MAL, containing maleimide, was more efficient in GSH depletion compared to orid-liposome. TLR2 pep-orid-liposome 5:1 showed comparable efficacy to orid-liposome-MAL, ranking them as the most effective among all groups. In the case of TLR2 pep-orid-liposome 1:1, majority of maleimide was bond to TLR2 pep, resulting in a GSH depletion capacity similar to that of orid-liposome without maleimide, and thus less efficient than 5:1 variant, which possesses more amount of free maleimide. Taken together, the presence of free maleimide enhances GSH exhaustion. TLR2 pep-orid-liposome 5:1 could simultaneously trigger inverse changes in GSH and ROS, thus further ameliorating AML killing ability of the original ROS targeting drug, oridonin in occurrence.
To validate the synergistic effect at molecular level, separate additions of oridonin and liposome-MAL were made to an excess of GSH solution (1 mM) across concentration gradients. After 4-hour incubation, we quantitatively measured the residual GSH levels. Subsequently, maintaining a constant concentration of liposome-MAL at 1 mM, we continued to add oridonin in concentration gradients to assess its synergistic ability in depleting GSH. The parameter CI was subsequently calculated using the CompuSyn software, which is quantifying the interaction between drugs. In the results presented in Table S2, it is evident that the CI is less than 1.0 (CI<1.0 was defined as synergism; CI>1.0 was defined as antagonism) for all concentration gradients, indicating a synergistic interaction between the oridonin and liposome-MAL. However, it is worth noting that our design is not simply the straightforward combination of two drugs. In fact, during the synthesis of the nano-medicine, the ratio of liposome-MAL to oridonin is fixed (10: 2: 1), and it involves a change in the drug formulation. Therefore, using the CI index or evaluating oridonin, liposome-MAL and orid-liposome-MAL in comparison is not rigorous. Additionally, oridonin induces explicit cytotoxic effects in AML cells, while liposome-MAL does not exhibit the characteristic (Figure S10), which only caused slightly changes in GSH level. Consequently, it was determined that a significant efficiency improvement between oridonin and liposome-MAL.
TLR2 pep-orid-liposome showed potent efficacy for AML therapy in luc-Molm13 xenograft NSG mouse model in vivo
To evaluate the efficacy of TLR2 pep-orid-liposome in vivo, we established an AML xenograft mouse model using luciferase-Molm13 cells in NSG mice. Mouse model has been verified to be powerful for hematologic tumor research in several studies[43]. Figure 4A illustrates the experimental setup, where 5×103 luc-Molm13 cells were injected into NSG mice via tail vein. 6 days after injection, 25 mice were randomly divided into five groups (n = 5). These groups received intravenous treatments of oridonin, orid-liposome, orid-liposome-MAL, and TLR2 pep-orid-liposome at a dosage of 5 mg/kg (equivalent to the active ingredient of oridonin), with PBS serving as the control, administered every other day. The tumor-bearing mice were monitored by living imaging, and the results were presented in Fig. 4B. Not surprisingly, the statistical outcomes demonstrated that TLR2 pep-orid-liposome exhibited the best efficacy in reducing tumor burden, as evidenced by the total flux (summed up fluorescence of dorsal and ventral) among the 5 groups (Fig. 4C), with a significant difference observed compared to the other treatment groups. These mice survived for a total of 25 days and naturally died, which we defined as the survival endpoint as illustrated in Fig. 4D. Results indicated that TLR2 pep-orid-liposome significantly prolonged the survival of tumor-bearing mice compared to the control group. In contrast, oridonin alone did not demonstrate sufficient efficacy at the same dosage. During the progress, the actual body weight variation of mice was closely monitored. Figure S15 indicates no significant difference in body weight changes across these treatment groups.
Rhodamined TLR2 pep-orid-liposome efficiently accumulated and retained in vivo
To explore the bio-distribution of TLR2 pep-conjugated drug, we prepared rhodamine-labeled orid-liposome and TLR2 pep-orid-liposome. The liposome formulation process is identical to the previously described protocol, with the incorporation of fluorescent rhodamine into these formulations. Fluorescent liposomes (rhdamined orid-liposome or TLR2 pep-orid-liposome) were intravenously administered into tumor-bearing Balb/c nude mice, which had been intraperitoneally inoculated with HL60 cells. The bio-distribution of orid-liposome or TLR2 pep-orid-liposome in various organs and tumor was quantitatively evaluated at three distinct time points (12, 24, and 48 hours post-injection) by an in vivo optical imaging system (Mean ± SD, n = 3). As illustrated in Figure S16, TLR2 pep-orid-liposome reached a maximum concentration in tumors as early as 12 hours post-administration, which exhibited greater efficient than that of orid-liposome at 24 hours. Besides of the AML tumors, TLR2 pep-orid-liposome predominantly accumulated in the kidney and liver. Notably, significant fluorescence was retained in the liver at 48 hours, aligning with the liver's role in liposome clearance. These observations indicate that in presence of TLR2 targeting peptide, TLR2 pep-orid-liposome is capable more extendedly of accumulating and retaining in AML, thereby efficiently delivering drug to the target site.
To further provide evidence supporting enhanced tumor accumulation of TLR2 pep-orid-liposome, a detailed analysis comparing the fluorescence ratios (tumor/liver) was conducted. The results clearly demonstrate that TLR2 pep-orid-liposome exhibited significantly higher and faster drug accumulation in tumors compared to the orid-liposome group, particularly at the 12-hour (Figure S17). Specifically, the fluorescence ratio for TLR2 pep-orid-liposome was 0.49 ± 0.05, in contrast to 0.08 ± 0.01 for the orid-liposome group at this time point. These findings robustly support the notion that TLR2 pep-orid-liposome facilitates delivery of the drug specifically to tumor sites, thus minimizing off-target effects in the liver while maximizing therapeutic efficacy against AML cancer cells.
TLR2 pep-orid-liposome treatment not showed obvious toxicity in vivo
Bone marrow suppression is a frequent adverse events associated with many chemotherapeutic drugs. To evaluate the hematological toxicity of TLR2 pep-orid-liposome, blood cell analysis was performed on peripheral whole blood from treated mice. This analysis focused on monitoring levels of hemoglobin (Hb), white blood cells (WBC), and platelets (PLT). The findings indicated that, at the administered dosage, TLR2 pep-orid-liposome did not cause significant hematological inhibition when compared to the control group, as shown in Fig. 5A. Moreover, it demonstrated enhanced efficacy in mitigating reductions in Hb and PLT levels.
To assess potential organ toxicity, an extensive toxicity analysis was carried out.TLR2 pep-orid-liposome and various control groups (n = 6) were administered to Balb/c mice every other day for a total of six doses. Following this regimen, a comprehensive evaluation of hepatic, renal, and myocardial toxicity was conducted. The dosage for each drug across all groups was standardized at 5 mg/kg to ensure consistency in the assessment. Specifically, the obtained serum samples were subjected to rigorous analysis for myocardial enzymes (CK, CK-MB, and LDH-L), liver function (ALT, AST) and renal function (Urea nitrogen, Cr). As presented in Fig. 5B to D, The results clearly demonstrated that TLR2 pep-orid-liposome did not exhibit any evident toxicity. Moreover, HE staining analysis confirmed that no significant organ toxicity associated with this drug (Figure S18).
TLR2 pep-orid-liposome induced apoptosis of AML patient primary cells ex vivo
Ex vivo cytotoxicity assays utilizing leukemic cells isolated from patients provide a valuable approach for assessing the therapeutic efficacy of antitumor agents, especially for hematological malignancies. Primary leukemic cells were obtained from de novo diagnosed AML patients; specific diagram had been presented in Fig. 6A. The baseline information, including AML subtype, was presented in Table S2. The cells were treated with PBS, oridonin, orid-liposome, orid-liposome-MAL and TLR2 pep-orid-liposome at a concentration of 4 µM for 24 hours. Patient samples were divided to two groups based on TLR2 expression to evaluate the advantage of TLR2 pep-orid-liposome. The expression of TLR2 was detected by flow cytometry, and the positive expression of all samples was counted. The average value was taken, and the expression of TLR2 above 80% was defined as high expression, while below 80% was defined as low expression (Fig. 6B and S19). As depicted in Fig. 6C and S20, in all patient samples, oridonin exhibited a certain level of killing effect compared to the control group. However, TLR2 pep-orid-liposome significantly enhanced the killing effect of oridonin. These results, obtained from freshly patient samples, suggested the potential for achieving a clinical response in AML patients with high TLR2 expression using TLR2 pep-orid-liposome. Of particular note is that the proportion of apoptotic cells in the TLR2 pep-orid-liposome group is significantly and positively correlated with the expression level of TLR2 as showed in Figure S21 (p < 0.05).
Moreover, to evaluate the potential cytotoxicity of TLR2 pep-orid-liposome, we performed an apoptosis analysis following drug treatments on PBMCs from two healthy donors. As expected, our results (Fig. 6D) showed a modest induction of cell death in normal cells, indicating a favorable safety profile for the targeting drug with minimal impact on healthy cells.