Aptamer Modeling
To perform the comparative analysis among the MUC1 targeted aptamers, we utilized the “UNAFold” web server to generate the 2D of the aptamers which represents the Gibbs free energy change associated with the folding and hybridization of nucleic acid sequences [41]. During the 2D structure prediction, the targeted aptamers, namely S1.6, 5TR1, MA3, S2.2, and STRG2, provided the dG score of -2.27, -2.00, -8.62, -1.57, and − 2.67, respectively (Table 1). A lower dG score suggests a more stable structure or stronger hybridization between nucleic acid strands [41]. Figure 1A–E represent the 2D structures of the aptamers. In addition, Fig. 1F–J depict the first predicted 3D model for each aptamer obtained from the Xiao Lab web server [42, 43]. These 3D models were minimized and refined by Maestro 12.5 software for molecular docking.
Molecular Docking
In the molecular docking process to screen the aptamers against the MUC1 protein, we utilized the HDOCK SERVER, renowned for its proficiency in DNA-protein molecular docking. This approach provided the 3D model data visualized in Fig. 1K–O, illustrating the docking simulations between various aptamers and MUC1. Also, the HDOCK SERVER provided docking scores and confidence scores for the DNA-protein complexes [44]. As the lowest docking score means better interaction, examining Table 1, it is evident that the docking score of S1.6 is better than that of MA3, S2.2, and STRG2 while being slightly lower than 5TR1. These scores provide insight into the binding affinity and strength between the aptamers and the protein.
In addition to docking scores, confidence scores were also obtained to estimate the reliability of the predicted complexes. Higher confidence scores indicate a higher level of confidence in the predicted interactions. The maximum confidence score is 1.00, and in our case, 5TR1 showed the best score which is 0.9492 and the second best is S1.6 exhibited a confidence score of 0.9390.
Table 1
Aptamer sequences and scoring metrics
Aptamer Name | Aptamer Sequence 5’ to 3’ | dG score | Docking Score | Confidence Score | Affinity (nM) |
5TR1 | GGGAGACAAGAATAAACGCTCAAG AAGTGAAAATGACAGAACACAACAT TCGACAGGAGGCTCACAACAGGC | -2.00 | -296.36 | 0.9492 | 47.3 |
MA3 | AACCGCCCAAATCCCTAAGAGTCG GACTGCAACCTATGCTATCGTTGAT GTCTGTCCAAGCAACACAGACACA CTACACACGCACA | -8.62 | -279.99 | 0.9308 | 38.3 |
S 1.6 | GGGAGACAAGAATAAACGCTCAAG CAACAGGGTATCCAAAGGATCAAAT TCGACAGGAGGCTCACAACAGGC | -2.27 | -286.72 | 0.9390 | 0.2131 |
S 2.2 | GCAGTTGATCCTTTGGATACCCTGG | -1.57 | -251.94 | 0.8848 | 0.135 |
STRG2 | GAGACAAGAATAAACGCTCAAGGCT ATAGCACATGGGTAAAACGACTTCG ACAGGAGGCTCACAACAGGC | -2.67 | -279.06 | 0.9296 | 18.6 |
This table displays the following information: the names of the aptamers, their dG scores of 2D modeling, as well as the binding affinity, docking score, and confidence score of the lipo-aptamer and MUC1 interaction.
Aptamer Selection
A comprehensive analysis of various parameters was conducted including docking scores, confidence scores, binding affinity, and dG scores, to compare different aptamers and visualized through radar charts (Figure S1, 2). Despite our study showing only a slightly superior outcome of 5TR1 in terms of docking and confidence scores, S1.6 demonstrated significantly better binding affinity and dG score. Notably, while 5TR1 exhibited a binding affinity of 47.3 nM, S1.6 displayed a substantially lower value of 0.2131. This considerable disparity emphasizes the remarkable superiority of S1.6 over 5TR1.
Although MA3 exhibits a better dG score in our study, it falls short in other parameters such as docking score, confidence score, and binding affinity when compared to S1.6. As a result, S1.6 emerged as a more favorable choice over MA3.
During our study, S2.2 exhibited lower scores in all parameters except for binding affinity. Additionally, the difference between S2.2 and S1.6 in terms of binding affinity is only 0.078, which can be considered negligible. Therefore, once again, S1.6 proves to be the superior choice over S2.2.
Both S1.6 and STRG2 lack prior research, making them suitable candidates for further study (Figure S3). Furthermore, considering the binding affinity, docking, and confidence scores, S1.6 outperforms STRG2. Considering all the parameters assessed, it can be concluded that S1.6 is the most suitable aptamer among the options considered.
Liposome Preparation
Figure 2A illustrates the schematic method for preparing an aptamer-conjugated liposome encapsulated with doxorubicin(DOX). To synthesize the liposomes, we focused on three key factors: size, PDI (Poly Dispersity Index), and DOX encapsulation. We varied the molar ratios of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), cholesterol (Chol) and DSPE-PEG-Mal (N-[(3-Maleimide-1-oxopropyl)aminopropyl polyethyleneglycol-carbamyl] distearoylphosphatidyl-ethanolamine) to determine the optimal combination. In our approach, we added 0.5 mg of DOX to the solvent and measured its absorption, which yielded a value of 0.595.
Table 2 presents the absorption values of several DOX-encapsulated liposomes along with their encapsulation rates. After purification using a syringe filter and a PD-10 column, we identified the formulation with the highest absorption value of 0.455, corresponding to a DOX amount of 0.412 mg within the liposomes as shown in Fig. 2B. We measured the DOX concentration using a Nanodrop spectrophotometer at a wavelength of 480 nm. Importantly, we achieved a high encapsulation rate of 73.39% with this liposome formulation, making it suitable for further experiments.
The size analysis revealed that our DOX-encapsulated liposomes had an average size of approximately 84.86 d.nm, meeting our target size of less than 100 d.nm. We also confirmed the appropriate synthesis of the liposomes through transmission electron microscopy (TEM) imaging as shown in Fig. 2C.
In summary, based on the absorption, encapsulation rate, and size analysis, we synthesized liposomes that effectively encapsulate DOX for our further experiments.
Table 2
Characteristics of liposomes based on lipid molar ratio.
Molar Ratio | PBS (ml) | Abs (Before) | Abs (After) | Encapsulation rate | Size (d.nm) | PDI |
15:9:1.0 | 2 | 0.612 | 0.060 | 9.80% | 47.79 | 0.164 |
15:9:1.5 | 2 | 0.595 | 0.086 | 14.45% | 50.73 | 0.147 |
12:8:1.0 | 2 | 0.623 | 0.046 | 7.38% | 51.60 | 0.161 |
12:8:1.5 | 2 | 0.602 | 0.111 | 18.44% | 51.98 | 0.163 |
10:6:1.0 | 2 | 0.586 | 0.208 | 35.49% | 75.05 | 0.106 |
10:6:1.5 | 2 | 0.620 | 0.455 | 73.39% | 84.86 | 0.166 |
The table provides information on the molar ratios utilized for liposome synthesis. It also includes the absorption scores before and after purification of doxorubicin (DOX) encapsulation by liposome. Additionally, the table presents the encapsulation rate of DOX, the size of the liposomes, and their PDI (Poly Dispersity Index) scores.
Aptamer Conjugation
The efficiency of aptamer conjugation was determined using Nano drop spectrophotometer, and the results are depicted in Fig. 2D. Before purification, the concentration of nucleic acid was measured as 152.5 ng/µl, while after purification, it reduced to 121.3 ng/µl, indicating a conjugation rate of 79.54%. To confirm aptamer conjugation, dynamic light scattering (DLS) measurements were performed to assess changes in size and charge. The graphical representation clearly illustrates the distinction between conjugated and non-conjugated samples. Figure 2E shows the alignment of aptamer-conjugated liposomes, while Fig. 2F displays random and scattered patterns for non-conjugated samples.
Gel electrophoresis was conducted to further validate the conjugation process. Figure 2G presents the results for our five samples, where liposome aptamer non-conjugated (LA-N) and liposome-doxorubicin (DOX) aptamer non-conjugated (LDA-N) showed bands similar to the aptamer alone (S1.6). In contrast, there were no bands observed for the conjugated liposome-aptamer and liposome-DOX-aptamer, indicating the absence of free aptamer molecules.
Zeta potential measurements were performed to assess the surface charge of the liposomes. The values obtained were − 0.26, -7.18, and − 5.39 for liposomes, liposome-aptamer, and liposome-DOX-aptamer, respectively. The negatively charged aptamer and positively charged doxorubicin contribute to the observed differences in zeta potential.
Regarding stability, the liposome-DOX-aptamer complex exhibited consistent size measurements at both 4℃ and 25℃, indicating its stability over time is detailed in Figure S4.
Cell line selection
The expression level of MUC1 was confirmed of using an anti-human MUC1 antibody through flow cytometry. While MCF7 cells are known to be MUC1-positive, previous literature suggested that MDA-MB-231 cells also exhibit MUC1 positivity. Surprisingly, our investigation revealed that MDA-MB-231 cells express MUC1 with a tandem repeat at extremely low levels as shown in Fig. 3F. The MUC1-targeting antibody used in our study, generated using a synthetic peptide representing a region near the beginning of the human MUC1 protein, potentially corresponding to the tandem repeat region, indicated negative MUC1 expression in MDA-MB-231 cells as determined by flow cytometry analysis. Consequently, we selected MDA-MB-231 cells as the negative cell line for our experiment, as our aptamer S1.6 specifically targets the tandem repeat region of the MUC1 extracellular domain.
In our study, we employed a primary antibody, rabbit anti-human MUC1, which can bind to human MUC1. The secondary antibody used was a fluorescence dye-conjugated antibody, specifically Goat anti-rabbit, enabling binding to the rabbit-derived primary antibody. This indirect staining method allowed us to investigate the specificity of the primary antibody for MUC1 to get better results at the binding of the anti-MUC1 antibody. The histogram data from Fig. 3 showcase three categories: "Non-treated" represents cells without any antibody treatment (Fig. 3D,H), "Control" denotes cells treated solely with the secondary antibody (Fig. 3C,G), and "Anti- MUC1 ab treated" signifies cells treated with both the primary and secondary antibodies (Fig. 3B,F). By examining the fluorescence intensity depicted on the X-axis of the graph, a shift towards the right indicates the binding of our fluorescence dye-conjugated antibody to the target antigen on the cells. Notably, the graph shifts in the "Anti- MUC1 ab treated" category is more pronounced compared to the "Control" and "Non-treated" groups, highlighting the specific binding of the primary antibody to MUC1 and the absence of nonspecific binding from the secondary antibody.
Cellular Association Study
The binding capacity of liposome was assessed using confocal microscopy imaging. In the case of cells alone, no doxorubicin (DOX) signal was detected (Fig. 4A4,B4,C4,D4) while only the nucleus was visible (Fig. 4A7,B7,C7,D7). With lipo-dox, a reduced presence of DOX was observed compared to lipo-dox-apt in both the positive and negative cell lines at both time points (Fig. 4A5,B5,C5,D5). Additionally, the presence of lipo-dox in both the positive and negative cell lines was similar at both time frames (Fig. 4A8,B8,C8,D8), indicating that cells uptake liposomes consistently over time.
However, a noticeable difference was observed between the positive and negative cell lines when treated with lipo-dox-apt at both time points (Fig. 4A6,B6,C6,D6). Furthermore, significant disparities were observed between lipo-dox and lipo-dox-apt (Fig. 4A8,A9,B8,B9). The absorption of liposomes increased over time, as indicated by the divergence in absorption rates between the 1-hour and 3-hour time points in the positive cell line for lipo-dox-apt. This emphasizes the enhanced binding affinity of liposomes mediated by the S1.6 aptamer compared to liposomes alone.
Based on these results, it can be concluded that the S1.6 aptamer is an effective ligand for specifically targeting MUC1, as demonstrated by the distinct binding patterns observed between the positive and negative cell lines.