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• The optical properties of chiral drugs bear a significant relationship to their pharmacological effects [1,2]. Chiral isomers can exhibit different pharmacological, pharmacodynamic, metabolic, and toxicological activities in living organisms [3,4]. The sedative effects of R-thalidomide and the teratogenicity of S-thalidomide are examples of this phenomenon. Consequently, the acquisition and administration of a single enantiomer of a chiral drug have emerged as a challenging task that requires attention. Chiral anticancer drugs are no exception to this rule. Several promising methods, such as high-performance liquid chromatography (HPLC) [5], chiral membrane separation [6], and asymmetric synthesis [7], have been employed to obtain single enantiomers, however, there are still several obstacles to overcome. These methods can be time-consuming, require specialized equipment, and operators, exhibit low separation efficiency, and have a limited variety of raw materials for chiral synthesis. Additionally, pure enantiomers of chiral anticancer drugs obtained through an arduous process may suffer continually from poor stability, racemization, low water solubility and bioavailability, strong side effects, and ineffective targeting [8]. Consequently, a novel approach to couple drug pharmacological property enhancement and chiral separation is critical and requires immediate development.

  Drug delivery systems (DDSs) have become an increasingly popular topic in the field of drug delivery due to their ability to effectively target specific disease sites [9]. DDSs can overcome the challenges associated with immune clearance and nonspecific binding, allowing for the targeted delivery of drugs [10]. Research has shown that diseases exhibit tissue-specific imbalances in pH, temperature, and enzyme expression [11]. For example, tumor tissue typically shows a higher temperature of 41–43 ℃ in comparison to normal tissue, which is at 37 ℃ [12]. Additionally, the pH of tumor sites is lower at 6.5 than that of normal self-tissue, which is 7.4 [13]. Tumor cells also overexpress various enzymes such as acetylcholinesterase (AChE) and hyaluronidase (HAase). For instance, AChE has been found to play a role in the growth of breast cancer cells, where its activity in tumor tissue is nearly double that observed in normal breast tissue [14]. These unique microenvironmental characteristics present an opportunity to design stimuli-responsive DDSs. Among the available drug-delivery vehicles such as made of nanoparticles [15], liposomes [16], hydrogels [17], and other materials, supramolecular nanoparticles have emerged as one of the promising delivery methods due to their ability to release drugs in response to various external stimuli such as enzymes [18], temperature [19], pH [20], redox [21], and light [22]. It is this characteristic that has made them a significant component of DDSs [13]. For instance, Eva Beňová et al. have developed a drug delivery system that is responsive to changes in pH, which has been studied for the co-delivery of 5-fluorouracil and naproxen [23]. Chen et al. have proposed a mechanism for preparing a lignin nanocarrier that is triggered by changes in pH and applied for Ibuprofen oral delivery [24]. Our research group has prepared various supramolecular nanocarriers that are responsive to changes in pH, temperature, and enzymes to release antitumor drugs at specific targets [25-27]. Although these nanocarriers have exhibited promise in enhancing drug pharmacological activity and targeting, their application in drug stereoisomerization has not been studied. This is likely due to the difficulty of implementing supramolecular nanocarriers that integrate chiral separation and targeted-release properties.

  Chiral separation and targeted release are critical factors in the design and preparation of nanocarriers. Oxaliplatin (OXA), a widely studied anticancer drug for breast cancer [28,29], poses several challenges in conventional chemotherapy, including poor bioavailability, short half-life, severe toxicities, and insufficient targeting. Furthermore, the study revealed that the enantiomer (R,R)-OXA exhibits anticancer activity, whereas (S,S)-OXA is deemed ineffective [30]. Therefore, the identification of OXA enantiomers and the subsequent enhancement of their pharmacological effects are of significant value in boosting drug efficacy. In light of this, the current study employed (R,R)/(S,S)-OXA enantiomers as drug models to explore this phenomenon.

  The separation of chiral compounds is dependent upon chiral environments, which require chiral ligands for their construction [31,32]. Pillar[n]arenes are a group of macrocyclic host molecules that possess upper and lower edges that can be modified with ease [33]. This characteristic offers an advantage for incorporating chiral auxiliaries into the structure of these compounds. The derivatives of pillar[n]arenes, such as (D/L)-alanine-appended pillar[5]arene (D/L-Ala-P5), (D/L)-tyrosine modified pillar[5]arene (D/L-Tyr-P5), L-cysteine decorated pillar[5]arene (L-Cys-P5), and other optically active derivatives, have been utilized as chiral "hosts" in the separation of racemic drugs [34], enantiomeric identification [35], the detection of chiral drugs [36], and other areas. Furthermore, a range of functionalized drug nanocarriers based on derivatives of pillar[n]arenes have been designed and prepared due to their unique rigid structure and remarkable host-guest properties [37-40]. Consequently, pillar[n]arenes have emerged as an effective platform molecule for the development of functional nanomaterials.

  Cancer cells have a heightened demand for specific vitamins such as d-biotin (vitamin B7), vitamin B12, and folic acid (vitamin B9) due to their rapid division [41]. The overexpression of d-biotin-receptors on the surface of fast-growing cancerous cells such as MCF-7, compared to normal cells, presents an opportunity for targeted drug delivery [42,43]. The strong and specific interaction between d-biotin and d-biotin-receptors could facilitate the receptor-mediated endocytosis of biotinylated nanoparticles in cancer cells [44,45]. Additionally, d-biotin presents the potential to serve as a chiral ligand due to its preferred chiral optical properties. The electron-deficient quaternary ammonium salt moiety of myristoyl chloride choline (MCC) has been identified as a promising guest for pillar[n]arenes. Furthermore, MCC can be specifically cleaved into smaller fragments by acetylcholinesterase (AChE), which is overexpressed in cancer cells, offering the possibility of AChE-responsive drug release in cancer cells [14]. Based on these considerations, we propose a research program that involves designing and synthesizing d-biotin-derived pillar[5]arenes (d-biotin-P5) as chiral ligands and targeting agents. Then, a self-assembly method between d-biotin-P5 and MCC will be utilized to prepare chiral nanocatchers. Finally, we will apply the chiral nanocatchers to selectively capture and release the OXA enantiomers, thus demonstrating its potential for targeted drug delivery.

  The study aimed to develop a chiral nanocatcher, which was named d-biotin-P5⊃MCC NCs, via host-guest interaction between d-biotin-P5 and MCC. The chiroptical and AChE-responsive disassembly properties of d-biotin-P5⊃MCC NCs were utilized to selectively capture and release chiral antitumor drugs, specifically oxaliplatin ((R,R)/(S,S)-OXA). The results indicated that d-biotin-P5⊃MCC NCs preferentially captured (R,R)-OXA enantiomers with an enantiomer excess (ee) of 58.8% and an encapsulation efficiency of 25.6%. Additionally, the release rate of OXA from OXA-captured NCs was assessed at 90% with AChE addition within 72 h, simulating the tumor microenvironment. DFT simulation was conducted to elucidate the interaction mechanism between (R,R)/(S,S)-OXA and d-biotin-P5⊃MCC NCs, i.e., d-biotin-P5⊃MCC NCs presented a higher affinity to (R,R)-OXA. The results from cell experiments demonstrated that OXA-captured NCs significantly improved the intracellular uptake of OXA, and OXA present in d-biotin-P5⊃MCC NCs was effectively released to MCF-7 cells. Furthermore, cellular viability assay (MTT) showed that OXA-loaded NCs exhibited a superior inhibitory effect on MCF-7 breast cancer cells at lower concentrations compared to free OXA. Additionally, OXA-loaded NCs reduced free OXA's cytotoxicity against HEK 293 human embryonic kidney cells. Collectively, these findings suggest that d-biotin-P5⊃MCC NCs are well-suited for targeted cancer therapy due to their low side effects on normal tissues, receptor-mediated endocytosis, and satisfactory inhibition of tumor cells. It is noteworthy that such a system may increase the therapeutic use of racemic drugs, which can avoid the traditional chiral resolution process or decrease the synthetic cost compared to the use of enantiomer drugs. Further investigations on d-biotin-P5⊃MCC NCs may broaden the study of chiral supramolecular nanomaterials in enhancing the physicochemical properties of antitumor drugs.

  The present study describes the design of chiral nanocatchers using a host-guest complex and their aggregates, as illustrated in Fig. 1. Specifically, d-biotin-P5 was synthesized for the first time as a host through an esterification reaction, as outlined in Scheme S1 (Supporting information). The primary objective of this synthesis was to produce a chiral receptor and targeting reagent capable of selectively capturing drug enantiomers and targeting tumor cells. The identity of d-biotin-P5 was confirmed through the use of ¹H NMR, ¹³C NMR, and MS, as demonstrated in Figs. S1-S4 (Supporting information). Note that the host molecules we obtained exhibited optical rotation after anchoring d-biotin in pillar[5]arene, as shown in Fig. S5 (Supporting information), which confirms the achievement of d-biotin-P5. We selected MCC as a guest molecule due to its favorable match with pillar[n]arene. The methods for preparing the chiral nanocatchers, which we named d-biotin-P5⊃MCC NCs, are described in the Supporting information. The successful production of d-biotin-P5 and the selected MCC as a guest molecule presents a promising avenue for selectively capturing drug enantiomers and targeting tumor cells.

  Figure 1

  

  Figure 1.  Schematic illustration of the preparation, selective capture, and targeted-delivery properties of the chiral nanocarrier.

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  The interaction between d-biotin-P5 and MCC was investigated via ¹H NMR. To this end, the ¹H NMR spectra of MCC, d-biotin-P5, and their mixtures were recorded with 1 equiv. amount of each compound set to 1.0. As depicted in Fig. S6 (Supporting information), the obtained results demonstrate that the resonance signals H1, H2, and H3 of MCC shifted high-field upon the addition of d-biotin-P5. In contrast, the Ha and Hb protons of d-biotin-P5 exhibited downfield chemical shifts. These observations suggest that the electron cloud density of MCC and d-biotin-P5 increased and decreased, respectively, due to inclusion-induced shielding effects. The binding mode between d-biotin-P5 and MCC was deduced to be the electron-deficient quaternary ammonium groups of MCC drilling into the hydrophobic cavity of d-biotin-P5 to form a d-biotin-P5⊃MCC inclusion complex. The results indicate that self-assembly occurred, consistent with prior reports [46,47].

  The admixture of d-biotin-P5 and MCC resulted in a Tyndall effect opalescence that exhibited a light blue and clear hue, indicating the formation of macroaggregates between d-biotin-P5⊃MCC self-assemblies (Figs. 2a and b inset). To determine the optimal molar ratio between d-biotin-P5 and MCC, the optical transmittance of the mixture solutions was recorded (Fig. S7). The curve representing the optical transmittance at 294 nm versus the d-biotin-P5/MCC ratio exhibited a trend of decline and subsequent increase, reaching a valley at the d-biotin-P5/MCC ratio of 0.5. This trend indicates that the optimal molar ratio of the amphiphilic assembly is 1:2 (Fig. 2a). The size and morphology of the aggregates formed by d-biotin-P5 and MCC were subsequently analyzed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The DLS results indicated that the aggregates had a narrow size distribution with an average diameter of 93.4 nm (Fig. 2b). The TEM images revealed that the aggregates had a uniform spherical morphology with a diameter of ~80 nm. Notably, the aggregates presented a vesicular structure, with the thickness of the hollow vesicles estimated to be ~9 nm (Fig. 2c).

  Figure 2

  

  Figure 2.  Characterizations of d-biotin-P5⊃MCC NCs. (a) The dependence of the optical transmittance at 294 nm was obtained from Fig. S7 (Supporting information) versus the various molar ratios between d-biotin-P5 and MCC. Inset: the images of MCC solutions absented (i) and presented (ii) d-biotin-P5, respectively. (b) DLS results (Inset: the image of Tyndall effect of d-biotin-P5⊃MCC NCs solutions), (c) TEM images, and (d) CD spectra of d-biotin-P5⊃MCC NCs.

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  The formation of aggregates may be comprehended by analyzing the interaction between d-biotin-P5 and MCC molecules. According to the rule of host-guest complex interaction, a self-assembly was first formed between d-biotin-P5 and MCC. The self-assembly was subsequently integrated to generate a large aggregate through hydrophobic interaction between the tail of MCC. The aggregate then curved to a spherical nanostructure, which caused a reduction in optical transmittance. An increase in the concentration of d-biotin-P5 beyond the optimal molar ratio leads to the MCC residue entering the cavity of d-biotin-P5, ultimately causing the dissolution of the aggregates and an increase in optical transmittance. The supramolecular nanoparticles are abbreviated as d-biotin-P5⊃MCC NCs in the remaining part of the article.

  The circular dichroism of d-biotin-P5⊃MCC NCs was characterized using a circular dichroism spectrometer. As illustrated in Fig. 2d, the peaks observed at 225 nm and 312 nm correspond to d-biotin and the inherent chirality of pillar[5]arene, respectively. These findings confirm the successful preparation of chiral d-biotin-P5⊃MCC NCs. DLS analysis revealed that the d-biotin-P5⊃MCC NCs exhibited an average diameter of 94.6 nm, alongside a zeta potential of -20.1 mV over a period of seven days, indicating a high level of stability for the d-biotin-P5⊃MCC NCs (Fig. S8 in Supporting information).

  Supramolecular nanocarriers are highly promising for achieving controlled cargo release in response to stimulus-disassembly [48]. In this context, d-biotin-P5⊃MCC NCs, which utilize the MCC motif as the building block, offer a potential solution for enzyme-responsive disassembly. In a previous report, MCC was shown to be specifically cleaved into small fragments including myristic acid and choline by acetylcholinesterase (AChE). Therefore, the AChE-responsive capability of d-biotin-P5⊃MCC NCs was investigated. As demonstrated in Fig. S9 (Supporting information), the Tyndall effect of d-biotin-P5⊃MCC NCs solutions disappeared, and the optical transmittance at 294 nm of d-biotin-P5⊃MCC NCs increased from 50.23% to 88.16%. The average diameter of the nanocarriers increased after the addition of AChE in d-biotin-P5⊃MCC NCs for 1 h, indicating that most of the nanocarriers were disrupted. TEM images of d-biotin-P5⊃MCC NCs treated with AChE showed no spherical vesicles. These results confirm that d-biotin-P5⊃MCC NCs feature AChE-stimuli disassembly properties, which endow the nanocarriers with the ability to encapsulate and release cargos in response to an environment where AChE is over-expressed. Furthermore, it is worth noting that numerous tumor lesion sites are associated with imbalances in the expression and activity of specific enzymes. This makes the obtained d-biotin-P5⊃MCC NCs highly suitable for controlling the release of anticancer drugs.

  The chiral anticancer drugs (R,R)-oxaliplatin ((R,R)-OXA) and (S,S)-oxaliplatin ((S,S)-OXA) were chosen as the molecular model (Fig. 3a). Symmetrical CD signal peaks observed in (R,R)-OXA and (S,S)-OXA indicate their distinct chiroptical properties (Fig. 3b). The interaction between d-biotin-P5 and OXA enantiomers was evaluated using fluorescence titration and CD test, which revealed that OXA interacts with d-biotin-P5, causing fluorescence quenching and CD signal reduction of d-biotin-P5, as shown in Figs. S10 and S11 (Supporting information). The association constants between (R,R)/(S,S)-OXA enantiomers and d-biotin-P5 were determined using the Benesi and Hildebrand method, with values of K_{(R, R)-OXA} = 3.63 × 10⁵ L/mol and K_{(S, S)-OXA} = 2.18 × 10⁵ L/mol, respectively. The selective coefficient was calculated to be 1.66, indicating that d-biotin-P5 exerted a stronger effect towards (R,R)-OXA. These findings support the feasibility of d-biotin-P5 selectively interacting with OXA enantiomers.

  Figure 3

  

  Figure 3.  The study of d-biotin-P5⊃MCC NCs selectively captured OXA enantiomers. (a) The chemical structures of (S,S)-OXA and (R,R)-OXA. (b) CD spectra of (S,S)-OXA and (R,R)-OXA. (c) UV–vis spectra of d-biotin-P5⊃MCC NCs (blue line), OXA-captured NCs (red line), and OXA (black line) were measured before and after d-biotin-P5⊃MCC NCs captured 0.1 mg/mL racemic OXA solution. (d) TEM images of OXA-captured NCs. HPLC files of racemic OXA (e), and OXA-captured NCs (f).

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  The process of capturing OXA enantiomers by d-biotin-P5⊃MCC NCs was monitored using UV–vis spectroscopy. As illustrated in Fig. 3c, the absorption curve of OXA-captured NCs displayed a significant reduction in peak at the wavelength of 268 nm, with a pronounced redshift compared to free OXA, indicating successful encapsulation of OXA into d-biotin-P5⊃MCC NCs. The increase in nanoparticle size from 93.4 nm to 140 nm, as evidenced by TEM images and DLS results, further confirmed the effective entrapment of OXA into the chiral nanocarriers (Fig. 3d and Fig. S12 in Supporting information). The loading content and encapsulation efficiency of d-biotin-P5⊃MCC NCs were calculated as 83.6% and 25.6%, respectively, following the capture of OXA. These results demonstrate the compatibility of d-biotin-P5⊃MCC NCs as carriers for OXA.

  To validate the selective capture of OXA enantiomers by d-biotin-P5⊃MCC NCs, we conducted an HPLC analysis to track the OXA content before and after the capture of 0.1 mg/mL racemic OXA solution by d-biotin-P5⊃MCC NCs. The racemic solutions containing (R,R)-OXA and (S,S)-OXA had retention times of 6.6 min and 7.2 min, respectively, with an ee of nearly 1.2%, as shown in Fig. 3e. Upon capturing the racemic OXA solution, we observed that the OXA-captured NCs exhibited a relative peak area of 79.4% for (R,R)-OXA and 20.6% for (S,S)-OXA, respectively, indicating that d-biotin-P5⊃MCC NCs possess obvious enantiomeric differential capture performance for racemic OXA (Fig. 3f). The ee was further calculated as 58.8%, which confirms that d-biotin-P5⊃MCC NCs preferentially capture (R,R)-OXA enantiomers. It is noteworthy that OXA-captured NCs showed a reduced peak intensity of (R,R)-OXA and (S,S)-OXA compared to racemic OXA, which may be attributed to the inability of d-biotin-P5⊃MCC NCs to fully encapsulate OXA. The encapsulation rate has been calculated in previous tests. We have attempted to enhance the selective capture efficiency of d-biotin-P5⊃MCC NCs by reducing the concentration of the OXA racemic solution. As evidenced by Fig. S13 (Supporting information), the ee has significantly improved to ~90.0%, while the encapsulation efficiency has remained at ~6%, which may affect the following antitumor effect. This suggested the need for further balancing between the selective capture efficiency and the encapsulation efficiency.

  To further comprehend the chiral capture behaviors of d-biotin-P5⊃MCC NCs, an achiral nanovehicle was prepared by substituting d-biotin-P5 with a glycine-derivative-pillar[5]arene (Gly-P5, without of chiral sites) as a building block, as delineated in Scheme S2 (Supporting information). In this regard, Gly-P5 was synthesized for the first time, which did not exhibit chiroptical properties (Figs. S14 and S15). Subsequently, the nonchiral nanovehicles, Gly-P5⊃MCC NCs, were prepared using the same procedures as d-biotin-P5⊃MCC NCs (Fig. S16 in Supporting information). The morphology of the Gly-P5⊃MCC NCs revealed a spherical particle with a size of 100 nm, and no chiroptical properties were detected, as displayed in Fig. S17 (in Supporting information). Furthermore, UV–vis spectrum and HPLC were carried out to determine how Gly-P5⊃MCC NCs capture OXA enantiomers. The absorption curve of OXA-captured Gly-P5⊃MCC NCs showed a diminished peak of OXA at the wavelength of 285 nm, as depicted in Fig. S18 (Supporting information). The HPLC analysis demonstrated 3.4% ee, indicating that Gly-P5⊃MCC NCs lacked the selective capturing property.

  The observed differences in chiral selective trapping in d-biotin-P5⊃MCC NCs may be attributed to their varying affinities for (R,R)-OXA and (S,S)-OXA enantiomers. Specifically, the selective encapsulation of (R,R)-OXA in the nanocatchers could result in chiral segregation. To confirm this hypothesis, a ¹H NMR analysis was conducted to examine the interaction between d-biotin-P5 and (R,R)/(S,S)-OXA. As illustrated in Fig. S19 (Supporting information), the low-field characteristic peak of d-biotin-P5 remained unaltered, indicating that OXA failed to infiltrate the cavity of d-biotin-P5. The characteristic peaks of the methylene belonging to d-biotin-P5 shifted to the high-field, conceivably due to the proximity of OXA molecules to the branched chain of d-biotin-P5, leading to a higher electron cloud density, as well as an enhanced shielding effect. The characteristic peaks of OXA enantiomers shifted to the low-field, with the difference being more pronounced between d-biotin-P5 and (R,R)-OXA. This could be due to the formation of hydrogen bonds between d-biotin-P5 and OXA enantiomers. To further elucidate the binding configurations between d-biotin-P5 and (R,R)/(S,S)-OXA, density functional theory (DFT) simulation was performed. Fig. 4 and Fig. S20 (Supporting information) presented the optimized geometries of d-biotin-P5 and d-biotin-P5 with the addition of (R,R)/(S,S)-OXA, respectively. There were more H-bonds in the interaction sites between d-biotin-P5 and (R,R)-OXA compared with d-biotin-P5 with adding (S,S)-OXA, in which the carbonyl groups in (R,R)-OXA were toward cavity of d-biotin-P5. The charge of the O atoms in carbonyl groups on the side chain of D-iotin-P5 increases from -0.550 e (mean value) to -0.590 e and -0.614 e, in which were to form the H-bonds. It suggested that the binding of (R,R)-OXA increased the charge population of the side chain of d-biotin-P5. The calculation results were consisted with the NMR change. The findings indicated that a hydrogen bond was formed and more stable between the oxygen atoms in the carboxide group of d-biotin-P5 and the chiral hydrogen atoms in (R,R)-OXA. Meanwhile, The binding energy between d-biotin-P5 and (R,R)/(S,S)-OXA was calculated as 28.7 and 46.3 kcal/mol, respectively, revealed that the configuration between d-biotin-P5 and (R,R)-OXA are more stable. These results were found to be in agreement with the previous fluorescence titration experiments, which confirmed that d-biotin-P5⊃MCC NCs demonstrate higher affinities for (R,R)-OXA [49,50]. Consequently, the chiroptical difference between d-biotin-P5⊃MCC NCs and (R,R)/(S,S)-OXA enables d-biotin-P5⊃MCC NCs to selectively capture (R,R)-OXA, ultimately leading to the separation of (R,R)/(S,S)-OXA. Meanwhile, the calculated binding energies for the interactions between Gly-P5 and (R,R)-OXA and (S,S)-OXA were determined to be 118.6 kcal/mol and 89.6 kcal/mol, respectively. These results indicate that the interactions between Gly-P5 and both (R,R)-OXA and (S,S)-OXA are relatively weak, suggesting an absence of significant chiral differentiation in binding affinity (Fig. S21 in Supporting information).

  Figure 4

  

  Figure 4.  Optimized geometries of d-biotin-P5 with adding (R,R)-OXA.

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  The enzyme-stimulated release behavior of OXA-captured NCs was investigated with the presence and absence of AChE, which was done to mimic the physiological conditions of normal cells and the microenvironment of tumor cells [51]. As shown in Fig. S22a (Supporting information), it was observed that the OXA release rate had a rapid initial increase, followed by a plateau, once AChE was added, with ~50% of OXA released in 10 h and ~90% released in 72 h. This high release efficiency of OXA under AChE stimuli indicated that d-biotin-P5⊃MCC NCs possess the ability to effectively release anti-tumor drugs in cancerous lesions where AChE is over-expressed. In contrast, the absence of AChE resulted in only 26% release of OXA in 72 h, suggesting that OXA-captured NCs were stabilized under simulated physiological conditions. In summary, these findings illustrate the potential of d-biotin-P5⊃MCC NCs as a promising platform for targeted drug delivery in AChE-overexpressing tumors. HPLC was simultaneously employed to test the percentage of release OXA enantiomers after treating OXA-captured NCs with AChE. The results, as depicted in Fig. S22b (Supporting information), showed that OXA-captured NCs treated with AChE exhibited a relative peak area of 76.1% for (R,R)-OXA and 23.9% for (S,S)-OXA, respectively. This finding revealed the dominance of the captured (R,R)-OXA. These results indicated that the d-biotin-P5⊃MCC NCs, which possess AChE-responsive performance, have the capability to selectively capture and release (R,R)-OXA from racemic OXA.

  The biocompatibility and cytotoxicity analysis of biomaterials are crucial for their safety and practical application. In this regard, ex vitro hemolysis assays were carried out using red blood cells (RBCs) and d-biotin-P5⊃MCC NCs. As illustrated in Figs. S23a and b (Supporting information), RBCs displayed negligible hemolytic signs even treated them with d-biotin-P5⊃MCC NCs at a high concentration of 300 µg/mL, indicating d-biotin-P5⊃MCC NCs exhibited good blood compatibility. Quantitative analysis demonstrated that a trace amount of hemoglobin was released, and the percentage of hemolysis was less than 5% for all the treatments, indicating that the d-biotin-P5⊃MCC NCs were highly hemocompatible. Thus, our results suggest that d-biotin-P5⊃MCC NCs possess the potential to be used as biomaterials [52].

  The cytotoxicity activity of OXA-captured d-biotin-P5⊃MCC NCs was evaluated using an MTT assay. The MCF-7 and HEK293 cell lines were subjected to free OXA, d-biotin-P5⊃MCC NCs, and OXA-captured NCs at concentrations of 10, 25, and 50 µg/mL. As depicted in Fig. S23c (Supporting information), free OXA exhibited a slightly dose-dependent antiproliferative activity against MCF-7 cells. Conversely, OXA-captured d-biotin-P5⊃MCC NCs displayed antitumor activity for MCF-7 cells equivalent to that of free OXA. Note that the antitumor activities of OXA-captured NCs at lower concentrations of 10 µg/mL were stronger than free OXA. The findings suggest that d-biotin-P5⊃MCC NCs have the potential to enhance the therapeutic effect of OXA. The observed enhancement in efficacy could be attributed to the fact that when OXA is loaded into d-biotin-P5⊃MCC NCs, it prolongs the circulation time of OXA. Additionally, the d-biotin-based targeting properties of d-biotin-P5⊃MCC NCs facilitate the entry of OXA into tumor cells, thus increasing the effective accumulative concentration of OXA and enhancing its therapeutic effect. To confirm the speculation, MCF-7 cells were pre-treated with d-biotin for one hour. This was done to block the d-biotin receptors' activities. OXA-captured NCs groups exhibited a significantly higher relative cell viability. These findings indicated that the d-biotin-P5⊃MCC NCs are indeed capable of displaying d-biotin-targeted properties. With regard to HEK293 cell lines, the relative cell viability of d-biotin-P5⊃MCC NCs and OXA-captured NCs groups was significantly greater compared to that of the free OXA group. This observation suggests that d-biotin-P5⊃MCC NCs are capable of reducing the toxicity of OXA to normal cells (Fig. S23d in Supporting information).

  To ascertain the targeting properties of d-biotin-P5⊃MCC NCs further, confocal laser scanning microscope (CLSM) images were obtained after incubating MCF-7 cells and d-biotin-pretreated MCF-7 cells with free OXA and OXA-captured NCs, in accordance with relevant reports [53,54]. As illustrated in Figs. 5a and b, a distinct red fluorescence was observed in MCF-7 cells after incubation with OXA and OXA-captured NCs, respectively, indicating that OXA was able to permeate the tumor cells. Notably, the fluorescence intensity of the OXA-captured NCs group, at the low relative concentration of encapsulated OXA, was almost equivalent to that of the OXA group, implying that NCs could enhance the delivery of more OXA into the tumor cells. This phenomenon is likely due to the d-biotin-based targeting properties of d-biotin-P5⊃MCC NCs, which facilitate the receptor-mediated endocytosis of OXA into tumor cells, increasing the effective accumulative concentration of OXA. To examine the targeting efficacy of d-biotin for MCF-7 cells, the cells were first subjected to a treatment with d-biotin for 1 h to inhibit d-biotin-receptor activity. Following this, the cells were incubated with OXA-captured NCs. As depicted in Fig. 5c, a negligible fluorescence intensity was observed, indicating that the uptake of OXA-captured NCs was significantly restricted. The results were consistent with the cytotoxicity test's findings. A control group was established with HEK293 cells exposed to OXA-captured NCs, and a CLSM test was conducted. As illustrated in Fig. 5d, slight fluorescence intensity was observed, suggesting that HEK293 cells exhibited no significant uptake effect on OXA@d-biotin-P5⊃MCC NCs. All the above results demonstrated that the introduction of d-biotin had significantly enhanced the specific targeting ability of OXA-captured NCs to cancer cells that overexpressed the d-biotin receptor.

  Figure 5

  

  Figure 5.  Cellular image study of OXA-captured NCs. CLSM images of MCF-7 cells were obtained by incubating MCF-7 cells with free OXA (a), OXA-captured NCs (b), and culturing the pretreated MCF-7 cells with OXA-captured NCs (c) for 24 h. (d) CLSM images of HEK 293 cells after incubating HEK 293 cells with OXA-captured NCs for 24 h. The red fluorescence signals were from the cells pre-treated by PI and green from the cells pre-treated by Calcein-AM, respectively (scale bar, 250 µm).

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  In summary, the purpose of this study was to create a new type of nanocarrier called d-biotin-P5⊃MCC NCs. These were made by combining d-biotin-P5 with myristoyl choline chloride (MCC), which is responsive to enzyme hydrolysis. D-Biotin-P5⊃MCC NCs were designed to capture chiral antitumor drugs, oxaliplatin ((R,R)/(S,S)-OXA), in a selective manner. The NCs preferentially captured (R,R)-OXA enantiomers, with ~60% ee and an encapsulation efficiency of ~26%. DFT simulation revealed that d-biotin-P5⊃MCC NCs exhibited higher affinity to (R,R)-OXA than (S,S)-OXA. In addition to their chiral capture abilities, the NCs were decorated with Biotin, which targets cancer cells. They also displayed acetylcholinesterase (AChE)-triggered disassembly behavior, enabling them to release OXA. The release rate of OXA from OXA-captured NCs was assessed at ~90% with AChE addition in 72 h. The findings of the cell image studies conducted on MCF-7 breast cancer cells suggest that the use of d-biotin-P5⊃MCC NCs to delivery OXA significantly enhances the intracellular uptake of OXA. The study further reveals that OXA present in d-biotin-P5⊃MCC NCs can be efficiently released to MCF-7 cells. In addition, the use of OXA-loaded NCs at a lower concentration has exhibited a better inhibitory effect on MCF-7 breast cancer cells than free OXA. Moreover, it has been observed to reduce the cytotoxicity of free OXA in HEK 293 human embryonic kidney cells. The development of chiral drug delivery nanocatchers, as presented here, will provide new perspectives on the use of racemic drugs. The nanocatchers are expected to simplify the traditional process of chiral resolution, reduce the cost of synthesizing single-enantiomer drugs, and expand the range of therapeutic applications for racemic drugs. Moreover, the study may broaden the scope for the application of chiral multifunctional materials in cancer treatment.

  Chiral nanocatchers would appear to hold great promise for both chiral separation and targeted delivery. Despite the extensive research conducted on nano-drug carriers for drug delivery and controlled release, the use of chiral nanocatchers as chiral separating agents is still in its nascent stages. A critical challenge in the development of chiral nanocatchers is to attain an optimal balance between chiral capture efficiency and drug loading and encapsulation rate. As presented in this study, we found that reducing the concentration of the racemic feed solution increases the chiral capture efficiency of nanocatchers. However, this reduction also negatively impacts the loading and encapsulation rate, which diminishes the antitumor effect. To address this issue, we propose several approaches to advance the development of chiral nanocatchers. Firstly, designing and synthesizing the novel chiral hosts that enable chiral nanocatchers to adapt to selective-capture requirements. Secondly, selecting appropriate functional guests can modulate the responsive properties of chiral nanocatchers to meet a broader range of therapeutic needs. These insights will serve as a guide in the design and preparation of chiral nanocatchers for the separation and targeted delivery of a broader range of chiral anticancer drugs, as well as promote their application in our future research.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Cui-Ting Yang: Writing – original draft, Investigation, Data curation, Conceptualization. Dan-Dan Wang: Visualization, Software, Investigation, Data curation. Shuai Chen: Methodology, Conceptualization. Jian-Mei Yang: Formal analysis, Conceptualization. Jun-Nan He: Methodology. Jun-Hui Zhang: Resources, Conceptualization. Xiao-Qing Liu: Resources. Jin Zhang: Writing – review & editing, Resources, Funding acquisition, Data curation. Lei Zhang: Software, Methodology, Data curation. Yan Zhao: Writing – review & editing, Methodology, Funding acquisition.

  Acknowledgments

  This work was supported by the National Natural Science Foundations of China (Nos. 22464022, 22461048, and 22364023), Scientific Research Fund Project of Yunnan Education Department (No. 2023j0204), Yunnan Normal University Doctoral Research Initiation Program (No. 01100205020503180), Xing Dian Talent Support Program Foundations (No. 01100208019916016), Yunnan Normal University Graduate Research Innovation Fund Project (No. YJSJJ23-B87), and Yunnan Basic Research Funding Program (Nos. 202401AT070128, 202301AT070074, and 202201AU070056), which are gratefully acknowledged.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2025.110820.

  
  
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• 

  Figure 1  Schematic illustration of the preparation, selective capture, and targeted-delivery properties of the chiral nanocarrier.

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  Figure 2  Characterizations of d-biotin-P5⊃MCC NCs. (a) The dependence of the optical transmittance at 294 nm was obtained from Fig. S7 (Supporting information) versus the various molar ratios between d-biotin-P5 and MCC. Inset: the images of MCC solutions absented (i) and presented (ii) d-biotin-P5, respectively. (b) DLS results (Inset: the image of Tyndall effect of d-biotin-P5⊃MCC NCs solutions), (c) TEM images, and (d) CD spectra of d-biotin-P5⊃MCC NCs.

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  Figure 3  The study of d-biotin-P5⊃MCC NCs selectively captured OXA enantiomers. (a) The chemical structures of (S,S)-OXA and (R,R)-OXA. (b) CD spectra of (S,S)-OXA and (R,R)-OXA. (c) UV–vis spectra of d-biotin-P5⊃MCC NCs (blue line), OXA-captured NCs (red line), and OXA (black line) were measured before and after d-biotin-P5⊃MCC NCs captured 0.1 mg/mL racemic OXA solution. (d) TEM images of OXA-captured NCs. HPLC files of racemic OXA (e), and OXA-captured NCs (f).

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  Figure 4  Optimized geometries of d-biotin-P5 with adding (R,R)-OXA.

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  Figure 5  Cellular image study of OXA-captured NCs. CLSM images of MCF-7 cells were obtained by incubating MCF-7 cells with free OXA (a), OXA-captured NCs (b), and culturing the pretreated MCF-7 cells with OXA-captured NCs (c) for 24 h. (d) CLSM images of HEK 293 cells after incubating HEK 293 cells with OXA-captured NCs for 24 h. The red fluorescence signals were from the cells pre-treated by PI and green from the cells pre-treated by Calcein-AM, respectively (scale bar, 250 µm).

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