A nanoconjugate compound, targeted drug delivery system and preparation method and application thereof

Abstract

This application proposes a nanoconjugated compound, a targeted drug delivery system, its preparation method, and its application. The nanoconjugated compound comprises surface-functionalized vinylphosphonate-based hydroxyapatite nanoparticles, and a molecule connected to the surface of the surface-functionalized vinylphosphonate-based hydroxyapatite nanoparticles via covalent bonds and / or electrostatic bonds. Ftaxilide, which showed no detectable anti-tuberculosis activity in the Bactect™ MGIT 960 system, was modified onto hydroxyapatite, resulting in enhanced anti-mycobacterial activity in the Bactect™ MGIT 960 system; the minimum inhibitory concentration against Mycobacterium tuberculosis reached 2 µg / mL; demonstrating potential clinical application value.

Description

Technical Field

[0001] This application belongs to the field of nanomedicine delivery systems and tuberculosis treatment technology, and particularly relates to a nanoconjugated compound, a targeted drug delivery system, its preparation method and application. Background Technology

[0002] Traditional anti-tuberculosis treatments suffer from poor bioavailability, systemic toxicity, and ineffective targeting of infected tissues, while also facing increasing drug resistance and treatment failures due to drug delivery systems. Therefore, tuberculosis (TB) caused by Mycobacterium tuberculosis remains a global health challenge.

[0003] Ftaxilide belongs to the class of cyclic aromatic compounds, which are widely used in pharmaceuticals, agrochemicals, cosmetics, personal care, polymers, materials science, energy, biotechnology, and diagnostics. Aryl carboxamide derivatives are among these pharmacologically active cyclic aromatic molecules; in particular, Ftaxilide's structural features exhibit significant biological properties, including antibacterial, antioxidant, and antituberculosis properties. Due to this pharmacological potential, Ftaxilide is considered a promising antimycobacterial agent, exhibiting pharmacological activity against tuberculosis. However, Ftaxilide did not demonstrate any antimycobacterial activity in the BACTEC™ MGIT 960 system, requiring further structural modification to enhance its antibacterial effect against Mycobacterium tuberculosis.

[0004] Hydroxyapatite (HA) nanoparticles have attracted attention as biocompatible carriers for drug delivery. However, unmodified HA has limitations in terms of drug loading efficiency and controlled release capability. Therefore, surface functionalization of HA nanoparticles is necessary. Summary of the Invention

[0005] This application provides a nano-conjugated compound targeted drug delivery system, its preparation method, and its application, to address the problems existing in related technologies. The technical solution is as follows: In a first aspect, embodiments of this application provide a nanoconjugated compound comprising surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles and Ftaxilide connected to the surface of the surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles by covalent bonds and / or electrostatic bonding.

[0006] In one embodiment, the surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles are prepared by mixing and reacting a phosphate solution with a calcium / phosphorus molar ratio of 1:(1.5-1.8) and a calcium precursor solution in an alkaline solution to obtain surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles. The calcium precursor solution contains vinylphosphonic acid; the amount of vinylphosphonic acid is 1-2% of the amount of calcium ions.

[0007] In one embodiment, the phosphate is disodium hydrogen phosphate; the calcium precursor is calcium nitrate and / or calcium chloride containing calcium ions.

[0008] In one embodiment, the pH of the alkaline solution is adjusted by adding ammonia to make the pH of the solution 10-14.

[0009] Secondly, embodiments of this application provide a method for preparing nano-conjugated compounds, comprising the following steps: Surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles were mixed with Ftaxilide in an ethanol solution, and ferric chloride was added to carry out the reaction. After the reaction was completed, sodium bisulfite was used to quench the reaction to obtain the nano-conjugated compound.

[0010] In one embodiment, the mass ratio of surface-functionalized vinylphosphonic hydroxyapatite nanoparticles to Ftaxilide is (15-20):1.

[0011] In one embodiment, the mass-to-volume ratio of Ftaxilide to ethanol is 1 mg: (8-10) mL.

[0012] In one embodiment, the amount of ferric chloride added is 1-1.2 times the mass of the surface-functionalized vinylphosphonic hydroxyapatite nanoparticles.

[0013] In one embodiment, the reaction further includes a post-processing step: After the reaction was completed, the solid and liquid phases were separated, and the mixture was washed to obtain the nano-conjugated compound.

[0014] Thirdly, embodiments of this application provide a targeted drug delivery system comprising the aforementioned nanoconjugated compound.

[0015] Fourthly, embodiments of this application provide the application of the nano-conjugated compound in the preparation of Mycobacterium tuberculosis drugs.

[0016] In one embodiment, the nanoconjugated compound has a minimum inhibitory concentration of 2 µg / mL against Mycobacterium tuberculosis in the BACTEC™ MGIT 960 system.

[0017] The advantages or beneficial effects of the above technical solutions include at least the following: The nanoconjugated compound of this application comprises surface-functionalized vinylphosphonic hydroxyapatite nanoparticles and Ftaxilide connected by covalent bonds and / or electrostatic bonds. The hydroxyapatite and Ftaxilide form an inorganic-organic hybrid structure, and the surface of the phosphonic hydroxyapatite nanoparticles is modified with vinylphosphonic acid groups to form a conjugation effect with Ftaxilide. The binding with vinylphosphonic hydroxyapatite enhances the therapeutic potential of ftaxilide against mycobacteria. The nanoconjugated compound has improved drug delivery and physicochemical stability.

[0018] The method for preparing the nano-conjugated compound of this application involves modifying the hydroxyapatite with vinylphosphonic acid groups during the preparation process, and then combining it with Ftaxilide. The preparation method is simple and the product is easy to separate and purify.

[0019] The application of the nano-conjugated compound of this application: Ftaxilide did not detect anti-mycobacterial activity in the BACTEC™ MGIT 960 system, but after preparing the nano-conjugated compound of this application, the minimum inhibitory concentration against Mycobacterium tuberculosis reached 2 µg / mL in the BACTEC™ MGIT 960 system; it has potential clinical application value.

[0020] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0021] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0022] Figure 1 This is a flowchart illustrating the preparation process of the nanoconjugated material of this application; Figure 2 The UV-Vis spectra of HA, VPA-HA and nanoconjugates; Figure 3 The direct and indirect band gap energies of nanoconjugated materials; Figure 4 Infrared spectra of HA, VPA-HA and nanoconjugates; Figure 5 Computer-simulated NMR spectra of Ftaxilide and nanoconjugates; Figure 6Particle size distribution curves of VPA-HA and nano-conjugates; Figure 7 Zeta potential and pH for HA, VPA-HA and nanoconjugates; Figure 8 SEM and EDX images of VPA-HA and nanoconjugates; Figure 9 SRD diagram of VPA-HA and nanoconjugate; Figure 10 The optimized structure of Ftaxilide with nanoconjugates is shown in HOMO-LUMO view; Figure 11 The molecular electrostatic potential of Ftaxilide and nanoconjugate; Figure 12 PES and RMS analysis results are shown in the figure. Figure 13 Analysis of MGIT activity in mycobacteria under the influence of different substances in the MGIT 960 system; Figure 14 The graph shows the MTT assay results for different substances against mycobacteria. Detailed Implementation

[0023] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0024] In recent years, nanotechnology-based drug delivery systems and computer simulation modeling methods have provided promising avenues for optimizing drug design and release behavior. However, there remains a need for experimentally validated integrated nanoformulations that combine surface engineering, targeted delivery, and corresponding computational optimization of drug interactions. This invention addresses these limitations by providing a novel surface-functionalized vinylphosphonate-based hydroxyapatite nanoconjugate loaded with ftaxilide, validated by in vitro antimycobacterial and cytotoxicity assays.

[0025] This application provides a nanoconjugated compound comprising surface-functionalized vinylphosphonic hydroxyapatite nanoparticles and Ftaxilide connected to the surface of the surface-functionalized vinylphosphonic hydroxyapatite nanoparticles by covalent bonds and / or electrostatic bonding.

[0026] Ftaxilide is considered a promising antimycobacterial agent, but it has not shown any antimycobacterial activity in the BACTEC™ MGIT 960 system, making it difficult to determine its therapeutic efficacy against mycobacterial diseases. This application loads ftaxilide onto surface-functionalized vinylphosphonate-based hydroxyapatite nanoparticles, forming a drug delivery system and enhancing its therapeutic potential through synergy with vinylphosphonate-based hydroxyapatite (VPA-HA). In this embodiment, the vinylphosphonate-based hydroxyapatite is formed by attaching vinylphosphonate groups to the calcium ions of hydroxyapatite, thereby modifying the hydroxyapatite surface with vinyl groups.

[0027] As one implementation method, the surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles are prepared as follows: A phosphate solution with a calcium / phosphorus molar ratio of 1:(1.5-1.8) and a calcium precursor solution were mixed and reacted in an alkaline solution to obtain surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles. The calcium precursor solution contains vinylphosphonic acid; the amount of vinylphosphonic acid is 1-2% of the amount of calcium ions.

[0028] Surface functionalization of vinylphosphonic acid-based hydroxyapatite is carried out during the preparation of hydroxyapatite. The preparation of hydroxyapatite by means of phosphate solution and calcium precursor in alkaline solution is a conventional method in the art. In this application, vinylphosphonic acid is added in this process, wherein the phosphorus of vinylphosphonic acid is attached to calcium, and vinylphosphonic acid becomes part of hydroxyapatite, thereby achieving vinyl modification, and the hydroxyl group on vinylphosphonic acid becomes the site for connection with ftaxilide.

[0029] In one embodiment, the phosphate is disodium hydrogen phosphate; the calcium precursor is calcium nitrate and / or calcium chloride containing calcium ions.

[0030] In one implementation method, the pH of the alkaline solution is adjusted by adding ammonia water to make the pH of the solution 10-14.

[0031] This application also provides a method for preparing nano-conjugated compounds, including the following steps: Surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles were mixed with Ftaxilide in an ethanol solution, and ferric chloride was added to initiate the reaction. The reaction was then quenched using sodium bisulfite to obtain the nano-conjugated compound. The preparation process is as follows: Figure 1 As shown.

[0032] The Ftaxilide structure is as follows. ; The carboxyl groups can form ligands with metal ions. Ferric chloride is introduced during the reaction primarily to aid monitoring through observable color changes and thin-layer chromatography (TLC). During this process, in the presence of sodium bisulfite, Fe³⁺… + Ions may undergo transient interactions and partial reduction, which are then removed in subsequent washing steps.

[0033] In one embodiment, the mass ratio of surface-functionalized vinylphosphonic hydroxyapatite nanoparticles to Ftaxilide is (15-20):1.

[0034] As one implementation method, the mass-to-volume ratio of Ftaxilide to ethanol is 1 mg: (8-10) mL.

[0035] In one implementation method, the amount of ferric chloride added is 1-1.2 times the mass of the surface-functionalized vinylphosphonic hydroxyapatite nanoparticles.

[0036] As one implementation method, the reaction also includes a post-processing step: After the reaction was completed, the solid and liquid phases were separated, and the mixture was washed to obtain the nano-conjugated compound.

[0037] This application also provides a targeted drug delivery system comprising the aforementioned nanoconjugated compound. The aforementioned nanoconjugated compound is used as the Ftaxilide drug delivery system to kill Mycobacterium tuberculosis.

[0038] This application also provides the use of the aforementioned nanoconjugated compound in the preparation of Mycobacterium tuberculosis drugs.

[0039] As one implementation method, the minimum inhibitory concentration of the nanoconjugated compound against Mycobacterium tuberculosis in the BACTEC™ MGIT 960 system is 2 µg / mL.

[0040] The following provides a further explanation using specific implementation methods.

[0041] The following materials are used in the embodiments of this application: Calcium nitrate [Ca(NO3)2, 99%] and ethanol [CH3CH2OH] 99% were purchased from Sigma Aldrich (Dorset, UK). Diammonium hydrogen phosphate [(NH4)2HPO4, 99%], vinylphosphonic acid (VPA) [C2H3PO3H2, 99%], sodium bisulfite [NaHSO3, 99%], ferric chloride [FeCl3], and ammonia (NH4OH 30%) were supplied by VWR International (UK) and Ftaxilide. Deionized water was used in all experiments.

[0042] Example 1 Measure 500 mL of 0.3 M diammonium hydrogen phosphate and 500 mL of 0.5 M calcium nitrate solution, and add 100 mL of 0.05 M vinylphosphonic acid solution to the calcium nitrate solution. Before the reaction, adjust the pH of the diammonium hydrogen phosphate and calcium nitrate solutions to above 10 using ammonia water. Use a T-connector to mix the diammonium hydrogen phosphate solution and the calcium nitrate solution containing vinylphosphonic acid solution. Pump the diammonium hydrogen phosphate solution and the calcium nitrate solution containing vinylphosphonic acid solution into an 8 m long spiral tube reactor from both ends of the T-connector at a rate of 20 mL / min. The reaction is carried out at 70 °C in the reactor for 5 min. After the reaction is complete, filter, wash the solid phase and dry to obtain vinylphosphonic acid-based hydroxyapatite particles, denoted as VPA-HA.

[0043] Example 2 Vinylphosphonic hydroxyapatite and ftaxilide were added to ethanol and mixed thoroughly. FeCl3 was then added, and the mixture was stirred continuously at 50°C for 5-6 hours. The reaction was monitored using thin-layer chromatography, and the color was observed to change from yellow to brown. 15% w / v sodium bisulfite was added to the reaction mixture while stirring. The pH was maintained in the range of 5-6 throughout the reaction. The mixture was repeatedly washed with deionized water to remove residual ions and then air-dried under ambient conditions to obtain the nano-conjugated compound.

[0044] Material characterization: (1) The nano-conjugated compound prepared in Example 2 was subjected to UV-Vis spectroscopy, and the results are as follows: Figure 2 As shown.

[0045] UV-Vis absorption spectra, such as Figure 2 As shown; the UV-Vis spectrum of pure hydroxyapatite (HA) is as follows. Figure 2 The curve in section a is shown; the UV-Vis spectrum of vinylphosphonic hydroxyapatite is shown in the figure. Figure 2 The curve shown in Figure b; the UV-Vis spectrum of the nanoconjugated material is shown in Figure b. Figure 2 As shown by curve c; The maximum absorption peak at 250-300 nm and the absorption spectra of HA and VPA-HA particles confirmed the existence of nanoconjugation; the maximum absorption peak at 250 nm of the nanoconjugated material confirmed the presence of ftaxilide and nanoparticles. These peaks also represent the nanoconjugation phenomenon, indicating the formation of inorganic-organic hybridization.

[0046] The obtained data were used to plot Tauc plots using Origin software to calculate the band gap of the synthesized nanoconjugated particles. The Tauc plots were obtained by plotting the energy (eV) along the x-axis and the (αhv)1 / 2 and (αhv)2 along the y-axis. The band gap energy was calculated by plotting tangents to a linear fit. The results are as follows: Figure 3 As shown. Figure 3 Figure a shows the direct band gap; Figure 3 Figure b shows the indirect band gap; like Figure 3 As shown in Figure a, the direct band gap energy of the nanoconjugated material is 5.29 eV. These direct band gap energies are due to electronic transitions (de-excitation) between the valence and conduction bands. The peaks shown in the low visible region are due to the presence of vinylphosphonic acid on the surface of pure HA particles. The UV-Vis absorption results of HA and VPA-HA particles and their band gap energies correlate with literature values ​​in the 250–300 nm range. The shift in λmax compared to the nanoparticles indicates electronic changes consistent with conjugation, which may be due to π–π* interactions or charge transfer between the ftaxilide and the nanoparticle surface. Weak n → π* interactions are associated with aromatic carbonyl groups (C=O), with absorption peaks in the 270–290 nm range. The direct and indirect band gap energies are consistent with these π–π* and n →π* interactions.

[0047]

[0048] (1); The energy required to upgrade HOMO to LUMO is given by the relationship E = hc / λ, where E = energy (in joules or eV, calculated as band gap), h = Planck's constant, c = speed of light, and λ = wavelength (in meters); these band gaps can complement nano-conjugation processes and DFT calculations.

[0049] (2) The nanoconjugates, ftaxilide, VPA-HA and HA prepared in Example 2 were subjected to Fourier transform infrared spectroscopy (FTIR) testing, and the results are as follows: Figure 4 As shown, Figure 4 In the table, e and f represent theoretical spectra from computer simulations; the peak absorption values ​​are shown in Table 1. Figure 4 In the FTIR spectrum, at 3190-3175, 2200-2000, 1692-1625, 1455-1416, and 1027-924 cm⁻¹ -1 Significant spectral bands were observed, which were attributed to -OH bending vibration, C=C stretching vibration, CO stretching vibration, Ca-O stretching-bending vibration, and PO bonds, indicating the presence of these functional groups in the prepared nanoconjugated material; this is consistent with PO4. 3-The presence of the relevant functional groups indicates the completion of the reaction, i.e., the formation of VPA-HA particles. The presence of characteristic peaks in the FTIR spectra confirms the occurrence of the nanoconjugation process, indicating the presence of shared functional groups and demonstrating the successful completion of the reaction under controlled conditions. All of these FT-IR spectra reveal the presence of ftaxilide compounds involved in the conjugation process.

[0050] To further verify this, the experimental spectra were compared with the computer-simulated theoretical spectra of ftaxilide and the nanoconjugated material, where the carbonyl stretching frequency of ftaxilide appeared at 1753 cm⁻¹. -1 And after nanoconjugation, it redshifted to 1683 cm⁻¹. -1 Consistent with enhanced conjugation or hydrogen bond formation ( Figure 4 (e and f).

[0051] For nanoconjugated materials, in the range of 3494-3742 cm⁻¹ -1 The region exhibits broad absorption bands corresponding to OH and NH stretching, indicating the formation of a hydrogen bond network or interaction with the hydroxyl groups of the nanosupport system. Fingerprint region (1000-1300 cm⁻¹) -1 The vibrational modes associated with CO and CN bonds in the 900-1000 cm⁻¹ exhibit enhanced intensity and subtle frequency shifts, demonstrating the involvement of these functional groups in the conjugation mechanism. -1 A new strong peak appeared within the range, confirming the structural modification after conjugation.

[0052] Table 1

[0053] (3) This application uses density functional theory to study the theoretical properties of ftaxilide and its nanoconjugates at the B3LYP / 6-311++G(d,p) computational level. 1 H NMR and 13 C10 NMR chemical shifts. All chemical shift values ​​are referenced to tetramethylsilane and reported in parts per million (ppm); NMR spectra are shown below. Figure 5 As shown.

[0054] Computer-simulated NMR spectroscopy of the compounds revealed a shift of the oxygen atom 26-O from 112.57 ppm in the ftaxilide to 7.47 ppm in the nanoconjugated form, indicating substantial coordination or covalent bond formation. The 25-O atom showed a shift from -94.58 ppm to -142.83 ppm, attributed to the deshielding effect of conjugation. Phosphorus resonances were detected at 249.15 and 287.22 ppm, while a calcium signal appeared at 1275.85 ppm, suggesting that phosphate or the calcium-containing moiety was involved in the conjugation process. The carbonyl carbon signal remained at approximately 173 ppm with slight perturbations, indicating that they were close to the conjugation site but not directly involved.

[0055] (4) Particle size analyzer (PSA) The average particle size of the synthesized nanoparticles was studied using a particle size analyzer, and the particle size distribution was as follows: Figure 6 As shown, the nanomaterials were dispersed in deionized water using an ultrasonic bath for 30 min; then, the particle distribution was examined using a colloidal solution; this method is effective for estimating the hydrodynamic diameter and polydispersity index of the synthesized products. Figure 6 The average particle size of the synthesized VPA-HA nanoparticles and nanoconjugates is shown.

[0056] Compared to the results described in the literature for VPA-HA nanoparticles, the synthesized samples exhibited a particle size range of 80±5 nm for VPA-HA and 200±5 nm for the nanoconjugates. The average particle size can be further reduced by changing the dispersion medium and increasing the sonication time, or by filtering the colloidal solution through a micromembrane.

[0057] (5) Zeta potential measurement Zeta potential measurements were performed on selected samples to investigate the colloidal properties of nanoconjugated materials (NC), VPA-HA, and HA, such as... Figure 7As shown. The lower zeta potential values ​​indicate the instability of the nanoparticles due to aggregation, with the large surface area exhibiting instability. The average zeta potential values ​​of the surface-functionalized nanoparticles ranged from -60 ± 5 mV to 70 ± 5 mV. In contrast, the zeta potential of VPA-HA (70 °C) prepared at pH 14 was -68.6 mV, and the zeta potential of the nanoconjugate prepared at pH 14 (50 °C) was -0.2 mV, which was expected to stabilize particle aggregation when the diluted solution was filtered through a nanomembrane and sonicated three times. Similarly, a zeta potential measurement of -43.48 mV was observed for HA, which is attributed to the moderate stability of the uncoated nanoparticles. Higher absolute zeta potential values ​​(above ± 60 mV) indicate better particle stability and anti-aggregation, while values ​​close to (± 20 to ± 60 mV) indicate reasonable stability, and values ​​close to zero (0 to ± 3 mV) indicate particle aggregation due to weak electrostatic repulsion. Zeta potential exploration and literature comparison showed that nanoparticles synthesized in solutions with different pH values ​​exhibited greater stability.

[0058] (6) Field emission scanning electron microscopy (FE-SEM) / EDX testing The morphological properties of VPA-HA were studied using FE-SEM (Sigma 500 VP / ZEISS, Gemini), and its elemental composition and quantitative analysis were determined by EDX coupled with FE-SEM; the results are as follows. Figure 8 As shown.

[0059] Figure 8 Figure (a) shows an FE-SEM image of VPA-HA prepared at pH 10; the hexagonal structure of the synthesized VPA-HA nanoparticles was confirmed by XRD analysis. Particle size was calculated using Image J software, ranging from 22 to 35 nm, with an average particle size of approximately 33.49 nm. Quantitative data from EDX spectroscopy showed that the weight percentages of Ca, P, and O were 27.82%, 15.25%, and 53.91%, respectively.

[0060] Figure 8(b) shows FE-SEM images of the nanoconjugated particles prepared at pH 5–6; these particles are highly aggregated with rough and porous surfaces. Measurements were taken from multiple regions of the images, and the calculated average particle size was approximately 15.70 nm. This is attributed to the rapid nucleation and growth processes during the synthesis of the nanoconjugated particles, resulting in the formation of numerous fine crystallites that lacked sufficient time or migration ability to organize into a well-defined structure. Similarly, FE-SEM analysis of ftaxilide revealed an ordered crystalline morphology characterized by plate-like particles with sharp edges and smooth surfaces; the average particle size was approximately 3.01 µm, larger and more uniform compared to the nanoconjugated particles and VPA-HA. This morphology suggests that ftaxilide underwent slower crystallization kinetics, possibly controlled by synthetic parameters such as lower supersaturation, longer aging time, or template growth conditions.

[0061] (7) X-ray powder diffraction (XRD) To determine the crystal phase, grain size, structural characteristics, and purity of the synthesized nanoparticles, a BRUKER D8 ADVANCE diffractometer was used at 20–80 nm. X-ray diffraction patterns were obtained within the range, and the results are as follows: Figure 9 As shown. The XRD spectrum of the synthesized VPA-HA matches the standard JCPDS spectrum (09-0432) obtained from Expert High Score software. Figure 9 a).

[0062] The XRD patterns of VPA-HA showed strong diffraction peaks at 25.84, 29.45, 32.35, 33.36, 34.26, 36.065, 39.571, 47.283, 49.086, 49.687, 51.39, and 52.89, corresponding to the crystal planes (200), (111), (002), (112), (112), (211), (210), (301), (302), (113), (310), (300), and (311). These results indicate that the synthesized VPA-HA nanoparticles have a hexagonal structure.

[0063] The XRD spectra of the nanoconjugated material and JCPDS (019-0272) were obtained using the same method, as shown in ( Figure 9 As shown in b), strong diffraction peaks were observed at 20.785, 25.846, 26.731, 28.103, 29.19, 29.57, 32.18, 33.94, 48.49 and 53.06, corresponding to the crystal planes (002), (102), (210), (300), (212), (310), (302), (320) and (004).

[0064] The grain size of the VPA-HA and nanoconjugate samples was evaluated using the Debye-Scherrer equation [D=0.9λ / βCosθ], where λ = 0.15406 nm, θ = diffraction peak obtained from XRD patterns, and β = FWHM calculated using Origin software.

[0065] (8) Computer computing research The physicochemical properties of the drug compound (Ftaxilide) and nanoconjugate (NC) were investigated at ADME, as shown in Table 2. Computational studies were performed on these compounds to determine their electronic properties and the factors leading to their variable biological behavior. The structures of the compounds were optimized using GaussView 6.0; further studies were conducted using FMO, MEP, and docking to determine their structure-activity relationships.

[0066] Table 2

[0067] FMO Analysis In quantum chemistry research, the most fundamental variables are the energies of HOMO (π-donor) and LUMO (π-acceptor); these molecular orbitals are called frontier molecular orbitals (FMOs). The reactivity and stability associated with the energy gaps of these orbitals are indicators of the chemical behavior of compounds. The HOMO-LUMO energy gap, which forms the basis of interaction ability or reactivity, is influenced by electron-withdrawing and electron-donating groups. Therefore, calculations of these energy gaps can reveal the inhibitory effects of these synthesized compounds. The high energy gap between HOMO and LUMO makes the compound rigid and stable, with low reactivity. Figure 10 As shown, dark red represents negative nodes in the HOMO, and positive nodes are marked in green. The biologically active part of the molecule, called the LUMO, can add nucleophilic groups, while the HOMO part can couple with electrophilic attracting groups to form chemical bonds. The band gap makes it easier to determine the kinetic stability and chemical reactivity of a molecule. ELUMO-EHOMO is an important indicator of stability. A smaller band gap indicates lower stability, while a larger band gap enhances the kinetic stability of the molecule. Evidence shows that the nanoconjugated material is the most stable compared to the standard sample, with a band gap of 0.18 eV, which confirms its lower chemical reactivity and greater kinetic stability.

[0068] MEP analysis Molecular electrostatic potential (MEP) represents a fundamental quantum mechanical property intrinsically linked to electron density distribution, providing comprehensive insights into electrophilic and nucleophilic reaction centers while elucidating mechanisms of intermolecular and intramolecular interactions. MEP analysis serves as a crucial computational tool for understanding molecular recognition phenomena, particularly drug-receptor binding affinity and enzyme-substrate specificity, through quantitative assessment of electrostatic interactions measured in volts. Three-dimensional MEP surfaces utilize a color-coded visualization scheme to represent different electrostatic potential magnitudes on the molecular surface. Positive electrostatic potential regions arise from the nuclear attraction exerted on the hypothetical test proton, while negative electrostatic potential regions arise from the cumulative attraction of electron density, typically depicted in red on the electrostatic potential map. Traditional color gradient classification systems identify nucleophilic sites as preferential red regions, while electrophilic sites are characteristically represented in blue. The electrostatic potential hierarchy follows a systematic progression: red < yellow < green < blue, corresponding to decreasing electron density and increasing positive potential. According to established computational conventions, the chromaticity scale provides a quantitative interpretation of electron distribution patterns. Red areas represent electron-rich environments favorable for nucleophilic attacks, yellow areas represent regions of moderate electron density, green areas represent electron-neutral regions with balanced charge distribution, and blue areas represent electron-deficient regions favorable for electrophilic interactions. For example... Figure 11 As shown.

[0069] Ftaxilide potential surface and RMS analysis Potential energy surface (PES) scans were performed by varying the scan coordinates from 175.36° to 575.36° in 20° increments, with geometric optimization at each point. The global minimum occurred at 175.36° (-894.2558 Hartree, RMS gradient: 0.000002 Hartree / Bohr), representing the most stable conformation. A local maximum at 275.36° (-894.2247 Hartree) generated an energy barrier of approximately 19.5 kcal / mol, with the highest RMS gradient norm (0.00230 Hartree / Bohr) indicating significant internal strain; beyond this, PES revealed local minima between 515.36° and 575.36° (-894.2503 to -894.2493 Hartree), approximately 3.5–4.0 kcal / mol higher than the global minimum. The intermediate region around 355.36°–375.36° exhibits flat energy behavior (-894.245 Hartree) and a low RMS gradient (~0.00044 Hartree / Bohr), indicating a shallow potential well. Figure 10 These results indicate moderate conformational flexibility of accessible metastable conformations. To calculate ΔEHartree, the local maxima are subtracted from the global minimum, denoted as Emax. Emin. The global minimum energy is -894.2558097 Hartree (at 175.36°), and the local maximum energy is -894.2247010 Hartree (at 275.36°).

[0070]

[0071] Potential energy surface and RMS analysis of nanoconjugated materials PES and RMS gradient analyses revealed complex conformational behaviors in nanoconjugates, such as Figure 12 As shown. The global minimum is located at 533.58° (-2775.0219 Hartree, RMS: 0.00034 Hartree / Bohr), with a closely associated stable conformation at 513.58° (-2775.0164 Hartree). Between 353.58° and 533.58°, the PES remains relatively flat with an energy difference below 0.02 Hartree (12.6 kcal / mol), indicating significant conformational flexibility, possibly due to extended π-conjugation or rotational degrees of freedom. A strain intermediate with an increased RMS gradient (0.00402 Hartree / Bohr) was identified at 433.58°. The overall RMS value fluctuates between 0.0003 and 0.0040 Hartree / Bohr, reflecting moderate internal motion. The nanoconjugated material exhibits enhanced conformational flexibility with multiple accessible minimums and a small energy barrier, potentially advantageous for applications requiring dynamic structural adaptation in drug delivery.

[0072] Comparison of nanoconjugates and ftaxilide Conformational analysis clearly shows that the nanoconjugated compounds remain more stable, while ftaxilide exhibits a rigid energy distribution with a single stable conformation and a large energy barrier of 19.5 kcal / mol. The nanoconjugated compounds possess multiple accessible minima, with energy differences below 12.6 kcal / mol, creating an enhanced, dynamic, and flexible system that can respond to biological conditions, making it ideal for drug delivery applications. In contrast, the high energy barrier of 275.36° in the natural compound creates a conformational bottleneck that limits its adaptability.

[0073] (9) MGIT analysis of nanoparticles and ftaxilide Antimycobacterial activity analysis using the MGIT 960 assay system like Figure 13As shown in Table 3, MGIT tubes containing control (no treatment), ftaxilide, nanoconjugates, and nanoparticles were monitored during the 16-day incubation period. Fluorescence trend is an important visual indicator of nodule growth in MGIT, where bright fluorescence indicates high oxygen consumption and active nodule growth, while dim or delayed fluorescence indicates partial inhibition, and no fluorescence indicates strong inhibition or no nodule growth (Table 3).

[0074] The control group showed strong fluorescence under UV irradiation, while the ftaxilide group remained inactive under UV light, with a minimum inhibitory concentration (MIC) exceeding the maximum test concentration. The nanoconjugated group exhibited excellent antibacterial efficacy, with an MIC of 2 µg / ml, while the MIC values ​​of VPA-HA and HA nanoparticles were 4 µg / ml, respectively. This indicates that although ftaxilide alone lacks significant anti-mycobacterial activity, its conjugation with nanoparticles greatly enhances its therapeutic efficacy against tuberculosis, demonstrating improved bioavailability and targeted delivery mechanism, enabling more effective elimination of Mycobacterium tuberculosis. Therefore, the nanocarrier system not only improves bioavailability but also possesses inherent antibacterial properties.

[0075] Table 3

[0076] Antimycobacterial activity of nanoconjugated materials Nanoconjugated compounds (CN), VPA-HA, and HA particles exhibited excellent antimycobacterial activity in the MGIT 960 system, as shown in Table 4. The MICs at different concentrations are shown in Table 5.

[0077] The detection time to date (TTD) was significantly delayed (>16 days), and fluorescence remained minimal throughout the incubation period, indicating strong growth inhibition. The growth curves contrasted sharply with the drug-free control and the natural compound ftaxilide, both of which showed rapid fluorescence onset and early positivity.

[0078] Table 4

[0079] Ftaxilide did not exhibit any measurable antimycobacterial activity under the test conditions. Its MGIT curve, including detection time to date (TTD) and fluorescence intensity, was very similar to the growth control, indicating no inhibition of Mycobacterium tuberculosis even at the highest test concentration (>16 µg / mL). The growth unit (GU) value of the growth control remained high (≥100), representing the positive threshold. Therefore, ftaxilide was classified as inactive because it exhibited a GU value comparable to the growth control, indicating no antibacterial efficacy (Table 4). Activity classification is an important parameter because higher activity indicates a small or no survival of Mycobacterium tuberculosis (inhibition), while lower activity tends to indicate continued tuberculosis growth despite treatment.

[0080] Table 5

[0081] (10) The cytotoxicity of nanoparticles and ftaxilide formulations was evaluated using the MTT assay. MTT assays were performed to assess the cytotoxicity of four substances: HA, nanoconjugate (NC), ftaxilide, and VPA-HA, with the following results: Figure 14 As shown in Tables 6 and 7. Each compound was prepared as a stock solution, serially diluted to a final working concentration of 10 µg / mL, 20 µg / mL, and 50 µg / mL, and applied to cultured cells under standard conditions. The absorbance at 570 nm was recorded, with the values ​​directly corresponding to cell viability.

[0082] Table 6

[0083] Table 7

[0084] The untreated control group showed the highest activity (mean = 1.0130 ± 0.0711), serving as baseline. Pure HA showed a dose-dependent decrease in absorbance: 0.8640 ± 0.0829 (10 µg / mL), 0.7395 ± 0.0215 (20 µg / mL), and 0.6375 ± 0.0390 (50 µg / mL). The nanoconjugated compounds followed a similar pattern: 0.8483 ± 0.0647 (10 µg / mL), 0.6767 ± 0.0370 (20 µg / mL), and 0.5525 ± 0.0475 (50 µg / mL). Ftaxilide showed initially high activity at 10 µg / mL (0.8601 ± 0.0923), but the decrease was more significant at 50 µg / mL (0.5582 ± 0.0560). VPA-HA exhibited a unique curve, with the highest activity at 10 µg / mL (0.9520 ± 0.0386), but a sharp decline at 20 µg / mL (0.7099 ± 0.1016) and 50 µg / mL (0.5713 ± 0.0516).

[0085] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0086] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0087] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A nanoconjugated compound, characterized in that, Includes surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles and Ftaxilide connected to the surface of the surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles via covalent bonds and / or electrostatic bonds.

2. The nanoconjugated compound according to claim 1, characterized in that, The preparation of surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles is as follows: A phosphate solution with a calcium / phosphorus molar ratio of 1:(1.5-1.8) and a calcium precursor solution were mixed and reacted in an alkaline solution to obtain surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles. The calcium precursor solution contains vinylphosphonic acid; the amount of vinylphosphonic acid is 1-2% of the amount of calcium ions.

3. The nanoconjugated compound according to claim 2, characterized in that, The phosphate is disodium hydrogen phosphate; the calcium precursor is calcium nitrate and / or calcium chloride, which are calcium ions.

4. The nanoconjugated compound according to claim 2, characterized in that, The pH of the alkaline solution is adjusted by adding ammonia water to make the pH of the solution 10-14.

5. A method for preparing the nano-conjugated compound according to any one of claims 1-4, characterized in that, Includes the following steps: Surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles were mixed with Ftaxilide in an ethanol solution, and ferric chloride was added to carry out the reaction. After the reaction was completed, sodium bisulfite was used to quench the reaction to obtain the nano-conjugated compound.

6. The method for preparing the nano-conjugated compound according to claim 5, characterized in that, The mass ratio of surface-functionalized vinylphosphonic hydroxyapatite nanoparticles to Ftaxilide is (15-20):1; The mass-to-volume ratio of Ftaxilide to ethanol is 1 mg: (8-10) mL; The amount of ferric chloride added is 1-1.2 times the mass of the surface-functionalized vinylphosphonic acid-based hydroxyapatite nanoparticles.

7. The method for preparing the nano-conjugated compound according to claim 5, characterized in that, The reaction also includes post-processing steps: After the reaction was completed, the solid and liquid phases were separated, and the mixture was washed to obtain the nano-conjugated compound.

8. A targeted drug delivery system, characterized in that, Including the nanoconjugated compound according to any one of claims 1-7.

9. The use of the nanoconjugated compound according to any one of claims 1-7 in the preparation of Mycobacterium tuberculosis drugs.

10. The application according to claim 9, characterized in that, The minimum inhibitory concentration of the nanoconjugated compound against Mycobacterium tuberculosis in the BACTEC™ MGIT 960 system was 2 µg / mL.

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