Drug-loaded microalgae motor system for colon-targeted delivery and preparation method and application thereof

Abstract

The application discloses a drug-loaded microalgae motor system for colon-targeted delivery and a preparation method and application thereof. The drug-loaded microalgae motor system comprises a Chlamydomonas reinhardtii microalgae motor core, the surface of the core is adsorbed with amino-modified ferroferric oxide nanoparticles through electrostatic adsorption, is wrapped with quercetin-modified chitosan quaternary ammonium salt, forms a nano-composite with magnetic responsiveness, and is encapsulated in a sodium alginate microsphere. The application utilizes Chlamydomonas reinhardtii as an antioxidant for adjuvant therapy, in a colon environment, the sodium alginate microsphere can be degraded in response to specific conditions, release the microalgae motor to autonomously move in intestinal fluid, and under the guidance of an external magnetic field, precisely target a lesion site in the intestinal tract. The application has good biocompatibility, biodegradability, gastric acid avoidance ability and magnetic / chemical dual driving targeting function, can effectively improve the treatment effect on colon diseases, and significantly reduce the systemic side effects of drugs, and expands the application of micro / nano motors in the biomedical field.

Description

Technical Field

[0001] This invention belongs to the fields of micro-nano technology and biomedicine, specifically relating to a drug-loaded microalgae motor system for colon-targeted delivery, its preparation method, and its application. Background Technology

[0002] Traditional drug delivery methods suffer from significant side effects due to systemic distribution and low bioavailability. Fe3O4 nanoparticles, with their superparamagnetism, magnetic responsiveness, and good biocompatibility, have become widely used materials in magnetically targeted drug delivery, enabling localization and retrieval via an external magnetic field. However, bare Fe3O4 is prone to aggregation and oxidation, and lacks functionalization sites, thus limiting its applications. Chitosan is an excellent modifying and protective layer material for magnetic nanoparticles, effectively preventing aggregation caused by dipole interactions between magnetic nanoparticles. It provides effective functional groups on the surface of magnetic nanoparticles, expanding their application range. Furthermore, the functionalization of chitosan allows magnetic chitosan nanoparticles to retain good magnetic properties while possessing high biocompatibility.

[0003] Alginate is a polysaccharide compound widely found in brown algae cells and is widely used in food and pharmaceuticals. Most alginates are soluble in water at varying temperatures. During the ionic gelation process of alginate, the selectivity of cations affects its bioactivity, resulting in higher drug release in vivo. Combining these two biocompatible materials can significantly improve drug encapsulation efficiency and release rate, making them excellent drug delivery carriers. For example, Chinese invention patent CN113633624A provides a method for preparing core-shell structured alginate-magnetic chitosan microspheres. Magnetic chitosan microspheres are prepared using an emulsification crosslinking method, and then core / shell structured alginate / magnetic chitosan microspheres are obtained using a D-phase emulsification method.

[0004] However, due to the combined effects of chitosan's solubility and cross-linking structure, Fe3O4 stability, the complex intestinal environment, and physiological barriers in the body, the release rate becomes uncontrolled, incomplete, and insufficiently targeted, ultimately reducing bioavailability. Furthermore, the amino groups (–NH2) on the chitosan molecular chain undergo protonation in the acidic environment of the stomach, generating positively charged -NH3. + This process disrupts intermolecular hydrogen bonds, causing chitosan to rapidly dissolve from a solid state into viscous solution microspheres. These microspheres are prematurely released in the acidic environment of the stomach (pH 1.0–3.0), preventing the drug from effectively entering the intestinal fluid. Furthermore, the drug delivery mechanism of chitosan-ferric oxide coated microspheres is primarily electrostatic adsorption or hydrogen bonding, which is difficult to achieve in the high-pH environment of the stomach. + Under high concentration and high ionic strength conditions, H +It will compete with the drug for the binding sites of chitosan, directly disrupting the drug-carrier interaction and triggering a burst release effect. Even if the microsphere structure does not completely disintegrate, the drug will be released in large quantities prematurely.

[0005] Therefore, the development of microspheres formed by encapsulating chitosan-ferric oxide with alginate as drug delivery carriers to target the intestines, avoid the dissolving effect of gastric acid, and improve the therapeutic effect on colonic diseases (such as colon cancer or colitis) is a technical problem that urgently needs to be solved. Summary of the Invention

[0006] The main objective of this invention is to provide a drug-loaded microalgae motor system for colon-targeted delivery, its preparation method, and its application, in order to overcome the shortcomings of the prior art.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] As the first objective of the invention, this invention provides a drug-loaded microalgae motor system for colon-targeted delivery, characterized in that it comprises a nanocomposite consisting of a microalgae motor core and magnetic nanoparticles; the nanocomposite is encapsulated in sodium alginate microspheres to obtain the drug-loaded microalgae motor system; the magnetic nanoparticles are uniformly distributed on the surface of the microalgae motor core through electrostatic adsorption; the magnetic nanoparticles include aminated iron tetroxide nanoparticles coated with quercetin-modified chitosan quaternary ammonium salt QCS-QT to form a magnetic nanocomposite Fe3O4@QCS-QT.

[0009] The synergistic effect of quercetin QT modified on the surface of QCS-QT and the endogenous hydrogen production of Chlamydomonas reinhardtii Cr together constructs a highly efficient reactive oxygen species neutralization system.

[0010] As one of the preferred embodiments, the core of the microalgae motor is Chlamydomonas reinhardtii GY-D55.

[0011] Preferably, the algal suspension of Chlamydomonas reinhardtii GY-D55 and the magnetic nanoparticles undergo physical adsorption and electrostatic self-assembly at the liquid-solid interface under vortex oscillation to form a magnetically responsive microalgal biohybrid Cr@NPS.

[0012] As one of the preferred embodiments, the magnetic nanocomposite Fe3O4@QCS-QT is attached to the surface of Chlamydomonas reinhardtii cells and biofilm structures.

[0013] As one of the preferred embodiments, the particle size of the drug-loaded microalgae motor system is 40-60 micrometers.

[0014] As a second objective of the invention, this invention provides a method for preparing a drug-loaded microalgae motor system, the specific steps of which include:

[0015] S1. Culture Chlamydomonas reinhardtii strain GY-D55 and pretreat it to obtain Chlamydomonas reinhardtii strain GY-D55 algal suspension;

[0016] S2. Preparation of Fe3O4@QCS-QT

[0017] Quercetin ethanol solution was added dropwise to chitosan quaternary ammonium salt aqueous solution and stirred to allow quercetin and chitosan quaternary ammonium salt to fully complex through intermolecular forces. After purification by dialysis, the QT-QCS complex solid was obtained.

[0018] The QT-QCS composite solid was redissolved in water and mixed with aminated Fe3O4 nanoparticles. The mixture was then ultrasonically treated to achieve coating through physical adsorption and electrostatic interaction, ultimately obtaining a magnetic nanocomposite Fe3O4@QCS-QT with a core-shell structure.

[0019] S3, Preparation of Cr@NPS

[0020] The algal suspension obtained in S1 is mixed with Fe3O4@QCS-QT provided in S2, and the mixture is vortexed to promote physical adsorption and electrostatic self-assembly of the two at the liquid-solid interface, thus obtaining the microalgal biohybrid Cr@NPS.

[0021] S4. Preparation of drug-loaded microalgae motor system

[0022] Cr@NPS was dispersed in a mixed aqueous solution of alginate and Ca-EDTA to form an aqueous solution. The aqueous solution was then mixed with an oil solution at a flow rate ratio of 1:3 to form monodisperse droplets. The monodisperse droplets were collected in a 1% acetic acid oil solution, allowed to stand, and then shaken to allow the alginate to crosslink and solidify in an acidic environment. Finally, after post-treatment, sodium alginate microspheres with uniform morphology were obtained.

[0023] As one of the preferred embodiments, in S1, the culture conditions of Chlamydomonas reinhardtii GY-D55 are an ambient temperature of 25±1℃, a light intensity of 4500 lux, a light-dark cycle of 12:12 h, a constant speed oscillation of 140 rpm, and a total culture period of 7 days.

[0024] Preferably, the concentration of Chlamydomonas reinhardtii GY-D55 in the algal suspension is 7~8×10⁻⁶. 6 Cells / mL; OD750 value is 0.8.

[0025] In one preferred embodiment, the concentration of the quercetin ethanol solution in S2 is 5~20 mg / mL;

[0026] Preferably, the concentration of the chitosan quaternary ammonium salt aqueous solution is 1~3% (w / w).

[0027] Preferably, the complexation reaction includes a reaction mixture obtained by continuous stirring at 1000 rpm for 8 to 24 hours.

[0028] Preferably, the dialysis purification includes: passing the reaction mixture through a 3500 Da dialysis bag and dialyzing it with ultrapure water at 0-4°C for 24-72 hours to remove unreacted quercetin molecules and small molecule byproducts; then freeze-drying to obtain the QT-QCS complex solid.

[0029] Preferably, the volume ratio of the algal suspension to the aminated Fe3O4 nanoparticles is 2:1, wherein the concentration of the aminated Fe3O4 nanoparticles is 1 mg / mL.

[0030] The concentration of the QT-QCS complex was 2 mg / mL; in the reaction system, the QT-QCS complex was in excess, thereby ensuring that QCS-QT could be fully coated on the surface of the aminated Fe3O4 nanoparticles.

[0031] As one of the preferred embodiments, in S3, the volume ratio of the algal suspension to the magnetic nanocomposite Fe3O4@QCS-QT is (2~4):1.

[0032] As one of the preferred embodiments, in S4, the flow rate of the aqueous phase solution is 2 μL / min; the flow rate of the oil phase solution is 6 μL / min, forming the monodisperse microdroplets.

[0033] Preferably, the oil phase solution is a mixture of mineral oil and Span 80.

[0034] More preferably, the volume ratio of mineral oil to Span 80 is 95:5.

[0035] As a third objective of the invention, the present invention also provides a colon-targeting drug comprising at least the drug-loaded microalgae motor system as described above.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] 1. This invention utilizes the endogenous hydrogen production characteristic of Chlamydomonas reinhardtii, whose generated hydrogen gas can serve as an antioxidant for adjuvant therapy. Simultaneously, in the colonic environment, sodium alginate microspheres can degrade under specific conditions, releasing microalgal motors. These microalgal motors can move autonomously in intestinal fluid and, guided by an external magnetic field, precisely target intestinal lesions.

[0038] 2. The drug-loaded microalgae motor system provided by this invention has good biocompatibility, biodegradability, gastric acid avoidance ability, and magnetic / chemical dual-drive targeting function. It can effectively improve the treatment effect of colon diseases (such as colon cancer or colitis) and significantly reduce the systemic side effects of drugs, thus expanding the application of micro-nano motors in the biomedical field.

[0039] 3. The direct free radical scavenging ability of quercetin (QT) in the drug-loaded microalgae motor system provided by this invention, combined with the synergistic effect of endogenous hydrogen production from Chlamydomonas reinhardtii (Cr), jointly constructs a highly efficient reactive oxygen species (ROS) neutralization system. Effective inhibition of ROS demonstrates the application potential of this magnetic microalgae motor in treating oxidative stress-related diseases, such as inflammatory bowel disease. Magnetic targeting can further enrich this antioxidant effect at the lesion site, enhancing the therapeutic effect.

[0040] 4. This invention demonstrates that the drug-loaded microalgae motor system provided by this invention can effectively circumvent the upper digestive tract and achieve colon-targeted drug release by placing the microspheres in simulated gastric juice (SGF) and simulated intestinal juice (SIF). In the first few hours, the microspheres release slowly with a high drug retention rate; while in simulated colonic juice (SCF), they exhibit a significant burst release effect. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 This is a SEM image of the magnetic nanocomposite prepared in Example 1 of this invention.

[0043] Figure 2 This is a SEM image of the magnetic nanocomposite prepared in Comparative Example 1 of this invention.

[0044] Figure 3 This is a SEM image of the magnetic nanocomposite prepared in Comparative Example 2 of this invention.

[0045] Figure 4 This is a potential diagram of Cr@NPS prepared in Example 1 of the present invention.

[0046] Figure 5 This is a TEM image of Cr@NPS provided in Embodiment 1 of the present invention.

[0047] Figure 6 This is the EDS energy spectrum analysis diagram of Cr@NPS provided in Embodiment 1 of the present invention.

[0048] Figure 7 This is the QCS-QT spectrum of Cr@NPS provided in Embodiment 1 of the present invention.

[0049] Figure 8 This is a graph showing the absorbance variation of Cr@NPS in hydrogen production testing according to Example 1 of the present invention.

[0050] Figure 9 This is a comparison chart of cytotoxicity tests of Fe3O4 nanoparticles, QCS-QT complex, and Fe3O4@QCS-QT nanocomposite provided in Example 1 of this invention.

[0051] Figure 10 These are ROS laser confocal images of the Fe3O4@QCS-QT nanocomposite provided in Example 1 and the Cr@NPS microalgae motor provided in Example 2 of this invention.

[0052] Figure 11 This is a comparison chart of the DPPH scavenging capabilities of the Fe3O4@QCS-QT nanocomposite provided in Example 1 and the Cr@NPS microalgae motor provided in Example 2.

[0053] Figure 12 This is a comparison chart of the ABTS removal capabilities of the Fe3O4@QCS-QT nanocomposite provided in Example 1 and the Cr@NPS microalgae motor provided in Example 2.

[0054] Figure 13 This is a microscopic image of the Cr@NPS microalgae motor microspheres prepared in Example 2 of this invention.

[0055] Figure 14 This is the controlled-release curve of the Cr@NPS microalgae motor microspheres prepared in Example 2 of the present invention in simulated intestinal fluid, simulated gastric fluid, and simulated colonic fluid.

[0056] Figure 15 This is a comparison diagram showing the aggregation of the Cr@NPS microalgae motor prepared in Example 2 of the present invention in simulated intestinal fluid in a peristaltic pump circulation system with and without a magnetic field applied.

[0057] Figure 16 This is a comparison of the absorbance of the Cr@NPS microalgae motor prepared in Example 2 of the present invention at a wavelength of 750 nm in simulated intestinal fluid under conditions of applying a magnetic field and not applying a magnetic field. Detailed Implementation

[0058] Detailed embodiments of the invention are disclosed herein; however, it should be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore, the specific functional details disclosed herein should not be construed as limiting, but rather as the basis for the claims and as intended to teach those skilled in the art to employ the representative basis of the invention in different ways in any suitable detailed embodiment.

[0059] This invention provides a drug-loaded microalgae motor system for colon-targeted delivery, its preparation method, and its application. The drug-loaded microalgae motor system includes a Chlamydomonas reinhardtii (Cr) microalgae motor core, the surface of which is electrostatically adsorbed onto aminated iron tetroxide nanoparticles (Fe3O4) and encapsulated with quercetin-modified chitosan quaternary ammonium salt (QCS-QT) to form a magnetically responsive nanocomposite (Cr@NPS). The Cr@NPS nanocomposite is further encapsulated in sodium alginate microspheres.

[0060] This invention utilizes the endogenous hydrogen production characteristic of Chlamydomonas reinhardtii, whose generated hydrogen gas can serve as an antioxidant for adjuvant therapy. Simultaneously, in the colonic environment, sodium alginate microspheres degrade under specific conditions, releasing microalgal motors. These microalgal motors can move autonomously in intestinal fluid and, guided by an external magnetic field, precisely target intestinal lesions. This invention combines excellent biocompatibility, biodegradability, gastric acid avoidance, and dual magnetic / chemical targeting capabilities, effectively improving the treatment efficacy of colonic diseases (such as colon cancer or colitis), significantly reducing systemic drug side effects, and expanding the application of micro / nanomotors in the biomedical field.

[0061] The technical solution and its effects of the present invention will be described in detail below through specific embodiments.

[0062] Example 1

[0063] 1. Algae cultivation

[0064] The Chlamydomonas reinhardtii strain GY-D55 was derived from Shanghai Guangyu Biotechnology Co., Ltd. (China) and propagated using TAP medium. The culture conditions were: ambient temperature 25±1℃, light intensity 4500 lux, 12:12 h light / dark cycle, constant oscillation at 140 rpm, and a total culture period of 7 days.

[0065] 2. Preparation of Fe3O4@QCS-QT

[0066] A 10 mg / mL quercetin ethanol solution was added dropwise to an equal volume of 2.0% (w / w) chitosan quaternary ammonium salt aqueous solution under light-protected, room-temperature conditions. The mixture was then stirred continuously at 1000 rpm for 12 hours to allow quercetin and chitosan quaternary ammonium salt to fully complex via intermolecular forces. After the reaction was complete, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed against ultrapure water at 4°C for 48 hours to completely remove unreacted quercetin molecules and small molecule byproducts. The solution was then freeze-dried to purify and obtain the lyophilized product, the QT-QCS complex solid. In use, the lyophilized product is reconstituted in water at a concentration of 2 mg / mL and mixed with aminated Fe3O4 nanoparticles (1 mg / mL) at a volume ratio of 2:1 (QT-QCS complex in excess). The mixture is then sonicated (15-30 min) to allow it to be coated through physical adsorption and electrostatic interaction, ultimately obtaining a magnetic nanocomposite with a core-shell structure.

[0067] See Figure 1 The image shows a TEM image of the magnetic nanocomposite prepared in this embodiment. As can be seen from the image, the magnetic nanocomposite particles are uniformly distributed on the surface of the microalgae.

[0068] 3. Preparation of Cr@NPS

[0069] First, *Chlamydomonas reinhardtii* was pretreated by centrifugation (2000 rpm, 5 min, 4°C) and resuspending in PBS to provide a clean and physiologically compatible reaction interface. Subsequently, the algal suspension (OD750 value 0.8, 7.9 × 10⁻⁶) was... 6 The Fe3O4@QCS-QT nanocomposite (cells / mL) was mixed with Fe3O4@QCS-QT nanocomposite at a volume ratio of 2:1 and then vortexed for 5 min to promote physical adsorption and electrostatic self-assembly of the two at the liquid-solid interface, ultimately forming a magnetically responsive microalgal biohybrid.

[0070] 4. Potentiometric measurement and transmission electron microscopy

[0071] Diluted sample suspensions were prepared and their electrokinetic properties were evaluated by dynamic light scattering analysis on the Malvern Zetasizer Nano-ZSE platform.

[0072] The morphology, size, and elemental composition of Cr@NPS were analyzed using a Thermo Fisher Scientific Talos F200X G2 field emission transmission electron microscope. The diluted sample was dropped onto an ultrathin carbon film copper mesh, dried, and observed under bright-field imaging at 200 kV. Elemental point analysis and surface distribution scanning were performed using the integrated energy dispersive spectroscopy system.

[0073] See Figure 4The figure shows the dot plot of Cr@NPS. As can be seen from the figure, the Fe3O4 particles wrapped by QCS-QT are positively charged, while the Cr in the microalgae itself is negatively charged. The two can combine through electrostatic interaction.

[0074] See Figure 5 The TEM image of Cr@NPS provided in this embodiment is shown in the figure. The figure clearly shows that the iron oxide nanoparticles were successfully attached to the cell surface and biofilm structure of Chlamydomonas reinhardtii, confirming the core structure of the "algae-nanocomposite" constructed through electrostatic adsorption.

[0075] See Figure 6 The following are EDS energy dispersive spectroscopy (EDS) spectra of Cr@NPS provided in this embodiment. Figure a shows the HAADF plot of Cr@NPS, figure b shows the C element distribution, figure c shows the N element distribution, figure d shows the O element distribution, figure e shows the Fe element distribution, figure f shows the P element distribution, and figure g shows the distribution of all elements. The EDS energy dispersive spectroscopy detected characteristic signal peaks of Fe and O elements, and these peaks co-located with the C and N element signals in the algal organic matrix in terms of spatial distribution, providing direct elemental evidence for the magnetic nanocomposite encapsulated by chitosan quaternary ammonium salt.

[0076] These results validate the successful loading of nanofunctional components onto microalgae carriers from both morphological and elemental perspectives, laying a crucial structural foundation for their subsequent targeted movement and controllable delivery under magnetic field drive.

[0077] 5. Fourier transform infrared spectroscopy

[0078] The Fourier transform infrared (FT-IR) spectra of each sample were measured using a Fourier transform infrared spectrometer, with a spectral range of 500–4000 cm⁻¹. -1 Potassium bromide particles were used, and the scan was performed four times with a resolution of 4 cm. -1 .

[0079] See Figure 7 The image shows the QCS-QT spectrum of Cr@NPS provided in this embodiment. The spectrum exhibits absorption peaks of characteristic QT functional groups (such as phenolic hydroxyl OH and carbonyl C=O), and these peaks show shifts or intensity changes relative to the characteristic peaks of QCS (such as quaternary ammonium groups), confirming that quercetin has been successfully grafted onto the chitosan quaternary ammonium salt molecular chain via chemical bonding. In the spectrum of the Fe3O4@QCS-QT complex, not only are the main characteristic absorption peaks of QCS-QT retained, but also significant absorption peaks are observed in the low wavenumber region (approximately 580 cm⁻¹). -1The absorption peaks of the Fe-O bond stretching vibrations, characteristic of Fe3O4 particles, appeared nearby, providing crucial chemical bonding evidence for the successful QCS-QT coating of Fe3O4 nanoparticles. The final Cr@NPS spectrum clearly integrated the characteristic absorption peaks of Chlamydomonas reinhardtii's own polysaccharides, proteins, and other biomolecules with the characteristic peaks of the Fe3O4@QCS-QT nanocomposite, indicating that the nanoparticles were successfully loaded onto the microalgae through surface interactions, forming a complete functionalized magnetic delivery complex.

[0080] 6. Hydrogen determination

[0081] First, prepare a methylene blue-platinum probe solution with a concentration of 1 mg / mL. The methylene blue-platinum probe solution consists of 150 mg of methylene blue (Maclean, CAS No.: 28983-56-4) dissolved in 42 mL of ethanol solution to obtain a methylene blue-ethanol solution. Then, add 8 mL of colloidal Pt (Xianfeng Nano, XFJ116 7440-06-4) with a concentration of 1 mg / mL to the methylene blue-ethanol solution to obtain the methylene blue-platinum probe solution.

[0082] Subsequently, the methylene blue-platinum probe solution was injected into different suspensions. After the solutions underwent hydrogen production treatment for 24 and 48 hours, the absorbance changes were monitored using a UV-Vis spectrophotometer 15 minutes after addition. In the presence of a platinum catalyst, blue methylene blue was reduced to a colorless state by hydrogen gas, thus achieving rapid detection.

[0083] See Figure 8 The graph shows the absorbance variation of Cr@NPS in the hydrogen production test provided in this embodiment. Figure 3 It can be seen that the characteristic peak of the MB probe at 664 nm in both the Cr-only group and the Cr@NPS group decreased significantly, and the MB color gradually faded. This indicates that the addition of magnetic composite nanoparticles does not affect the hydrogen release of Cr, and the amount of hydrogen released increases with time.

[0084] 7. Biocompatibility and antioxidant properties

[0085] RAW264.7 cells were planted at a density of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 96-well plates and cultured for 24 hours. Different concentrations of the test drug were then added. After a specified culture time, 10 μL of CCK-8 solution was added to each well, and the plates were incubated at 37°C for 0.5 hours. Finally, the absorbance of each well was measured at 450 nm using a microplate reader, and the relative cell viability was calculated.

[0086] See Figure 9The figure shows a comparison of cytotoxicity tests of Fe3O4 nanoparticles, the QCS-QT complex, and the Fe3O4@QCS-QT nanocomposite. As can be seen, neither the Fe3O4 nanoparticles nor the QCS-QT complex exhibited significant cytotoxicity within the tested concentration range, and cell viability remained at a high level. This lays a safe material foundation for the subsequent construction of the composite. Compared with the single components, the constructed Fe3O4@QCS-QT nanocomposite and Cr@NPS microalgal motor maintained the highest cell viability at all tested concentrations, indicating that the successful composite encapsulation strategy effectively reduced the potential biological risks of each component and demonstrated excellent biocompatibility. These results demonstrate that the entire system, from the initial materials to the final functionalized microalgal motor, possesses good cellular safety, providing in vitro safety evidence for its further application in the biomedical field, particularly as a targeted delivery system.

[0087] See Figure 10 The figures show confocal laser images of ROS in the Fe3O4@QCS-QT nanocomposite and Cr@NPS microalgal motor. Image a shows the fluorescence intensity of the control group; image b shows the fluorescence intensity of PBS; image c shows the fluorescence intensity of QCS-QT; image d shows the fluorescence intensity of Fe3O4; image e shows the fluorescence intensity of Fe3O4@QCS-QT; image f shows the fluorescence intensity of Cr; and image g shows the fluorescence intensity of Cr@NPS. Compared with other groups, the ROS fluorescence intensity in cells or tissues treated with Cr@NPS was significantly reduced, indicating that the nanocomposite can effectively scavenge reactive oxygen species and possesses significant antioxidant stress resistance. This antioxidant effect stems from the synergistic effect of the direct free radical scavenging ability of quercetin (QT) in the composite material and the endogenous hydrogen production of Chlamydomonas reinhardtii (Cr), jointly constructing a highly efficient reactive oxygen species neutralization system. The effective inhibition of ROS demonstrates the potential of this magnetic microalgal motor in treating oxidative stress-related diseases such as inflammatory bowel disease. Its magnetic targeting can further enrich this antioxidant effect at the lesion site, enhancing the therapeutic effect.

[0088] Comparative Example 1

[0089] The difference between this comparative example and Example 1 is that in step 2, the volume ratio of the QT-QCS composite solid to the aminated Fe3O4 nanoparticles is different. In this comparative example, the volume ratio of the microalgae Cr suspension to the Fe3O4@QCS-QT nanocomposite solution is 1:1.

[0090] See Figure 2 The image shows a SEM image of the magnetic nanocomposite prepared in this comparative example. As can be seen from the image, the magnetic nanoparticles are aggregated and unevenly dispersed on the surface of the microalgae.

[0091] Comparative Example 2

[0092] The difference between this comparative example and Example 1 is that in step 2, the volume ratio of the QT-QCS composite solid to the aminated Fe3O4 nanoparticles is different. In this comparative example, the volume ratio of the microalgae Cr suspension to the Fe3O4@QCS-QT nanocomposite solution is 3:1.

[0093] Figure 3 Comparative Example 2 shows the SEM image of the magnetic nanocomposite prepared in this comparative example. The magnetic nanoparticles account for a small proportion, and there is little adsorption on the surface of the microalgae, with only a portion adsorbed on the flagella.

[0094] Further comparison Figures 1-3 When the volume ratio of the microalgal suspension of Cr to the Fe3O4@QCS-QT nanocomposite solution is 2:1, the magnetic nanoparticles are uniformly distributed on the surface of the microalgae; however, when the volume ratios are 1:1 and 3:1, the magnetic nanoparticles cannot be uniformly dispersed on the surface of the microalgae, or the adsorption on the surface of the microalgae is low.

[0095] Example 2

[0096] This embodiment provides a method for preparing microspheres, the specific steps of which include:

[0097] A 50 μm narrow-necked PDMS cross-shaped droplet chip was used with a pressure pump. An aqueous solution containing 1% sodium alginate and 120 mM Ca-EDTA (the Cr@NPS prepared in Example 1 was centrifuged and then dispersed in this aqueous solution) was flowed at a rate of 2 μL / min, while the flow rate of the droplet-generating oil (95% (volume percentage) mineral oil and 5% (volume percentage) Span 80) was controlled at 6 μL / min. Monodisperse droplets were generated under these conditions. The monodisperse droplets were collected into centrifuge tubes containing a 1% acetic acid oil phase solution. The sodium alginate was cross-linked and solidified in an acidic environment by standing and gentle agitation. The 1% acetic acid oil phase solution was prepared by adding 5 μL of glacial acetic acid to 500 μL of the droplet-generating oil and agitating until homogeneous.

[0098] After treatment with a demulsifier and repeated centrifugation and washing to remove the oil phase, uniform sodium alginate microspheres were finally obtained and dispersed in PBS buffer for subsequent applications.

[0099] See Figure 13 The figure shows a microscopic image of the Cr@NPS microalgae motor microspheres prepared in this embodiment. As can be seen from the figure, Cr@NPS is coated in sodium alginate, and the size of the microspheres is about 50 micrometers.

[0100] Furthermore, this embodiment also monitors the generation of reactive oxygen species (ROS) using the DCFH-DA probe. When DCFH-DA enters the cell, its acetyl groups dissociate and transform into non-fluorescent DCFH. The newly formed DCFH is rapidly oxidized by intracellular ROS into DCF with strong fluorescence intensity, and the fluorescence intensity is directly proportional to the ROS content. The specific operation is as follows: RAW264.7 cells are inoculated at 5 × 10⁻⁶ cells per cell. 4 Seeds were inoculated at a density of 1 probe / well in the lower chamber of a 24-well transfilter. Different suspensions were added to the upper chamber of the transfilter and incubated in the dark for 24 hours. The control group was cultured in basal DMEM medium, while the other groups were exposed to 100 µM LPS in the dark for 12 hours. The samples were then washed twice with PBS, and 10 µM DCFH-DA dye was added. The samples were incubated at 37°C in the dark for 30 minutes. After incubation, the samples were washed three times with serum-free medium to remove excess DCFH-DA probes. Imaging was performed using a confocal laser scanning microscope (CLSM).

[0101] Mix 2 mL of 0.1 mM DPPH ethanol solution with 0.5 mL of sample solution. After homogenizing the mixture, react it at room temperature in the dark for 30 min, and then measure the absorbance at 517 nm using a UV-Vis spectrophotometer.

[0102]

[0103] Where Ac is the absorbance of the DPPH and ethanol mixture, and As is the absorbance of the sample and the DPPH mixture.

[0104] ABTS radical stock solution: Transfer 10 mL of 7 mM ABTS to 0.176 mL of 40 mM potassium persulfate and react at room temperature in the dark for 14–16 h. Before measurement, dilute the ABTS radical stock solution by mixing 0.5 mL of the sample solution with 3 mL of the diluted ABTS. Homogenize the mixture and react at room temperature in the dark for 30 min. Then, measure the absorbance at 734 nm using a UV-Vis spectrophotometer, using the same formula as DPPH.

[0105] See Figure 11 and Figure 12 Fe3O4 particles coated with QCS-QT exhibit good DPPH and ABTS scavenging capabilities. Meanwhile, pure microalgal Cr also possesses some antioxidant capacity, but it was found that the antioxidant capacity is significantly enhanced after microalgal Cr is combined with composite iron tetroxide particles.

[0106] Example 3

[0107] To simulate the intestinal fluid environment, a peristaltic pump circulation system equipped with silicone tubing was constructed. (See [reference needed]). Figure 15The inner diameter of the tubing is 2 mm, and the flow rate of intestinal fluid is 8 mL / min.

[0108] A Cr@NPs suspension with an OD750 value of 0.8 was injected into the peristaltic pump circulation system, and a permanent magnet was installed in the pipeline to generate a local magnetic field.

[0109] The magnetic control capability was qualitatively evaluated by comparing the retention of particles in the region before and after the application of a magnetic field. Subsequently, the retained particles under the action of the magnetic field were collected, and their absorbance at a wavelength of 750 nm was measured for resuspension and quantification. The OD750 value was used as an indicator to accurately quantify the active targeting and retention efficiency of the system.

[0110] See Figure 14 The figures show the controlled-release curves of the Cr@NPS microalgae motor in simulated intestinal fluid (artificial small intestinal fluid, source leaf, R22156-100mL), simulated gastric fluid (artificial gastric fluid, source leaf, R22155-500mL), and simulated colonic fluid (artificial colonic fluid, source leaf, R22152-500mL); the temperature was simulated human body temperature of 37℃. As can be seen from the figures, the sodium alginate microspheres in simulated intestinal fluid, with a size of approximately 50 micrometers, effectively prevent premature absorption or destruction in the stomach and small intestine, while ensuring efficient drug release in the colonic environment due to physical retention and specific degradation. Their size is highly matched to the colon-targeted design. The release curves clearly show that in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), the microspheres release slowly in the first few hours, with a high drug retention rate; while in simulated colonic fluid (SCF), they exhibit a significant burst release effect. This three-stage release behavior strongly validates that the system can effectively bypass the upper digestive tract and achieve colon-targeted drug release. The results ultimately demonstrate that a smart delivery system capable of responding to the colonic environment and releasing the internal "Chlamydomonas reinhardtii-nanocomplex" on demand has been successfully constructed through sodium alginate microsphere encapsulation, providing a key controlled-release basis for targeted treatment of colonic diseases (colon cancer, colitis, etc.).

[0111] Figure 15 This is a comparison of the aggregation of the Cr@NPS microalgae motor prepared in this embodiment in simulated intestinal fluid in a peristaltic pump circulation system with a silicone tube under the conditions of applying a magnetic field (b) and not applying a magnetic field (a). Figure 16 This is a comparison of the absorbance of the Cr@NPS microalgal motor prepared in this embodiment at 750 nm wavelength in simulated intestinal fluid under conditions of applied and unexposed magnetic fields; Figure 15 and Figure 16As can be seen, in this embodiment, the aggregation of magnetic complexes can be clearly observed after 15 minutes of guidance with an external magnetic field (magnetic field strength 2000~2500 Gauss). Compared with the group without external magnetic field guidance (Nomagnet), the OD750 value of the group with magnetic field guidance (With magnet) is significantly increased. This directly proves that the constructed magnetic microalgal motor can achieve efficient and specific targeted retention in simulated intestinal fluid under the guidance of an external magnetic field.

[0112] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims.

Claims

1. A drug-loaded microalgae motor system for colon-targeted delivery, characterized in that, The system comprises a nanocomposite consisting of a microalgal motor core and magnetic nanoparticles, wherein the nanocomposite is a magnetically responsive microalgal biohybrid; the drug-loaded microalgal motor system is obtained by encapsulating the nanocomposite in sodium alginate microspheres; wherein the sodium alginate microspheres release slowly in simulated gastric and intestinal fluids, but exhibit a significant burst release effect in simulated colonic fluid; The magnetic nanoparticles are uniformly distributed on the surface of the microalgae motor core through electrostatic adsorption. The magnetic nanoparticles are a magnetic nanocomposite Fe3O4@QCS-QT formed by encapsulating aminated iron tetroxide nanoparticles with chitosan quaternary ammonium salt QCS-QT modified by quercetin. The preparation method includes: firstly, adding quercetin ethanol solution to chitosan quaternary ammonium salt aqueous solution and stirring to react, so that quercetin and chitosan quaternary ammonium salt undergo a complexation reaction through intermolecular forces, followed by dialysis purification to obtain QT-QCS composite solid; then, redissolving the QT-QCS composite solid in water, adding aminated Fe3O4 nanoparticles and mixing, and ultrasonically treating it to complete the encapsulation through physical adsorption and electrostatic interaction; The core of the microalgae motor is Chlamydomonas reinhardtii, which is capable of endogenously producing hydrogen. The hydrogen produced works synergistically with quercetin to remove reactive oxygen species. The particle size of the drug-loaded microalgae motor system is 40-60 micrometers; The preparation method of the sodium alginate microsphere system includes: dispersing the nanocomposite in a mixed aqueous solution containing alginate and Ca-EDTA to form an aqueous solution, forming monodisperse microdroplets using droplet microfluidics, and collecting the monodisperse microdroplets in an acetic acid oil solution to allow the alginate to crosslink and solidify in an acidic environment.

2. The drug-loaded microalgae motor system according to claim 1, characterized in that, The core of the microalgae motor is Chlamydomonas reinhardtii GY-D55; Under vortex oscillation, the algal suspension of Chlamydomonas reinhardtii GY-D55 and the magnetic nanoparticles undergo physical adsorption and electrostatic self-assembly at the liquid-solid interface to form a magnetically responsive microalgal biohybrid Cr@NPS.

3. The drug-loaded microalgae motor system according to claim 2, characterized in that, The magnetic nanocomposite Fe3O4@QCS-QT is attached to the surface of Chlamydomonas reinhardtii cells and biomembrane structures.

4. A method for preparing a drug-loaded microalgae motor system for colon-targeted delivery as described in any one of claims 1-3, characterized in that, The specific steps include: S1. Culture Chlamydomonas reinhardtii strain GY-D55 and pretreat it to obtain Chlamydomonas reinhardtii strain GY-D55 algal suspension; S2. Preparation of Fe3O4@QCS-QT; Quercetin ethanol solution was added dropwise to chitosan quaternary ammonium salt aqueous solution and stirred to allow quercetin and chitosan quaternary ammonium salt to fully complex through intermolecular forces. After purification by dialysis, the QT-QCS complex solid was obtained. The QT-QCS composite solid was redissolved in water and mixed with aminated Fe3O4 nanoparticles. The mixture was then ultrasonically treated to achieve coating through physical adsorption and electrostatic interaction, resulting in a magnetic nanocomposite Fe3O4@QCS-QT solution with a core-shell structure. S3. Preparation of Cr@NPS; The algal suspension obtained in S1 is mixed with the Fe3O4@QCS-QT solution provided in S2, and the two are physically adsorbed and electrostatically self-assembled at the liquid-solid interface by vortex oscillation to obtain the microalgal biohybrid Cr@NPS. S4. Preparation of a drug-loaded microalgae motor system; Cr@NPS was dispersed in a mixed aqueous solution of alginate and Ca-EDTA to form an aqueous solution, and monodisperse microdroplets were formed by mixing the aqueous solution with the oil solution at a flow rate ratio of 1:

3. The monodisperse microdroplets were collected in a 1% acetic acid oil solution, and after standing, the solution was shaken to allow the alginate to crosslink and solidify in an acidic environment. Finally, after post-treatment, sodium alginate microspheres with uniform morphology were obtained.

5. The preparation method according to claim 4, characterized in that, In S1, the culture conditions for Chlamydomonas reinhardtii GY-D55 were: ambient temperature of 25±1℃, light intensity of 4500 lux, light-dark cycle of 12:12 h, constant speed oscillation of 140 rpm, and a total culture period of 7 days. The concentration of Chlamydomonas reinhardtii GY-D55 in the algal suspension was 7~9×10⁻⁶. 6 Cells / mL.

6. The preparation method according to claim 4, characterized in that, In S2, the concentration of quercetin ethanol solution is 5~20 mg / mL; The concentration of the chitosan quaternary ammonium salt aqueous solution is 1~3% w / w; The complexation reaction involves continuous stirring at 1000 rpm for 8 to 24 hours to obtain a reaction mixture; The dialysis purification process includes: passing the reaction mixture through a 3500 Da dialysis bag and dialyzing it with ultrapure water at 0-4°C for 24-72 hours to remove unreacted quercetin molecules and small molecule byproducts; then freeze-drying to obtain the QT-QCS complex solid. The concentration of the QCS-QT complex was 2 mg / mL; The concentration of aminated Fe3O4 nanoparticles was 1 mg / mL.

7. The preparation method according to claim 4, characterized in that, In S3, the volume ratio of the algal suspension to the Fe3O4@QCS-QT solution is (2~4):1; In S4, the flow rate of the aqueous phase solution is 2 μL / min, and the flow rate of the oil phase solution is 6 μL / min, forming the monodisperse microdroplets; And / or, the oil phase solution is a mixture of mineral oil and Span 80; wherein the volume ratio of mineral oil to Span 80 is 95:

5.

8. The application of a drug-loaded microalgae motor system as described in any one of claims 1-3 or a drug-loaded microalgae motor system prepared by the preparation method as described in any one of claims 4-7 in the preparation of targeted colon drugs.

9. A colon-targeting drug comprising the drug-loaded microalgae motor system as described in any one of claims 1-3 or the drug-loaded microalgae motor system prepared by the preparation method as described in any one of claims 4-7.

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