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Alginate lyase immobilized Chlamydomonas algae microrobots: minimally invasive therapy for biofilm penetration and eradication
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Xiaoting Zhanga, Huaan Lib, *, Lu Liua, Yanzhen Songa, Lishan Zhanga, Jiajun Miaoa, Jiamiao Jianga, Hao Tiana, Chang Liuc, *, Fei Pengd, *, Yingfeng Tua, *
Acta Pharmaceutica Sinica B | 2025, 15(6) : 3259 - 3272
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Acta Pharmaceutica Sinica B | 2025, 15(6): 3259-3272
• ORIGINAL ARTICLE •
Alginate lyase immobilized Chlamydomonas algae microrobots: minimally invasive therapy for biofilm penetration and eradication
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Xiaoting Zhanga, Huaan Lib, *, Lu Liua, Yanzhen Songa, Lishan Zhanga, Jiajun Miaoa, Jiamiao Jianga, Hao Tiana, Chang Liuc, *, Fei Pengd, *, Yingfeng Tua, *
Affiliations
  • aNMPA Key Laboratory for Research and Evaluation of Drug Metabolism & Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
  • bGuangdong Provincial Key Laboratory for Research and Evaluation of Pharmaceutical Preparations & Guangdong Provincial Engineering Center of Topical Precise Drug Delivery System, Center for Drug Research and Development, Guangdong Pharmaceutical University, Guangzhou 510006, China
  • cSport Science College, Beijing Sport University, Beijing 100091, China
  • dSchool of Materials Science and Engineering, Sun Yat-Sen University, Guangzhou 510275, China
doi: 10.1016/j.apsb.2025.03.034
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Bacterial biofilms can make traditional antibiotics impenetrable and even promote the development of antibiotic-resistant strains. Therefore, non-antibiotic strategies to effectively penetrate and eradicate the formed biofilms are urgently needed. Here, we demonstrate the development of self-propelled biohybrid microrobots that can enhance the degradation and penetration effects for Pseudomonas aeruginosa biofilms in minimally invasive strategy. The biohybrid microrobots (CR@Alg) are constructed by surface modification of Chlamydomonas reinhardtii (CR) microalgae with alginate lyase (Alg) via biological orthogonal reaction. By degrading the biofilm components, the number of CR@Alg microrobots with fast-moving capability penetrating the biofilm increases by around 2.4-fold compared to that of microalgae. Massive reactive oxygen species are subsequently generated under laser irradiation due to the presence of chlorophyll, inherent photosensitizers of microalgae, thus triggering photodynamic therapy (PDT) to combat bacteria. Our algae-based microrobots with superior biocompatibility eliminate biofilm-infections efficiently and tend to suppress the inflammatory response in vivo, showing huge promise for the active treatment of biofilm-associated infections.

Microrobots  /  Biological orthogonal reaction  /  Biofilms  /  Chlamydomonas reinhardtii  /  Alginate lyase  /  Microalgae  /  Antibacterial therapy  /  Photodynamic therapy
Xiaoting Zhang, Huaan Li, Lu Liu, Yanzhen Song, Lishan Zhang, Jiajun Miao, Jiamiao Jiang, Hao Tian, Chang Liu, Fei Peng, Yingfeng Tu. Alginate lyase immobilized Chlamydomonas algae microrobots: minimally invasive therapy for biofilm penetration and eradication[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (6) : 3259 -3272 . DOI: 10.1016/j.apsb.2025.03.034
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1.　Introduction
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With the widespread use of various medical devices (central venous catheters, urinary catheters, etc.), the safety issue of invasive treatment becomes increasingly important. The surfaces of medical devices that have been the foci of device-related infections show the presence of large numbers of slime-encased bacteria1,2. Biofilm-associated microorganisms generally cause a great deal of infections3. Over 600 million people annually suffer from catheter-related bloodstream infections (CRBSI), catheter-associated urinary tract infections (CAUTI), and ventilator-associated pneumonia (VAP)4,5. Also, non-device-related chronic biofilm diseases were commonly seen in patients with cystic fibrosis6, chronic wound infections7, and chronic prostatitis8.
Biofilms are three-dimensional multicellular communities with extracellular matrix (ECM) including exopolysaccharide, extracellular DNA (eDNA), RNA, lipids, and extracellular membrane vesicles. These components enable bacteria to adhere irreversibly to indwell medical instruments and protect microorganisms against environmental assaults6,9,10. As a hyper secreted exopolysaccharide of ECM, alginate contributes to the formation of structured and heterogeneous biofilms6. The functions of alginate include facilitating cell adhesion, aggregation, cell-to-cell, and cell-to-biofilm connections, supporting biofilm development by providing strength and rigidity through calcium chelation11,12. Therefore, biofilms on medical devices allow bacteria to become resistant to antibiotic treatment, block the flow of urine or bloodstream through the catheter, and prevent eradication from innate immune cells, resulting in persistent infection, infectious complications, and devastating device malfunction (intravascular catheter dysfunction, biliary tube obstruction, crystalline encrustations on urinary stents, etc.) in clinical practice, which required surgical debridement and device removal8.
A recent study has demonstrated that alginate lyase can efficiently degrade the secreted alginate of Pseudomonas aeruginosa (P. aeruginosa) by breaking the glycosidic bond through a β-elimination reaction, resulting in the generation of oligomers with 4-deoxy-L-erythro-hex-4-enepyranosyluronate acid at the nonreducing end6. Therefore, alginate lyase has the potential to disrupt the biofilm architecture, enhance antibiotic distribution, and promote existing therapy effects by hydrolyzing the negatively charged alginate11,13. Furthermore, the degradation products of alginate, reported as alginate oligosaccharides (AOs), have been found they show various physiological activities, including immunomodulatory, antihypertensive, and anticoagulant properties14. However, biofilm-associated infections need a therapeutic strategy with penetrating properties, and alginate lyase can only damage the surface of biofilms and subsequently show a potential anti-bacterial effect. Wu et al.15 designed a pH-sensitive system, which exhibited better biofilm penetration and eradication via electrostatic interactions. Additionally, Mayorga-Martinez et al.16 used aqua sperm micromotors with rapid velocity and snake-like undulatory locomotion for biofilm damage, highlighting the importance of a drug's capability to infiltrate the biofilms for effective eradication.
Compared with traditional passive particle systems, micro/nanomotors, especially for biohybrid motors/robots received strong attention in various fields, due to their ability for precise navigation and tissue permeability17,18. Based on living systems (including bacteria, microalgae, sperms, etc.), biohybrid microrobots have been widely applied in cargo transportation19, wireless actuation20, anti-bacterial therapy21, tumor-targeting22 and various other aspects in biomedicine. Notably, biohybrid micromotors based on Chlamydomonas reinhardtii (C. reinhardtii) microalgae have a promising prospect in biomedicine, since showing much more adaptability to the complicated microenvironment23, minimal invasiveness24, and good biocompatibility25. Due to the existing eukaryotic flagella (≈12 μm long), as linear motors containing an inner core of microtubules and associated motor proteins (the axoneme), C. reinhardtii demonstrate efficient locomotion for versatility in diverse aqueous environments without any toxic fuel26. Also, the cell wall of C. reinhardtii, provides facile surface modification, and the amino-terminal residue and structure (such as 4-hydroxyproline (4-HP)-rich glycopeptides)27-29 can be exploited to immobilize cargoes such as polypeptides26, liposomes30, and other materials via combinations of covalent or noncovalent interactions26. Furthermore, the abundant chlorophyll in C. reinhardtii, as a natural photosensitizer, can generate reactive oxygen species (ROS) under specific laser irradiation31.
Because of quorum sensing32, a cell-to-cell communication system used by bacteria to collaborate, reproduce, and protect from biofilms, bacteria can eventually evolve to exhibit antibiotic resistance to new drugs33. Nevertheless, among the non-antibiotic treatments, photodynamic therapy (PDT) is minimally invasive, site-specific, and can be used to eradicate various bacteria, even those resistant to antibiotics34. PDT utilizes photosensitizers and lasers to trigger ROS, such as singlet oxygen (1O2) and superoxide anion (O2−). ROS disrupts the eDNA, protein, and membrane assembly directly, leading to bacteria suffering oxidative stress and death35. Studies showed that the infrared light (wavelength range: 495–630 nm) of PDT can penetrate 3–6 mm depth underneath the skin, covering the epidermis (100 μm thick) and dermis (1–4 mm thick) to fight bacterial infections36. Therefore, it is necessary to preserve the viability of C. reinhardtii for multiple applications30. Thus, the construction of biohybrid micromotors using bioactive substances directly without the usage of synthetic materials as cargo becomes increasingly focused, such as polypeptides and enzymes19. Hence, to preserve the respective activities of both alginate lyase and C. reinhardtii as much as possible, the bioorthogonal method without the requirement of copper catalysis is used for the construction of microrobots, in accordance with the idea of click chemistry, which is a technique for efficiently joining molecules together quickly and without unwanted by-products37. Streptavidin-biotin cross-linking, a type of bioorthogonal method, is commonly used in tissue engineering38 and has become one of the strongest known noncovalent interactions in nature, such as reinforcing the attachment for cargo delivery, tracking, and signal enhancement19,39,40.
Herein, we developed bioinspired microrobots consisting of enzyme-immobilized microalgae to facilitate deep biofilm penetration and treat bacterial infections. As illustrated in Scheme 1, the biohybrid microrobot was based on C. reinhardtii modified with active alginate lyase via the biotin-streptavidin-biotin self-assembly system (denoted as ‘CR@Alg microrobots’). Our self-propelled microrobots exhibit efficient biofilm penetration due to the alginate degradation with alginate lyase creating surface shortfalls on biofilms so that the depth of biofilms was much easier to be penetrated by the microrobots. Since ROS, generated by CR@Alg microrobots, acts as strong oxidants to damage micro-organisms directly, CR@Alg microrobots showed stable killing efficiency against P. aeruginosa in both of in vitro and in vivo models. The inherent properties of environmentally friendly microalgae are ingeniously combined with active alginate lyase to produce a self-propelled microrobot capable of biofilm penetration, providing a minimally invasive drug-free therapy to eradicate biofilms on implanted medical instruments in vivo.
2.　Materials and methods
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2.1.　 Fabrication of CR@Alg microrobots
C. reinhardtii were obtained from the Institute of Hydrobiology, Chinese Academy of Science (FACHB-355, Wuhan, China), and cultivated in Tris-acetate-phosphate (TAP) medium (Phyto Technology, T8224-10L, Lenexa, USA), at 23 ℃ under the cycles of 12 h sunlight and 12 h dark. C. reinhardtii were centrifuged at 500×g for 3 min (DLAB D3024R, Beijing, China), washed with DI water to remove any residual TAP medium, and subsequently resuspended in DI water for optical density (OD) for microalgae at the wavelength of 660 nm. Then, 1 mL of 6.0 × 107C. reinhardtii were incubated with Sulfo–NHS–Biotin (1 mg/mL, Thermo Fisher Scientific, A39256, Shanghai, China) for 1 h at 25 ℃. The resulting biotin-modified-C. reinhardtii (biotin-CR) were centrifuged (700×g, 3 min), washed with DI water and then redispersed in 500 μL of DI water, followed by dropwise addition of streptavidin (0.5 mg in DI water, APExBIO Technology LLC., B7921, Houston, USA) and subsequent reaction for 1 h at 25 ℃ to allow streptavidin conjugated to biotin-CR. After centrifugation at 700×g for 3 min and washing with DI water, streptavidin-biotin-CR were prepared. In parallel, 1 mg/mL of alginate lyase (Sigma–Aldrich, A1603, Shanghai, China) was incubated with Sulfo–NHS–Biotin (1 mg/mL), and the resulting solution was stirred for 1 h at 25 ℃. The obtained biotin-modified-alginate lyase (biotin-Alg) were concentrated by filtration (10 kDa molecular weight cut-off, Amicon Ultra 0.5 mL Filters, Merck Millipore, UFC5010, Massachusetts, USA) at 6000×g for 15 min and washed three times with DI water to remove excess NHS ester. CR@Alg microrobots were then synthesized by mixing streptavidin-biotin-CR with biotin-Alg for 1 h incubation via bioorthogonal chemistry. Scanning electron microscope (SEM) images were recorded on a Phenom emission scanning electron microscope (Nikon, Phenom ProX, Tokyo, Japan). Alginate lyase conjugating results were analyzed by flow cytometer (Becton Dickinson, LSR Fortessa X-20, New Jersey, USA).
2.2.　 Motion analysis of CR@Alg microrobots
The motion of CR@Alg microrobots and C. reinhardtii were analyzed in Phosphate buffers (PBS), TAP, DI water (H2O) and Tryptic Soy Polmyxin Brot Base (TSB) medium (Merck Millipore, 22092, Massachusetts, USA) in Petri dishes. The inverted fluorescence microscope (Nikon, Ti2-A, Tokyo, Japan) was used to record the motion of microrobots. Time-lapse images were recorded (time interval between each frame ΔT = 200 ms). The velocity of CR@Alg microrobots was evaluated at 0 and 24 h. Tracking image sequences were analyzed with the Image J plugin manual tracking.
2.3.　 Alginate lyase hydrolytic activity assay
Enzyme activity was performed as the method reported in the previous study41. Briefly, 400 μL of sodium alginate (Sigma–Aldrich, 180947, Shanghai, China) solution (10 mg/mL) in PBS (pH 6.5) was incubated with 100 μL alginate lyase (20 μg/mL), CR@Alg (5.0 × 106 algae/mL), C. reinhardtii (5.0 × 106 algae/mL) and PBS for 10 min at 37 ℃. Finally, 40 μL of 10 mol/L NaOH was added to stop the reaction and absorbance at 235 nm was measured on a spectrophotometer (SHIMADZU, UV-2600, Tokyo, Japan).
2.4.　 ROS detection
The ROS generation capability of CR@Alg microrobots upon laser irradiation was then investigated using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime Biotechnology, S0033S, Shanghai, China). It was reported that in the presence of ROS, the transferred 2′,7′-dichlorofluorescein (DCFH) by DCFH-DA would be rapidly oxidized and form fluorescent molecule (dichlorofluorescein, DCF). DCFH solution (5 μmol/L) and C. reinhardtii (5.0 × 106 algae/mL) were placed in a 1.5 mL EP tube, exposed to 638 nm laser irradiation (0.6 W/cm2) for varied time intervals (0, 10, 20, 30, 40, 50, 60, 70, 80, 90 min). The ROS generation as determined by DCF (excitation, Ex: 488 nm; emission, Em: 525 nm), was recorded as fluorescence spectrum using fluorescence spectroscopy (SHIMADZU, RF-6000, Tokyo, Japan). DCFH in the group of C. reinhardtii (5.0 × 106 algae/mL) was lasered by 638 nm laser irradiation for 30 min, for varied power density (0, 0.1, 0.3, 0.6, 0.9, 1.2 W/cm2), record the fluorescence spectrum using fluorescence spectroscopy. DCFH in the groups of C. reinhardtii, CR@Alg (5.0 × 106 algae/mL), PBS medium, and alginate lyase were lasered by 638 nm laser irradiation (0.6 W/cm2, 30 min) record the fluorescence spectrum using fluorescence spectroscopy (SHIMADZU).
2.5.　 DCFH-DA staining fluorescent images of P. aeruginosa
Pseudomonas aeruginosa (P. aeruginosa, Guangdong Microbial Culture Collection Center, ATCC 27853, Guangzhou, China) was first streaked onto a Luria broth (LB, Sigma–Aldrich, L3022, Shanghai, China) agar plate and cultured overnight at 37 ℃. A single colony was inoculated in 100 mL LB medium in a conical bottle and further cultured for 10 h at 37 ℃ on a shaker 200 rpm.
P. aeruginosa (1.0 × 106 CFU/mL) suspensions were placed in 96-well plates, and treated with TSB medium, 638 nm laser irradiation (0.6 W/cm2, 30 min), C. reinhardtii (dark), C. reinhardtii + Laser, CR@Alg (dark), CR@Alg + Laser, C. reinhardtii and CR@Alg (1.0 × 107 algae/mL). At 2.5 h after treatment, the bacterial cells were collected and stained with DCFH-DA at 37 ℃ for 80 min, followed by DAPI staining (Sigma–Aldrich, MBD0020, Shanghai, China). Then, bacterial cells were washed with PBS and the images were captured by a confocal laser microscope (Olympus, FV3000, Tokyo, Japan). “L” represents “under 0.6 W/cm2 power density laser irradiation for 30 min”.
2.6.　 3D fluorescent images of biofilm penetration
P. aeruginosa was washed with PBS, and then resuspended in TSB medium to a final concentration of 5.0 × 108 CFU/mL, followed by being placed in 96-well plates at 37 ℃ for 48 h to allow the formation of biofilms. The biofilms were treated with C. reinhardtii (dark), C. reinhardtii + Laser, CR@Alg microrobots (dark), CR@Alg microrobots + Laser, C. reinhardtii and CR@Alg (5.0 × 106 algae/mL), laser groups were under 0.6 W/cm2 power density laser irradiation for 30 min, respectively. At 1.5 h after treatment respectively, the amount of CR@Alg microrobots that remained in the biofilms was observed under a fluorescence confocal microscope (Nikon), to evaluate their penetrative effect. The 3D fluorescent images of C. reinhardtii in the red channel and biofilms with DAPI staining in the blue channel.
2.7.　 3D fluorescent images biofilm eradication
P. aeruginosa (200 μL, 1.0 × 108 CFU/mL per well) was incubated in TSB medium and placed in 96-well plates at 37 ℃ for 48 h to allow the formation of biofilms. The biofilms were treated with TSB medium, 638 nm laser irradiation (0.6 W/cm2, 30 min), C. reinhardtii (dark), C. reinhardtii + Laser, CR@Alg (dark), CR@Alg + Laser, C. reinhardtii and CR@Alg (5.0 × 106 algae/mL), respectively. At 3 h after treatment, the biofilms were washed with PBS gently, and then the bacteria were stained with Hoechst 33342 (Thermo Fisher Scientific, H1399, Shanghai, China). Finally, the destructive conditions within the biofilms and their 3D fluorescent images were taken on an inverted fluorescence microscope (Nikon).
2.8.　 Crystal violet staining measurements
P. aeruginosa biofilms formed in 96-well plates were exposed to C. reinhardtii or CR@Alg (density of 0, 5.0 × 105, 1.0 × 106, 5.0 × 106, 1.0 × 107 algae/mL), then treated with 638 nm laser irradiation (0.6 W/cm2, 30 min) or in the dark after 2 h of incubation. Followed by washing with PBS to remove the disintegrated biofilms after treating with C. reinhardtii and CR@Alg for 24 h, the biofilms were fixed with absolute methanol (150 μL) for 15 min. After discarding the absolute methanol and thoroughly drying, crystal violet (Beyotime Biotechnology, C0121, Shanghai, China) solutions (100 μL, w/w 1% in PBS) were added to each well for 10 min to stain the biofilms. After staining, the biofilms were washed with PBS to remove the excess crystal violet and thoroughly dried. The stained biofilms were dissolved by adding acetic acid (200 μL, v/v, 33% in PBS) and measured at 570 nm on a microplate reader (BioTek, 800 TS, Vermont, USA).
2.9.　 Animal experiment of the implanted catheter infective model
BALB/c mice (6 weeks, female) were supplied by Laboratory Animal Center of Southern Medical University. All experimental protocols were carried out under the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Southern Medical University (permit number: SMUL202312009).
To investigate the in vivo therapeutic effects of CR@Alg microrobots, subcutaneous murine P. aeruginosa-infected implanted models in BALB/c mice were used. To form catheters with adhering biofilms, commercial vacuum catheters (B. Braun, Venofix® A, Melsungen, Germany) were cut into 10 mm segments, sterilized with 75% ethanol, and then 1 mL of TSB medium containing 1.0 × 108 CFU/mL of P. aeruginosa were incubated with each 10 mm-catheter at 37 ℃ for 48 h. The catheters washed by PBS were implanted in the inner thigh of mice. After 24 h, all the mice were randomly divided into 6 groups (n = 4), treated with blank PBS without irradiation, only 638 nm laser irradiation (0.6 W/cm2, 30 min), C. reinhardtii (dark), C. reinhardtii + Laser, CR@Alg (dark), CR@Alg + Laser groups. 50 μL C. reinhardtii or CR@Alg (1.0 × 107 algae/mL) was injected to the infectious sites, including the interior and the surrounding of the implanted catheters so that the infected areas were fully infiltrated. After 1 h, when the wound heals, caused by the puncture of the syringe used to administer the drug, the infectious sites were treated with 638 nm laser irradiation (0.6 W/cm2, 30 min). The treatment was proceeded 3 times on Days 1, 3, and 5 after implantation surgery. On Days 2, 4, and 7, the mice were euthanatized, in parallel the implanted catheters and tissues around the infectious sites were obtained.
2.10.　 Bacteria counting by agar plate dilution method
To evaluate the eradication of biofilm-infection, bacteria of the recollected catheters from the infectious sites were counted by the typical agar plate dilution method. In detail, the catheters were completely immersed in 4 mL physiological saline in centrifuge tubes and sonicated for 30 min under a water bath to make the bacteria fully into suspension. Then the suspension was diluted and spread onto LB agar plates. After incubation for 20 h at 37 ℃, the bacterial plaques on the plate were counted.
2.11.　 Quantitative analysis of the level of inflammatory cytokines
The anti-inflammatory effect of CR@Alg was assessed by ELISA assays. Specifically, 1 mm3 infectious tissues from mice were separated and weighted. After being immersed in PBS (1:9), and thoroughly mashed under an ice bath, the tissues were centrifugated (900×g, 20 min, 4 ℃). Then the supernatants were collected to quantify the concentration of proinflammatory cytokines IL-1β, IL-6, and TNF-α by using corresponding ELISA kits. IL-1β ELISA kit was purchased from Elabscience (E-EL-M0037c, Wuhan, China) ELISA kits for IL-6 and TNF-α were supplied by Dakewe Biotechnology (1210602, 1217202, Shenzhen, China). Experiments were performed three times.
2.12.　 Statistical analysis
Data analysis was carried out via Student's t-test (two-tailed), and Image J (1.52a). Data was expressed as means ± standard deviation (SD) from several separate experiments. The asterisk was considered as statistical significance: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.
3.　Results and discussion
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3.1.　 Fabrication and characterization of CR@Alg microrobots
As the main component of microrobots, C. reinhardtii were first cultured in TAP medium and exhibited uniform ovoid morphology with 8.7 ± 0.29 μm average diameter (Supporting Information Fig. S1). As a typical biological orthogonal reaction, biotin-streptavidin-biotin system was utilized to introduce alginate lyase onto the surface of C. reinhardtii (Fig. 1A). In detail, Sulfo–NHS–biotin was first conjugated to alginate lyase and C. reinhardtii respectively, via the N-hydroxysuccinimide (NHS) ester reaction. As a mediator, each streptavidin tetramer has four independent biotin-binding sites. One biotin of biotin-modified C. reinhardtii (CR-biotin) was bound to one active binding site of streptavidin via strong noncovalent interaction. After biotin-modified alginate lyase (Alg-biotin) conjugating to other active biotin-binding sites of streptavidin-biotin-CR, CR@Alg microrobots were then successfully constructed. Compared to the bare C. reinhardtii with a smooth surface, the immobilized alginate lyase could be clearly observed on the surface of CR@Alg microrobots with roughness as shown in SEM images (Fig. 1B). Fluorescent Cy3-NHS ester was then used to label the anchored alginate lyase on C. reinhardtii. As illustrated in Fig. 1C, Cy3-labeled-alginate lyase emitted a strong fluorescent signal in yellow channel and was also colocalized with those of the autofluorescence of chlorophyll from C. reinhardtii in a red round shape. Then, to further perform the quantitative analysis, flow cytometry results were showed in Fig. 1D. 98.2% of the C. reinhardtii population emitted fluorescent signals in the Cy3 channel, indicating that a considerable amount of C. reinhardtii was modified with Cy3-labeled-alginate lyase. In Supporting Information Fig. S2, CR@Alg microrobots showed corresponding fluorescent peaks of Cy3-labeled-alginate lyase (Ex:550 nm, Em:570 nm), indicating the successful introduction of alginate lyase onto C. reinhardtii via the bioorthogonal chemistry.
Because of the modification with Sulfo–NHS–biotin via the biorthogonal method, alginate lyase retained the capacity of alginate degradation, normally resulting in dispersing the three-dimensional structure of the bacterial biofilm matrix. Therefore, the enzyme activity of alginate lyase was then evaluated according to the previous report41. As shown in Fig. 1E, around 74.9 ± 1.9 % of alginate lyase enzymatic activity was reserved after bioorthogonal reaction, indicating that the introduced alginate lyase with relatively high enzyme activity is possible to trigger the efficient degradation of alginate.
3.2.　 Motion analysis of CR@Alg microrobots
The unicellular photosynthetic C. reinhardtii normally use two eukaryotic flagella (12 μm long) for the locomotion and biological propulsion42. The flagellum consists of a distinctive microtubule structure43, controlled by each interlocking subunit via torque transmission to deform the shape actively. As puller microswimmers, microalgae presents breaststroke propelling motility, drawing the front site of fluid to the cell surface for the construction of a nutrient flux42. In this study, the motion of CR@Alg microrobots was recorded in PBS. As shown in Fig. 2A, time-lapse images and representative trajectories of C. reinhardtii and CR@Alg microrobots over 1, 3, 6 and 10 s intervals were clearly presented and demonstrated similar trends in their motion. The corresponding mean velocity distribution and values of C. reinhardtii and CR@Alg microrobots were measured to be 45.5 ± 10.6 and 40.8 ± 7.0 μm/s in PBS, respectively (Fig. 2B). Similar results of velocity and trajectories were obtained for CR@Alg microrobots and C. reinhardtii swimming in different media (H2O, PBS, TSB, and TAP media), reflecting that the motion capability of algae-based microrobots was barely affected after alginate lyase functionalization and showing adaptivity under diverse environments (Fig. 2C‒E). Our CR@Alg microrobots maintained a steady speed and achieved distances of 4.69 body lengths per second, providing an approach to attacking biofilms at a wider and deeper range.
Then, we further recorded the long-term motion behaviors of CR@Alg microrobots. Notably, Fig. 2F showed the velocity change in the motion of CR@Alg microrobots and 90% of microrobots preserved activity with steady velocity after 24 h of motility in PBS, indicating the great adaptivity of microalgae without TAP medium for a longer time and becoming viable for in vivo conditions. These results verified the negligible cytotoxicity of alginate lyase functionalization on C. reinhardtii and they still displayed fast movement capability. Also, it has been indicated that the biotin-streptavidin-biotin system, as a typical biological orthogonal reaction, does not compromise the intrinsic mobility of unmodified microalgae44. Furthermore, as shown in Supporting Information Fig. S3A‒S3C, the similar tracking trajectories were recorded and the corresponding mean velocity of CR@Alg microrobots after 30 min 638 nm laser irradiation with 0.6 W/cm2 were measured to be 41.96 ± 3.5 μm/s in PBS, reflecting that the motion capability of CR@Alg microrobots were barely affected by the generated ROS during PDT.
3.3.　 In vitro ROS generation
Since C. reinhardtii contain natural photosensitizers chlorophyll, their excellent ROS-producing capacity can be triggered by laser irradiation of particular wavelengths45. By promoting the ROS level to induce oxidative stress, the eDNA and protein in ECM can be disrupted directly and the bacterial innate redox homeostasis can be disturbed35. To investigate the ROS production property of CR@Alg, the level of ROS was determined by the fluorescence intensity change of a ROS probe, DCFH-DA. After incubating with sodium hydroxide, DCFH-DA was transformed to nonfluorescent DCFH, which can be oxidized by ROS to become a fluorescent DCF molecule rapidly46. C. reinhardtii (5 × 106 algae/mL) were incubated with DCFH under a 638 nm laser irradiation with varied power densities, followed by measuring the fluorescence intensity of DCF at 530 nm that represented the level of ROS. As shown in Fig. 3A, the fluorescence intensity of DCF increased to 1.2 × 105 a.u. within 30 min triggered by C. reinhardtii under the irradiation of 638 nm laser (0.6 W/cm2, Supporting Information Fig. S4A). However, it was found that the ROS production property of C. reinhardtii cannot be strengthened by increasing the power density of 638 nm laser irradiation. Subsequently, the fluorescence spectra showed the progression of ROS generation by C. reinhardtii over time under 638 nm laser irradiation (0.6 W/cm2). As shown in Fig. 3B and Supporting Information Fig. S4B, an obvious enhancement of DCF fluorescence intensity triggered by C. reinhardtii upon the laser irradiation (0.6 W/cm2) between 0 and 30 min was clearly observed, indicating massive production of ROS. Whereas the fluorescence intensity of C. reinhardtii under irradiation between 30 and 90 min remained at a slight increase.
Compared with the fluorescence spectra and the curve of C. reinhardtii, CR@Alg microrobots still performed great talent for ROS production (Fig. 3C and Supporting Information Fig. S4C). This was consistent with the activity of C. reinhardtii before modification of alginate lyase. Besides, the fluorescence intensity of TSB medium and single alginate lyase remained at a low level. To further investigate ROS produced by CR@Alg microrobots in biofilms, we detected the level of ROS by DCFH-DA staining in different groups. As depicted in Fig. 3D, the fluorescent images demonstrated that strong signals with green fluorescence were captured in C. reinhardtii + Laser and CR@Alg + Laser groups. Barely fluorescent signal was observed in CR@Alg group without laser treatment, and almost no significant fluorescent signal for PBS and PBS + Laser group because of C. reinhardtii absence. In addition, Image J was then utilized to quantitatively analyze DCF fluorescence intensity of P. aeruginosa in different groups (Supporting Information Fig. S5), indicating that the ROS level was obviously increased. The grey value of DCF signal in bacteria and DAPI signal colocalization analyzation (Fig. 3E) showed that the DCF fluorescent signal emitted by the ROS were almost at the same position as DAPI signal, proving that the ROS indeed diffused into bacterial cells. The fluorescence intensity of CR@Alg + Laser and C. reinhardtii + Laser groups were both higher than 160 a.u., while the fluorescence intensity of PBS, C. reinhardtii, and CR@Alg groups were all lower than 25 a.u., which might imply the antibacterial effect due to the high photosensitive activity of ROS production of C. reinhardtii upon the laser irradiation. The consistent results of DCF fluorescent signal can also be observed in P. aeruginosa biofilms (Supporting Information Fig. S6A and S6B). Notably, because of adaptive locomotion and long lifespan, the challenges of short diffusion distance and limited lifetime of ROS within tissues can be overcome effectively, for CR@Alg microrobots carrying photosensitiser, chlorophyll, to increase the range of action of ROS under light conditions47.
3.4.　 Enhanced penetration of biofilms
P. aeruginosa can produce highly structured heterogeneous biofilms, and the extra polymeric substance matrix prevents drug penetration, binding and repelling charged antibiotics6. Therefore, it is important to improve the abilities of microrobots to disrupt the structure and infiltrate into the biofilms. Immobilization of alginate lyase allowed CR@Alg microrobots to degrade alginate in biofilms, and thus disperse the dimensional structure of the matrix. Combined with the flexible swimming behavior and the activity of alginate degradation, CR@Alg microrobots can penetrae into the inner biofilms, mending the susceptibility of bacteria inside of the biofilms. To evaluate the biofilm penetration effect of CR@Alg microrobots, P. aeruginosa bacteria was placed in 96-well plates to allow the formation of 48 h-biofilm as a model with the thickness of approximately 60 μm depicted in Supporting Information Fig. S7. Leveraging autofluorescence from the chloroplasts in C. reinhardtii, the result of 3D fluorescent images showed that a certain number of CR@Alg microrobots efficiently penetrated through the biofilms (Fig. 3F). The results of quantitative fluorescence analysis on the stacked longitudinal sections of C. reinhardtii and CR@Alg density in biofilms were shown in Fig. 3G. Compared to C. reinhardtii group, around 1.9-fold CR@Alg microrobots invaded into the biofilms with alginate lyase immobilization. As the presented by laser irradiation, more than 2.3-fold CR@Alg microrobots penetrated into the biofilms. Taken together, these results were consistent with the previous reports that alginate lyase has proved to degrade alginate exopolysaccharide11. Furthermore, PDT has been demonstrated to damage the components of ECM35, and the generated ROS can also promote the penetration of microrobots into biofilms. CR@Alg microrobots upon laser irradiation herein exhibited excellent ability to disperse the architecture of biofilms and enhance bacterial biofilm penetration effect.
3.5.　 In vitro biofilm eradication
Encouraged by the results of penetration, the biofilm eradication ability of CR@Alg microrobots was further investigated. Compared with the passive particles, microalgae have higher mechanical energy to attack biofilms due to their flagellar oscillations, supporting actively swimming motion. Meanwhile, ROS generated from the chlorophyll of C. reinhardtii disrupted biomolecules of bacteria and resulted in bacteria death48,49. Their activity of biofilm eradication was detected by crystal violet and Hoechst staining methods. After different treatments, the mature biofilms were stained by crystal violet, and thus they can be observed intuitively at 570 nm to evaluate the enhanced PDT efficacy of CR@Alg microrobots. As illustrated in Fig. 4A, the biofilm-destructive effect of CR@Alg microrobots were substantially elevated upon 638 nm laser irradiation by comparing the photographs of crystal violet-stained biofilms. Biofilms became loose and porous after treating with CR@Alg and CR@Alg + Laser, and almost no biofilm was observed in wells at density of 5.0 × 106 and 1.0 × 107 algae/mL in CR@Alg + Laser group. In parallel, the stained biofilms of each group and different densities of C. reinhardtii were dissolved and then measured at 570 nm. Comparing the OD 570 nm values of each group, the CR@Alg microrobots exhibited the strongest penetrating and anti-bacterial effects under laser irradiation, generating 10.7- and 1.6-fold higher eradication effect than the control group and C. reinhardtii group at the density of 5.0 × 106 algae/mL (Fig. 4B and C and Supporting Information Fig. S8).
In a microcosmic perspective, 3D images of Hoechst staining DNA of bacterial cells demonstrated the eradication effect of CR@Alg + Laser treatment on biofilms intuitively (Fig. 4D). Notably, compared with the fluorescent images of other groups, the biofilms became the thinnest and the weakest after treating with CR@Alg + Laser. The results were further confirmed by the quantitative analysis of fluorescence intensity by Image J and showed that CR@Alg + Laser group was 1.4-fold lower than that of the control group (Fig. 4E). Taken together, the treatment of CR@Alg with laser irradiation demonstrates that the combination of ROS and alginate lyase dispersed the architecture of biofilms, and caused much more distinct destructive performance on P. aeruginosa bacteria in vitro.
3.6.　 In vivo anti-bacterial activity and anti-inflammatory
Before investigating the in vivo anti-bacterial effect, safety issues have also been considered. C. reinhardtii show good biocompatibility25 and their abundant chlorophyll can generate ROS under specific laser irradiation31 rather than photosensitizer of inorganic–organic hybrid materials with potential toxicity50. Based on the in vitro data, CR@Alg microrobots had been already validated the effect of ECM degradation by immobilized alginate lyase and the excellent locomotion, as an active natural photosensitizer delivering platform to enhance PDT at the depth of biofilms. We further demonstrated the short-term bacterial eradicating and anti-inflammatory effects of CR@Alg microrobots synergistic therapy on an implanted catheter infective mouse model (Fig. 5A). The body weights of BALB/c mice were recorded before implanting catheters with adhering biofilms on Day 0 and after the implantation. As shown in Supporting Information Fig. S9A and S9B, the body weights of the mice were decreased by 10% on Day 1 before treatment, redness and swell occurred at the surgical site of skin, which showed that the model of acute infection was established. Subsequently, the infected mice were randomly divided into 6 groups (n = 4) including PBS, PBS + Laser, C. reinhardtii, C. reinhardtii + Laser, CR@Alg, CR@Alg + Laser groups, and the mice were respectively administrated on Days 1, 3 and 5 (Fig. 5A). Compared with the infected sites on Day 0 (Supporting Information Fig. S10), the infected sites became rubor or swollen in all of groups before treatment on Day 1. As time passed, better healing of infected wounds was observed after treating with CR@Alg + Laser on Day 7, whereas the ulcer occurred in infected sites and became increasingly inflamed in PBS group (Fig. 5B). During the treatment, the body weights of the mice were monitored and the weights recovered to normal level more effectively under the treatment of CR@Alg microrobots, indicating no acute toxicity of CR@Alg microrobots (Supporting Information Fig. S9A and S9B).
Moreover, to clearly identify the effects of bacteria elimination by CR@Alg microrobots, the bacteria were obtained from implanted catheters, and evaluated quantitatively by using the agar plate dilution method. As shown in Fig. 5C, gradually serious pyosis in the implanted catheters of PBS group and PBS + Laser group were observed on Days 4 and 7, indicating that the infection and inflammations were mainly caused by bacteria without CR@Alg + Laser treatment. By contrast, the implanted catheters of CR@Alg + Laser group were obviously cleaner, and the pyosis almost disappeared on Day 7. On the other hand, the agar plate dilution method was employed to measure the colony forming unit (CFU) of implanted catheters and verify the biofilm eradication effect of CR@Alg microrobots quantitatively. As shown in Fig. 5D‒G, the number of live bacteria in the treated biofilms was considerably reduced, and bacteria were almost not detected on the surface of the catheters under the treatment of CR@Alg + Laser on Day 7, whereas the agar plate data of PBS group saw a gradual rise between Days 2 and 7. Besides, although the PBS + Laser group showed decreased bacterial counts on Day 2 and probably depended on the promoted microbicidal activity of phagocytes, triggered by photobiomodulation (PBM)51, this only laser therapy way failed to inhibit bacterial activity and hard to eradicate the bacterial biofilms for the rest of the time of this infective model. Furthermore, biofilms were degraded by CR@Alg, and the degradation products of alginate, reported as alginate oligosaccharides (AOs), have been found that they exhibit immunomodulatory physiological activities14. Also, once the bacteria lose the protection of biofilms, they are faced with the secondary metabolites52 generated by microalgae and phagocytes clearing behavior in vivo environment. Therefore, as shown in Fig. 5C and F, it was a combination approach of CR@Alg in vivo treatment to eliminate bacteria and becomes the basic function for enhancing anti-bacterial laser therapy. But compared with CR@Alg + Laser group, CR@Alg still needs laser irradiation to achieve a better bacterial-eliminating effect for the whole period of treatment. Accordingly, CR@Alg with laser therapy can enhance the anti-bacterial effect remarkably for the whole 7-day period of the bacterial biofilm-infective model, indicating that CR@Alg microrobots enhanced photodynamic effect and performed a biofilm elimination potency in vivo.
Since the lipopolysaccharide (LPS) in the gram-negative bacterium elicits inflammatory cell infiltration and up-regulates inflammatory cytokines level53, the intrinsic immune response of the implanted catheter mouse model was mainly triggered by bacterial infection. Furthermore, to investigate the antibacterial effect and anti-inflammatory effect, the tissues around the infected regions of the mice were harvested and stained with hematoxylin and eosin (H&E). It was found that neutrophils tended to aggregate in the infection sites. As depicted in Fig. 6A, fewer neutrophils were observed in the CR@Alg + Laser group, indicating that the inflammatory reaction was milder. Conversely, severe neutrophilic infiltration was observed in the PBS group and PBS + Laser group, which demonstrated obvious acute inflammation because of CR@Alg + Laser treatment absence after bacterial infection. We also quantitatively assessed the changes in inflammatory cytokines on Day 4 after different groups’ treatment two times as a surrogate of recovery in mice. As displayed in Fig. 6B‒D, the inflammatory cytokines levels of IL-1β, TNF-α, and IL-6 in the CR@Alg (dark) and CR@Alg + Laser groups were greatly reduced, compared to that of those in the PBS group. In contrast to bacterial flagellin54, further in vivo studies verified that CR@Alg microrobots prevent innate immune cells producing proinflammatory cytokines. As previous studies reported that microalgae can use a range of strategies to withstand stress, including phenolic compounds belonging to secondary metabolites52, which possess anti-inflammatory activity55. The secondary metabolites of C. reinhardtii can possibly inhibit inflammation and clean away the inflammatory chemokines. Therefore, this microalgae-based therapy would offer prospective benefits for the treatment of important diseases. These results also indicated that CR@Alg microrobots did not trigger the significant production of proinflammatory cytokines, consistent with the previous report30. Simultaneously, CR@Alg microrobots performed prominent antibacterial and anti-inflammatory effects in vivo, exhibiting great prospects for clinical application.
Moreover, the main organs of the mice in both the experimental and control groups were fixed, using H&E staining to evaluate the potential toxicity (n = 4). The results indicated that the administration of the CR@Alg microrobots did not induce any pathogenic pathological effect on the separated tissues from the BALB/c mice at the tested concentration when compared to the PBS group (Supporting Information Fig. S11). Also, CR@Alg microrobots did not result in significant harm to the mice, indicating the feasibility of their utilization in the treatment of in vivo bacterial infections.
4.　Conclusions
Less
In summary, we demonstrated biohybrid microalgae-based robots with a biofilm eradication ability and biocompatibility by immobilizing alginate lyase via the biological orthogonal method. This method preserved both microalgae and alginate lyase bioactivities, and thus the CR@Alg microrobots can be readily fabricated by a biotin−streptavidin−biotin approach, without compromising the motion behavior of algae and the bioactivity of alginate lyase. After the modification on the surface of C. reinhardtii with alginate lyase, CR@Alg microrobots were allowed to penetrate the bacterial biofilms much easier, due to the enhanced capability to degrade alginate of biofilm matrix. When the self-propelled CR@Alg microrobots swam into the depth of the biofilms, the alginate lyase also reached because of the bioconjugation with C. reinhardtii and continued to degrade the biofilms. Moreover, thanks to the abundant chlorophyll in C. reinhardtii, as a natural photosensitizer, CR@Alg microrobots generated a large amount of ROS under a 638 nm laser irradiation, to achieve synergistic bactericidal as well as anti-inflammatory effects in vivo in the implanted catheter mouse model associated with biofilm formation on urinary and central venous catheters. Therefore, CR@Alg microrobots offered an approach to healthcare for catheter-implanted patients who can just accept minimally invasive surgical or medical treatments. Our work developed minimally invasive CR@Alg microrobots with promising biofilm eradication ability, providing a potential strategy without antibiotics and surgical debridement to eliminate medical device-associated biofilm infections.
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Year 2025 volume 15 Issue 6
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doi: 10.1016/j.apsb.2025.03.034
  • Receive Date：2024-10-10
  • Online Date：2026-04-03
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  • Received：2024-10-10
  • Revised：2024-12-20
  • Accepted：2025-01-28
Affiliations
    aNMPA Key Laboratory for Research and Evaluation of Drug Metabolism & Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
    bGuangdong Provincial Key Laboratory for Research and Evaluation of Pharmaceutical Preparations & Guangdong Provincial Engineering Center of Topical Precise Drug Delivery System, Center for Drug Research and Development, Guangdong Pharmaceutical University, Guangzhou 510006, China
    cSport Science College, Beijing Sport University, Beijing 100091, China
    dSchool of Materials Science and Engineering, Sun Yat-Sen University, Guangzhou 510275, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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