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Bio-inspired biorthogonal compartmental microparticles for tumor chemotherapy and photothermal therapy

Journal of Nanobiotechnology volume 22, Article number: 498 (2024) Cite this article

Abstract

Microcarrier is a promising drug delivery system demonstrating significant value in treating cancers. One of the main goals is to devise microcarriers with ingenious structures and functions to achieve better therapeutic efficacy in tumors. Here, inspired by the nucleus-cytoplasm structure of cells and the material exchange reaction between them, we develop a type of biorthogonal compartmental microparticles (BCMs) from microfluidics that can separately load and sequentially release cyclooctene-modified doxorubicin prodrug (TCO-DOX) and tetrazine-modified indocyanine green (Tz-ICG) for tumor therapy. The Tz-ICG works not only as an activator for TCO-DOX but also as a photothermal agent, allowing for the combination of bioorthogonal chemotherapy and photothermal therapy (PTT). Besides, the modification of DOX with cyclooctene significantly decreases the systemic toxicity of DOX. As a result, the developed BCMs demonstrate efficient in vitro tumor cell eradication and exhibit notable tumor growth inhibition with favorable safety. These findings illustrate that the formulated BCMs establish a platform for bioorthogonal prodrug activation and localized delivery, holding significant potential for cancer therapy and related applications.

Graphical Abstract

Introduction

Cancer is a significant global public health issue that profoundly impacts human lives and well-being [1,2,3]. Chemotherapy is among the main methods of tumor treatment, which kills cells directly through chemotherapeutic drugs [4, 5]. However, systemic administration of the drugs leads to low tumor accumulation, resulting in suboptimal drug utilization [6, 7]. Besides, chemotherapy is commonly linked to significant toxicity and adverse effects and frequently falls short of achieving an optimal therapeutic outcome via standalone treatment [8, 9]. In recent years, phototherapies, including photothermal (PTT) and photodynamic therapy, have garnered significant interest due to their minimal invasiveness, high spatiotemporal selectivity, and controllability [10,11,12]. By combining phototherapies with traditional chemotherapy, various drug delivery carriers such as films, hydrogels, fibers, etc. have been developed [13,14,15,16]. Although with promising therapeutic effects, these carriers still face many problems that limit their further application. Simple mixing of the drug and carrier material tends to result in insufficient control of the drug release behavior [17,18,19]. Besides, therapeutic drugs with intrinsic toxicity released from these carriers inevitably cause damage to normal tissue [20, 21]. Therefore, there is a promising prospect in developing an ingenious microcarrier with smart drug release and novel therapeutic options for the treatment of tumors.

In this paper, inspired by the nucleus-cytoplasm structure of cells and the material exchange reaction between them, we developed a type of biorthogonal compartmental microparticles loading biorthogonal reaction agents with sequential release abilities from microfluidics (Fig. 1). The nucleus and cytoplasm in the cell have different structures and functions and work together to maintain the cell's normal functioning. Inspired by this specific structure, biomimetic drug delivery systems have been developed for disease treatment [22,23,24]. Recently, bioorthogonal chemistry as a highly effective tool in chemical biology, has attracted much attention in various biomedical areas [25,26,27,28,29]. Inverse electron-demand Diels–Alder (IEDDA) reactions are typically bioorthogonal reactions that do not require metal catalysis [30]. By employing tetrazine structure components in Diels–Alder cycloadditions with high-tension cycloalkenes, these reactions demonstrate exceptional potential in various applications such as precise biomolecular labeling, synthesis of antibody–drug conjugates, prodrug activation, etc. [31,32,33] In addition, microfluidic technology enables the preparation of structural tunable microcarriers, which have broad applications in drug delivery and disease treatment [34,35,36,37,38,39,40]. It combines essential operational components, such as sample mixing, reaction, and separation onto a microfluidic chip. In particular, microfluidic electrospray is a precisely controllable technology with a wide range of application prospects [41, 42]. This technology utilizes electrical shear stress to produce droplets that are subsequently ejected into a solidifying solution to fabricate microparticles [43, 44]. Nowadays, microfluidic electrospray is commonly employed to fabricate various microcarriers with desired features [45,46,47]. Therefore, inspired by the cell structure and employing the bioorthogonal reaction and microfluidic electrospray technology, it is conceivable that by constructing compartmental microcarriers, different biorthogonal drugs can be loaded separately, and sequential release can be achieved for efficient tumor therapy.

Fig. 1
figure 1

Cell-inspired biorthogonal compartmental microparticles (BCMs) encapsulated with biorthogonal reaction agents for tumor therapy. a Biomimetic construction of biorthogonal compartmental microparticles (BCMICG/DOX) loaded with TCO-DOX and Tz-ICG via microfluidic electrospray. b The therapeutic mechanism of the BCMICG/DOX. (i) After being injected into the tumor site, BCMs exert potent PTT effects upon 808 nm light irradiation based on the intrinsic photothermal performance of Tz-ICG. (ii) Subsequently, TCO-DOX and Tz-ICG are released gradually, initiating a bioorthogonal reaction that activates chemotherapy. As a result, the BCMICG/DOX could efficiently eliminate the cancer cells to prevent the tumor’s growth through a combination of PTT and bioorthogonal chemotherapy

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Herein, the biorthogonal compartmental microparticles (BCMs) were fabricated for biorthogonal drug loading, activation, and combination cancer therapy. The core and shell of the microparticles (BCMICG/DOX) encapsulated a prodrug cyclooctene-modified doxorubicin (TCO-DOX) and an activator tetrazine-modified indocyanine green (Tz-ICG), respectively. Upon application at the tumor site, the BCMICG/DOX exhibited efficient PTT capability due to the inherent photothermal properties of Tz-ICG. Subsequently, TCO-DOX and Tz-ICG were sequentially released from the core and the shell of the microparticles, triggering a specific inverse electron-demand Diels–Alder (IEDDA) reaction for DOX activation [48, 49]. This activated the efficacy of DOX, enabling effective chemotherapy. Notably, the strategy of prodrug modification could greatly reduce the systemic toxicity of DOX. Given these attributes, the BCMICG/DOX effectively eradicated tumor cells in vitro and significantly inhibited tumor growth through the synergistic combination of bioorthogonal chemotherapy and PTT, demonstrating favorable safety. These results underscored the potential of BCMs as optimal vehicles for drug delivery, providing a versatile and efficient therapeutic platform for diverse biomedical applications.

Results and discussion

In a typical experiment, BCMs were prepared through a two-step microfluidic electrospray process based on a glass capillary microfluidic device (Fig. 2a, d and Figure S1). Firstly, microparticles (CMICG) composed of methacrylate gelatin (GelMA) and Tz-ICG were fabricated as the core of BCMs. Briefly, the mixture of GelMA and Tz-ICG was pumped into a capillary microfluidic device to generate droplets, which were collected into liquid nitrogen and then further crosslinked by ultraviolet (UV) light to obtain the CMICG (Fig. 2a and Figure S1a). Microparticles collected in liquid nitrogen before UV polymerization were free from droplet fusion and cleaning processes, unlike traditional methods, where droplets are collected in an oil phase [50]. Microparticles without drug loading (CMB) were fabricated as a control. The successful preparation of the microparticles was verified by observing under confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). As shown in Fig. 2b and Figure S2–S3, both CMB and CMICG have regular spherical shapes, with a diameter of 77.6 ± 2.8 μm and 74.9 ± 3.5 μm, respectively. Owing to the loading of Tz-ICG, the CMICG displayed strong red fluorescence under the excitation light through CLSM, while it could not be observed in CMB (Fig. 2b). The spherical morphology of the microparticles was observed using SEM, with a notable decrease in average diameter observed following dehydration (Fig. 2c). Subsequently, the above core microparticles were mixed with sodium alginate (Alg) solution and pumped into another capillary microfluidic device to fabricate the BCMs after electrospray and crosslinking with CaCl2 (Fig. 2d and Figure S1b). The compartmental structure of the BCMs with TCO-DOX in the outer core and Tz-ICG in the inner layer was prepared and denoted as BCMICG/DOX. Of note, TCO-DOX was synthesized according to the previous reports. The 1H NMR spectra of DOX, (2E)-TCO-PNB, and TCO-DOX and the mass spectrum of TCO-DOX are shown in Figure S4–S8, indicating the successful synthesis of TCO-DOX. By careful design according to previous work [51], BCMs containing a single core were fabricated. It could be seen that the inner core and the outer layer of BCMICG/DOX displayed a red and green color, respectively, due to the fluorescence of Tz-ICG and TCO-DOX under CLSM (Fig. 2e). As shown in Fig. 2f, g and Figure S9, the BCMs had regular spherical shapes with a diameter of around 260 μm. These results verified the successful fabrication of biorthogonal BCMs.

Fig. 2
figure 2

Characterization of BCMs. a Schematic diagram of the preparation process of CMs. b Optical and fluorescent images of CMB and CMICG. Scale bar: 100 μm. c SEM images of CMB and CMICG. Scale bar: 25 μm. d Schematic diagram of the preparation process of BCMs. e Optical and fluorescent images of BCMICG/DOX. Scale bar: 250 μm. f Size distribution of BCMICG/DOX. g SEM image of BCMICG/DOX. Scale bar: 50 μm

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Indocyanine green (ICG) is a kind of photothermal reagent that is widely used in PTT [52, 53]. As a variant of ICG, it was hypothesized that Tz-ICG might also exhibit photothermal properties. Therefore, a thorough examination of the photothermal effects of Tz-ICG-loaded BCMs was conducted. Figure 3a illustrates the substantial temperature increase of both BCMICG and BCMICG/DOX under 808 nm laser irradiation, with a notable rise observed after 3 min (Δ42.2 °C for BCMICG and Δ41.1 °C for BCMICG/DOX). Conversely, BCMB and BCMDOX (without Tz-ICG) showed minimal temperature variations under the same conditions. Additionally, increasing the light power intensity notably enhanced the photothermal effect of BCMICG/DOX, as depicted in Fig. 3b. Furthermore, BCMICG/DOX exhibited consistent thermal stability after five near-infrared (NIR)-On/Off cycles, as shown in Fig. 3c, indicating the sustained photothermal effect of these microparticles. Thermal images visually depict the photothermal heating process induced by NIR in both BCMICG and BCMICG/DOX (Fig. 3d–f and Figure S10). These findings collectively underscore the robustness of the photothermal performance of Tz-ICG-loaded microparticles, laying the foundation for subsequent PTT investigations.

Fig. 3
figure 3

Photothermal effects and in vitro drug release of BCMs. a Photothermal heating curves of different BCMs under 808 nm laser irradiation (0.6 W cm−2). b Photothermal heating curves of BCMICG/DOX with different laser power densities. c Temperature variation of BCMICG/DOX with five 808 nm laser On/Off cycles (0.6 W cm−2, On for 100 s and Off for 300 s). d–f Infrared thermal images of BCMB, BCMICG, and BCMICG/DOX under irradiation (0.6 W cm−2). g DOX release profile. h Images showing ICG release

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As shown in Figure S11 and Figure S12, Tz-ICG and TCO-DOX have similar absorption and fluorescence spectra with ICG and DOX, respectively. Thus, ICG and DOX was used to load into the BCMs to monitor the drug loading and release processes. Through UV–vis–NIR spectrometer detection and calculating by the standard curves of ICG and DOX (Figure S11), the loading contents of BCMs were 0.15 μg/mg for DOX and 0.20 μg/mg for ICG. Then, the drug-release behaviors of BCMs were investigated. As shown in Fig. 3g, DOX loaded at the outer layer quickly released from BCMs with an 80.9% cumulative release content within 12 h. However, the ICG loaded in the core displayed much slower release behaviors, which could be directly seen from the images (Fig. 3h). Within 48 h, the ICG-loaded microparticle still had strong fluorescence intensity and lasted until 120 h. Such results demonstrated that the BCMs possess the ability to progressively release drugs, which could be beneficial to the gradual activation of drugs and longstanding therapy against cancer cells.

Following this, the in vitro anti-cancer efficacy of BCMICG/DOX was assessed using cell counting kit-8 (CCK8) assay and live/dead staining analysis. Initially, the cytotoxicity of TCO-DOX was evaluated both before and after activation by Tz-ICG. In Fig. 4a, free DOX demonstrated potent cytotoxicity with an IC50 of 0.339 μM, whereas TCO-DOX exhibited reduced cytotoxicity, with an IC50 of 8.146 μM. Upon bioorthogonal activation via IEDDA between TCO-DOX and Tz-ICG (Figure S13), the therapeutic efficacy of TCO-DOX improved, leading to a significant decrease in IC50 to 0.423 μM. Importantly, Tz-ICG showed minimal cytotoxicity (Figure S14), affirming the feasibility and effectiveness of the biorthogonal activation reaction. Next, BCMICG/DOX was added to the upper chamber of a 24-well transwell plate, where melanoma B16F10 cells were cultured in the lower chamber (Fig. 4b). Following a 5-min irradiation period, the BCMICG/DOX was then incubated with the B16F10 cells for 24 h before assessing cell viability using the CCK8 assay. As depicted in Fig. 4c, BCMB and BCMICG had negligible impact on cell proliferation, while BCMDOX exhibited slight inhibition of cancer cells. In contrast, treatment with BCMICG/DOX without irradiation demonstrated significantly stronger cytotoxicity against cancer cells compared to BCMDOX alone. Additionally, BCMICG with photothermal effects (BCMICG ( +)) displayed enhanced cell-killing performance compared to BCMICG without NIR irradiation. Importantly, the BCMICG/DOX ( +) treatment group exhibited the most effective inhibition of B16F10 cells, attributed to the combined effects of biorthogonal drug activation and photothermal therapy. Live/dead staining consistently supported these findings (Fig. 4d), confirming the outstanding in vitro anti-cancer effectiveness of BCMICG/DOX, achieved through the combination of PTT and biorthogonal-activated chemotherapy.

Fig. 4
figure 4

In vitro antitumor efficacy of BCMs. a CCK8 assay of B16F10 cells after incubation with different agents for 24 h. b Diagram of the treatment of BCMs against B16F10 cells. c CCK8 assay of B16F10 cells after incubation with different BCMs with or without irradiation for 24 h. d Live/dead staining of B16F10 cells after incubation with different BCMs with or without irradiation for 24 h (Scale bar: 100 μm). ( +) indicated with irradiation (808 nm, 0.6 W cm−2, 5 min). All the cell experiments had three independent replicates (n = 3). Data are presented as the mean ± SD. n. s.: no significance, *p < 0.05, **p < 0.01, ***p < 0.001

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The in vivo antitumor effect of BCMICG/DOX was further evaluated against a subcutaneous melanoma mouse model. The treatment schedule was schematized in Fig. 5a. Then, the in vivo photothermal ability of the microparticles was investigated and the results were displayed in Fig. 5b and Figure S15. The irradiation tumor sites of the BCMICG and BCMICG/DOX treatment groups showed significant temperature increases in 5 min, which contributed to the PTT. By contrast, in the PBS and BCMDOX treatment groups, the temperature increases at the tumor sites were within the safe range after irradiation. After that, the tumor volume of each group was recorded and the results indicated that the PBS and BCMICG treatment without irradiation cannot suppress the tumor growth due to the unlimited proliferation of melanoma B16F10 cells (Fig. 5c). Compared with the slight inhibition of tumors by the free DOX and BCMDOX treatments, the BCMICG/DOX group displayed much better anti-tumor efficacy because of the gradually released Tz-ICG and TCO-DOX and bioorthogonal reaction between them, thereby activating chemotherapy. Besides, the PTT of BCMICG could also evidently influence tumor growth. Of note, the BCMICG/DOX with NIR light irradiation led to the most tumor inhibition effects (Fig. 5d, e). These results demonstrated that the bioorthogonal chemotherapy and PTT could achieve potent anti-tumor effects for tumor therapy.

Fig. 5
figure 5

In vivo anti-tumor performance of BCMs. a Schematic illustration of the treatment processes. b Infrared thermal images of mice with different treatments and under irradiation for 5 min (0.6 W cm−2). c Tumor growth curves of mice after different treatments. d, e Photograph and tumor weights of B16F10 tumors after different treatments on day 16. Each experiment group has 5 mice (n = 5). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

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In addition, hematoxylin and eosin (H&E) and terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining analyses on tumor slices were examined to reveal the anti-tumor mechanisms. As anticipated, the H&E staining results indicated that the control and BCMICG groups without irradiation didn’t influence the proliferation of the tumor cells, which were densely populated without apparent cellular nuclear crumbles or disappears (Fig. 6a). In contrast, the BCMICG/DOX ( +) treatment resulted in significant damage to tumor cells in comparison with other groups. Besides, the TUNEL staining results also verified that the BCMICG/DOX ( +) treatment induced the highest apoptosis in tumor tissues (Fig. 6b), indicating the effectiveness of the combination of PTT and bioorthogonal chemotherapy. Moreover, the mice were all alive and their body weight increased during the experiments (Figure S16), except that obvious body weight loss could be seen in the DOX treatment group due to its systemic toxicity after intravenous injections. In addition, no significant histological variations were found in the tissue sections of the major organs in different treatments (Fig. 6c). These results confirmed the good biocompatibility of the BCMs.

Fig. 6
figure 6

H&E and TUNEL analyses. a, b H&E and TUNEL staining analyses of tumors after different treatments. c H&E staining analyses of major organs after different treatments. Scale bars: 50 μm

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Conclusion

In summary, we have fabricated bio-inspired biorthogonal compartmental microparticles (BCMICG/DOX) from microfluidics for step-wise loading and releasing of biorthogonal reaction agents for synergistic tumor therapy. TCO-DOX and Tz-ICG were encapsulated in the outer layer and core of the BCMICG/DOX, respectively. Thus, the drugs could be released from BCMICG/DOX in a step-wise mode. When applied at the tumor site, the quickly released TCO-DOX from the out layer of the microparticles could be gradually activated by the Tz-ICG released from the core through an IEDDA bioorthogonal reaction, thereby restoring DOX’s cytotoxicity and enabling potent chemotherapy. Additionally, the Tz-ICG encapsulated in the microcarriers exhibited efficient PTT ability under NIR light irradiation. Consequently, the resulting BCMICG/DOX showcased a substantial cytotoxic effect on tumor cells and effectively suppressed tumor growth through a synergistic combination of PTT and chemotherapy, with minimal systemic toxicity observed. Thus, the developed BCMs offer an effective and versatile delivery system for bioorthogonal drug activation and delivery, holding significant potential for tumor therapy. The limitations associated with the developed system involve the fabrication processes and the further assessment of the therapeutic efficacy. The two-step microfluidic electrospray process may be improved with the adoption of a one-step process for more effective mass production of the microparticles. Besides, additional testing, including large animal experiments, is necessary prior to the clinical application of this system.

Materials and methods

Materials

Calcium chloride (CaCl2), diisopropylethylamine (DIPEA), doxorubicin hydrochloride (DOX·HCl), and sodium alginate (Alg) were purchased from Macklin, while gelatin was obtained from Sigma-Aldrich. Me-tetrazine-ICG (Tz-ICG) and (E)-Cyclooct-2-en-1-yl (4-nitrophenyl) carbonate ((2E)-TCO-PNB) were acquired from Confluore Biotechnology Co. Ltd. The calcein-AM/propidium iodide (PI) staining assay kit was obtained from Meilunbio, Co., Ltd., and the CCK8 assay kit was procured from Beyotime Biotechnology Co. Ltd.

Synthesis of TCO-DOX

The compound (2E)-TCO-PNB (15 mg, 0.05 mmol) was initially dissolved in 2 mL of dimethylformamide. Following this, DIPEA (64.5 mg, 0.50 mmol) and DOX·HCl (35 mg, 0.06 mmol) were added to the solution. The mixture was stirred in darkness at 30 °C for 3 days. Subsequently, 10 mL of water was added, and the resulting mixture was extracted with EtOAc (4 × 50 mL). The combined organic phase was then washed sequentially with saturated NaHCO3 (3 × 50 mL), distilled water (3 × 50 mL), and saturated NaCl solution (3 × 50 mL). It was subsequently dried using anhydrous Na2SO4 for 2 h. Finally, the solvent was removed under reduced pressure, leaving behind a residue that underwent purification via column chromatography (CH2Cl2: MeOH = 98: 2) to yield the TCO-DOX (22 mg, 63.5%). 1H NMR (CDCl3, 400 MHz), δ (ppm): 14.03 (s, 1H), 13.30 (s, 1H), 8.10–8.06 (d, 1H), 7.83 (t, 1H), 7.46–7.40 (d, 1H), 5.90–5.00 (m, 6H), 4.80 (d, 2H), 4.57 (s, 1H), 4.20 (m, 1H), 4.13 (s, 4H), 3.92 (s, 1H), 3.77–3.64 (m, 2H), 3.37–3.28 (d, 1H), 3.15–3.00 (m, 2H), 2.48 (s, 1H), 2.41–2.31 (d, 1H), 2.28–2.16 (m, 1H), 2.11–1.77 (m, 10H).

Preparation of BCMs

The preparation of BCMs was a two-step process by using microfluidic electrospray technology. Firstly, the microparticles in the core were prepared. The pregel solution containing methacrylate gelatin (GelMA, 10 wt%) and Tz-ICG (10 mg mL−1) was pumped into a capillary microfluidic device. The orifice of the cylindrical glass capillary tube was 60 μm. Set the injection pump flow rate to 0.5 mL h−1 and the high voltage supply voltage to 6 kV. Then, the droplets generated under the high-voltage electric field were collected into liquid nitrogen and further crosslinked by UV light to obtain the microparticles. After that, the above microparticles were added to the Alg solution (2 wt%) containing TCO-DOX (0.5 mg mL−1). After the solution was homogenous, it was pumped into a capillary microfluidic device with an orifice of 150 μm. The flow rate and voltage were set as 0.5 mL h−1 and 4 kV. Subsequently, the BCMs were collected and solidified in a CaCl2 solution (2 wt%) through microfluidic electrospray. By changing the drugs loaded in the core or the outer layer, various types of BCMs could be fabricated.

Characterizations

A stereomicroscope (Olympus BX51, Tokyo, Japan) was utilized for optical imaging of the microparticles, and a field emission SEM (SU8010, Hitachi, Japan) was employed to examine the surface morphology and structure of the microparticles. Before imaging with an SEM, the microparticles underwent a sequential dehydration process using ethanol solutions with concentrations of 70%, 80%, 90%, and 100%, followed by supercritical drying.

The photothermal effect of BCMs

The photothermal properties of BCMs were assessed using 808 nm laser irradiation. Various BCMs underwent laser irradiation, and the resulting temperature rise was monitored over time using a thermal imager (FLIR E5-XT). Additionally, the photothermal behavior of BCMs was evaluated by adjusting the laser power intensity (0, 0.3, 0.6, and 1.0 W cm−2). Furthermore, the photothermal stability of BCMs was investigated through five “On–Off” cycles, during which the BCMs were subjected to 100 s of laser irradiation (laser On, 0.6 W cm−2) followed by natural cooling without irradiation for 300 s (laser Off).

Drug release study

Firstly, the absorption and fluorescence spectra of DOX, TCO-DOX, ICG, and Tz-ICG were investigated. The absorption spectra of DOX and TCO-DOX from 300 to 600 nm were measured by a UV–Vis-NIR spectrophotometer. The absorption spectra of ICG and Tz-ICG from 300 to 900 nm were also measured. The fluorescence spectra of DOX and TCO-DOX, ranging from 500 to 700 nm, were obtained using a Cary Eclipse fluorescence spectrophotometer with excitation at a wavelength of 480 nm. The fluorescence spectra of ICG and Tz-ICG from 790 to 860 nm were measured with 780 nm excitation. Then, to facilitate testing and operation, the release profiles of ICG and DOX were examined from BCMICG and BCMDOX, respectively. BCMDOX was placed into a 50 mL tube containing 20 mL of PBS for DOX release assessment. Subsequently, the tube underwent oscillation in a chamber with a velocity of 100 rpm at 37 °C. At predefined intervals, 1 mL of the release medium was withdrawn, and an equal volume of PBS was replenished. The released DOX in the collected solution was quantified using a UV–Vis-NIR spectrophotometer. Similarly, BCMICG was submerged in PBS and subjected to oscillation. At specified time points, BCMICG was observed under a CLSM to directly monitor the release of ICG.

In vitro antitumor study

In each well of the lower compartment of a 24-well transwell plate, 5 × 104 B16F10 cells were seeded and allowed to attach for 12 h. Following the attachment, various BCMs were introduced into the upper compartment of the transwell plate. The BCMICG or BCMICG/DOX treatment groups underwent partial exposure to NIR laser irradiation for 5 min, followed by a 24 h incubation period, while the remaining groups were kept in the dark for the entire 24 h. Subsequently, to assess the in vitro anti-tumor effects of BCMs on cancer cells, live/dead staining and a CCK8 assay were performed. For live/dead staining, the treated cells were incubated with the Calcein-AM/PI agent, and their viability was observed under a fluorescence microscope. Simultaneously, the CCK8 reagent was added to the cells for 2 h, and the absorbance at 450 nm was measured using a microplate reader.

Tumor inhibition in vivo

After inoculating the mouse’s right flank with 1 × 106 B16F10 cells via subcutaneous injection, tumor growth was permitted until the tumor volume approached 100 mm3. Subsequently, to assess the in vivo photothermal efficacy of the microparticles, PBS, BCMDOX, BCMICG, and BCMICG/DOX were intratumorally injected at the tumor sites. Following injection, the tumors were exposed to the laser for 5 min, and the resulting temperature elevations were monitored using an infrared thermal imager. The mice were then randomly divided into 7 groups (n = 5): control, free DOX, BCMDOX (no irradiation), BCMICG (no irradiation), BCMICG/DOX (no irradiation), BCMICG (with irradiation), and BCMICG/DOX (with irradiation). Record the body weight and tumor volume every 2 days. Finally, euthanasia was performed after 16 days, and primary organs and tumors were taken, fixed with 4% (v/v) paraformaldehyde, and cut into 5 μm-thick slices for H&E and TUNEL staining. The power intensity of the 808 nm laser was set at 0.6 W cm−2 for 5 min. The animal studies were permitted by the Ethical Committee of Wenzhou Institute, University of Chinese Academy of Sciences (approval WIUCAS23062105) and strictly according to the Laboratory Animal Care and Use Guidelines.

Statistical analysis

All statistical data are expressed as the mean ± standard deviations. Statistical evaluation was analyzed using unpaired Student’s t-test or one-way ANOVA, and a p-value < 0.05 was considered statistically significant. n. s.: no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Availability of data and materials

No datasets were generated or analysed during the current study.

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Acknowledgements

Not applicable.

Funding

This work was supported by the National Key Research and Development Program of China (2022YFA1105304), National Natural Science Foundation of China (52103196, 32201118, T2225003 and 52073060), Guangdong Basic and Applied Basic Research Foundation (2021B1515120054), Wenzhou Institute UCAS startup fund (WIUCASQD2023010), and Wenzhou Municipal Basic Scientific Research Project (Y20240114).

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Authors and Affiliations

  1. Department of Gastrointestinal Surgery, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, 325035, China

    Qingfei Zhang, Gaizhen Kuang, Yechao Zhou, Yuanjin Zhao & Weijian Sun

  2. Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, 325001, China

    Qingfei Zhang, Gaizhen Kuang, Li Wang & Lu Fan

  3. Department of Rheumatology and Immunology, Nanjing Drum Tower Hospital, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China

    Yuanjin Zhao

  4. Shanghai Xuhui Central Hospital, Zhongshan-Xuhui Hospital, and the Shanghai Key Laboratory of Medical Epigenetics, International Co-Laboratory of Medical Epigenetics and Metabolism (Ministry of Science and Technology), Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China

    Luoran Shang

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  1. Qingfei Zhang
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  2. Gaizhen Kuang
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  3. Li Wang
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  4. Lu Fan
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  5. Yechao Zhou
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  6. Luoran Shang
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  7. Yuanjin Zhao
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  8. Weijian Sun
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Contributions

Weijian Sun, Yuanjin Zhao, and Luoran Shang conceived the conceptualization and designed the experiment. Qingfei Zhang carried out the experiments and analyzed the data. Qingfei Zhang, Luoran Shang, and Yuanjin Zhao wrote the paper. Gaizhen Kuang, Li Wang, Lu Fan, Yechao Zhou, and Luoran Shang contributed to the scientific discussion of the article.

Corresponding authors

Correspondence to Luoran Shang, Yuanjin Zhao or Weijian Sun.

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The animal studies were permitted by the Ethical Committee of Wenzhou Institute, University of Chinese Academy of Sciences (approval WIUCAS23062105) and strictly according to the Laboratory Animal Care and Use Guidelines.

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The authors declare no competing interests.

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Zhang, Q., Kuang, G., Wang, L. et al. Bio-inspired biorthogonal compartmental microparticles for tumor chemotherapy and photothermal therapy. J Nanobiotechnol 22, 498 (2024). https://doi.org/10.1186/s12951-024-02778-w

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Journal of Nanobiotechnology

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