A two-stage nanoengineered microneedle platform for on-demand tumor-microenvironment-responsive therapy and enhanced wound healing against postoperative breast cancer recurrence

2.1 Synthesis and characterization of CS-β-CD NPs

Figure 2A illustrates the synthesis pathway for chitosan (CS) grafted with β-cyclodextrin (CS-β-CD). Specifically, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide/N-hydroxysuccinimide activated the carboxyl group of carboxymethyl-β-cyclodextrin (CMCD), which reacted with the amino group on the CS chain, yielding a CMCD-modified CS. According to the Fourier transform infrared spectroscopy (FTIR) results (Figure 2B), CS exhibited a characteristic peak of the amide I band at 1641 cm⁻¹, attributable to the C=O stretching vibration. On the other hand, the amide II band showed a characteristic peak at 1574 cm⁻¹, reflecting the plane-bending vibration of the N-H bond. At the same time, the vibrational peak of the amino group was observed at 1155 cm⁻¹. Compared to CS, the amide peak position of CS-β-CD showed a slight redshift with a significantly enhanced signal, potentially attributable to the formation of a new amide bond between the amino and carboxyl groups of CS and CMCD, respectively. Furthermore, CS-β-CD showed a carboxymethyl absorption peak near 1416 cm⁻¹, corresponding to the carboxymethyl group derived from CMCD. According to the X-ray diffraction (XRD) results (Figure 2C), CS exhibited a strong crystalline peak at about 20 °, indicating that it mainly exists in the form of type II crystals. Furthermore, the crystalline peaks of CMCD appeared at about 20 ° and 10 °, indicating that it contains both type I and type II crystals, with the latter being slightly more frequent. Conversely, the XRD image of CS-β-CD revealed that the diffraction peaks of type II crystals were significantly broader compared to those of CS. Additionally, the XRD map of CS-β-CD revealed that the diffraction peaks of type II crystals were significantly broader than those of CS, almost covering the entire region of the diffraction peaks of type II crystals in both CS and CMCD. It is also noteworthy that the crystallographic diffraction peaks of CS-β-CD were more shifted compared to those of CMCD, indicating that introducing CMCD not only somewhat maintained the original ordered structure of CS but also altered the order of part of the CS structure, generating a new ordered structure.

Herein, CS-β-CD NPs were successfully prepared through the ionic-crosslinking method. During synthesis, the negatively charged TPP interacted with the positively charged CS chains in CS-β-CD (Figure 2A). To determine the most suitable mixing ratio of CS-β-CD and TPP solutions for constructing CS-β-CD NPs with the most suitable particle size, 5 different ratios of CS-β-CD to TPP solutions (2:1, 3:1, 4:1, 5:1, and 6:1) (Figure 2D) were prepared. Particle sizes at different ratios were then characterized using a nanoparticle size analyzer. The average particle size gradually increased with the amount of TPP added dropwise (Figure 2E). As the volume ratio of CS to TPP decreased, the Zeta potential of CS-β-CD NPs decreased from + 25.71 to + 15.23 mV (Figure 2F). This trend is attributed to the increased TPP concentration, which promotes ion crosslinking between phosphate groups and protonated amino groups, resulting in larger NPs and more hydrogen bonding between molecules, thereby increasing particle size and decreasing zeta potential. Furthermore, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images (Figure 2J, K) revealed that at low magnification, CS-β-CD NPs exhibited aggregated particle morphology with a relatively uniform size distribution. At higher magnification, dispersed CS-β-CD NPs appeared as spherical NPs approximately 450 nm in diameter, consistent with the particle size analysis results.

In order to investigate the stability and pH responsiveness of CS-β-CD NPs, herein, CS-β-CD NPs were dispersed in PBS at pH values of 3.0, 5.5, 7.4, and 9.0 to explore their pH responsiveness. Changes in particle size were then measured using a dynamic light scattering particle size analyzer. According to the results (Figure 2H, I), the NPs maintained their original size at pH values of 7.4 and 9.0, indicating the stability of CS-β-CD NPs under slightly and moderately alkaline conditions. Conversely, the original peaks disappeared at pH values of 5.5 and 3.0, and new peaks with smaller size ranges appeared, suggesting that CS-β-CD NPs disintegrated under acidic conditions before decomposing into larger molecular chains. This outcome could be attributed to TPP protonates losing their negative charge under acidic conditions, leading to the dissolution of the crosslinked structure between TPP and CS and NP breakdown into large molecular chains (Figure 2G) Therefore, CS-β-CD NPs could be widely used in nano-loaded drug delivery systems for acidic TMEs.

The cytotoxicity of blank CS-β-CD NPs was evaluated by the HeLa cell line and the HUVEC cell line, respectively. Supplementary Figure 2, https://links.lww.com/MEDMAT/A8 shows the survival of HeLa cells (A) and HUVEC (B) treated with different concentrations of CS-g-CD NP for 48 hour. In the concentration range of 1–200 μg/mL, the survival rate of HeLa cells treated with CS-g-CD NP was more than 80 %, and the survival rate of HUVEC was more than 90%. The results of cell proliferation/death experiments showed that red cells did not appear after the introduction of CS-β-CD NPs (the surviving cells were green and the dead cells were red) (Figure 2L), indicating that CS-β-CD NPs did not cause cytotoxicity 5 days later, the number of surviving cells became more, which proved that CS-β-CD NPs could promote cell proliferation (Figure 2M).

2.2 Preparation of GelMA and CSMA

By improving the method reported in the literature, methacrylated gelatin (GelMA) and methacryloylated chitosan (CSMA) were successfully synthesized (Figure 3A, B). A supramolecular hydrogel tip matrix, GelMA-CS-β-CD-CSMA, composed of GelMA, CSMA, and CS-β-CD NPs, was then prepared (Figure 3C). Upon adding the dispersed CS-β-CD NP solution into the mixed solution containing aromatic amino acids, the gel incorporated amino acids such as tyrosine, phenylalanine, and tryptophan. These aromatic amino acids subsequently infiltrated the β-cyclodextrin cavity, forming host–guest inclusion complexes. Figure 3E shows the proton nuclear magnetic resonance (¹H NMR) spectra of GelMA and CSMA. In GelMA, peak “a” represents the proton signals of aromatic amino acid residues in gelatin. In contrast, peak “b” corresponds to distinct signals at 5.62 and 5.48 ppm, attributed to the hydrogen peaks of the MA’s C=C double bond^[43,44], indicating successful grafting of MA onto the gelatin molecular chain. Furthermore, the ¹H NMR spectrum of CSMA exhibited peaks at chemical shifts of 1.84, 1.92, 2.9, and 3.2–3.9 ppm, corresponding to the methyl protons of MA residues, methyl protons on CS sugar, acetyl-amino protons on CSMA sugar, and sugar ring protons on CSMA, respectively. It is also noteworthy that the chemical shift at 4.7 ppm corresponds to the proton peak of D₂O, while those at 5.49 and 5.58 ppm correspond to the 2 proton peaks of the MA C=C bond (Figure 3D)^[45]. Each material’s infrared spectra are presented in Figure 3D. In the FTIR spectrum of GelMA, the absorption peaks at 3483 cm⁻¹ corresponded to the N-H and O-H stretching vibrations, whereas the absorption peak at 1645 cm⁻¹ corresponded to the stretching vibration of the C=C bond in the amide I band. Furthermore, the absorption peak at 1550 cm⁻¹ corresponded to N-H bending vibration in the amide II band, implying an interaction between amino groups in gelatin molecules and MA through methyl acrylate ester units^[46,47]. In the FTIR spectrum of CSMA, the telescopic vibrational absorption peak of −NH₂ at 1699 cm⁻¹ disappeared. Furthermore, the absorption peaks at 3319, 1642, and 1445 cm⁻¹ corresponded to the telescopic vibrational peaks of N-H and O-H, telescopic vibrational peaks of the amide I band with the C=C bond, bending vibrational peaks of the amide II band with the N-H bond, and characteristic absorption peak of the amide III band, respectively^[48]. In summary, the disappearance of the −NH₂ absorption peak and formation of a characteristic peak of the amide bond indicate that MA was successfully acylated with the amino group of CS and grafted onto the CS molecular chain. Finally, in FTIR GelMA-CS-β-CD-CSMA spectra, a carboxymethyl absorption peak was observed at 1416 cm⁻¹, which originated from CMCD.

The results of the traditional inverted tube method showed that the GelMA-CS-β-CD-CSMA aqueous solution had photocuring gelation properties, that is, under ultraviolet light irradiation, GelMA-CS-β-CD-CSMA formed a supramolecular hydrogel (Figure 3F). Figure 3G shows the cross-section morphology of GelMA-CS-β-CD-CSMA. It can be seen from the figure that its interior is a loose porous structure, which can provide a convenient transmission channel for nutrients, making it easier for cells to adhere, grow, and migrate inside the hydrogel. Moreover, based on the rheological behavior graph (Figure 3H), the G′ (energy storage modulus) values of the 4 materials were greater than the respective G″ (loss modulus) values, indicating that the samples formed a stable gel compound. Furthermore, the MN had a larger G′ value, indicating that it resulted in a more stable network structure with a strong mechanical strength. This outcome could be attributed to the fact that the strain capacity of the final MN significantly improved with the continuous modification and crosslinking of the Gel and the addition of the Gel-PDA substrate part, resulting in a more stable structure.

2.3 Preparation and morphological characterization of MN

Figure 4A shows the MN preparation process. Herein, polydimethylsiloxane (PDMS) MN molds were utilized for the preparation of MNs in 4 steps: (1) First, the GelMA-CS-β-CD-CSMA solution was vacuum-filled into PDMS molds and exposed to UV light for gelation (Figure 4Ai); (2) second, the solution was naturally dried to form a needle tip structure (Figure 4Aii); (3) third, the Gel-PDA solution was added as a backing material through vacuum-assisted application (Figure 4Aiii); and (4) finally, the Gel-PDA solution was naturally dried and demolded to obtain 2-ended MN products (Figure 4Aiv).

To observe the morphology of the MNs, they were characterized using SEM, confocal laser scanning microscopy (CLSM), and super depth of field microscopy. Figure 4B depicts a macroscopic and microscopic view of an intact MN comprising 10 × 10 circular MNs with a conical tip portion and tip distance of ~500 µm. The tip portion of the MN was not clearly demarcated from the basal portion, indicating that the 2 portions were well connected. Under the bright-field image of the microscope, a uniformly arranged array of conical spikes in the MN can be observed (Figures 4Ci). To more effectively visualize the distribution of DOX within the MNs, the red dye rhodamine B (RhB) was added to GelMA-CS-β-CD-CSMA to simulate the drug. RhB was observed to be tightly attached to the MNs (Figure 4Ciii). To illustrate the MN’s 2-stage structure, DOX was simulated using RhB and examined with a super depth-of-field microscope. The MN exhibited a distinct 2-part structure (Figure 4Cv): a tip (red; height = 800 μm) and a base (thickness = 200 μm). Additionally, three-dimensional (3D) imaging (Figure 4Cvi) and cross-sectional views (Figure 4D) obtained via laser confocal microscopy revealed a uniform and continuous red fluorescence in the outer layer of the MN + RhB. Overall, these results demonstrate the successful fabrication of the GelMA-CS-β-CD-CSMA drug-loaded tip and its integration with the Gel-PDA base to form a 2-part MN. This structural design reduces drug wastage commonly associated with conventional 1-piece MNs.

2.4 Performance characterization of the MNs

To verify the puncture performance of the MN, an MN patch loaded with Rh B was pierced into the skin tissue of depilated rats. The superficial layer of the skin exhibited regular red dimples (Figure 5A). Subsequently, this skin tissue was processed by freezing, sectioning, and paraffin embedding before observation via CLSM. The results (Figure 5A) confirmed that the MNs were successfully embedded into the rat skin, showing uniform puncture point spacing and clear puncture depth. Moreover, the red RhB localized in the deeper skin layers showed minimal diffusion, indicating that the UV-cured crosslinked 3D mesh structure limited tissue fluid penetration and inhibited fluorescent dye diffusion. This suggests that osmotic pressure could facilitate the slow release of the drug after the MNs loaded with the drug are inserted into the skin tissue. Next, the mechanical properties of the MNs were characterized using a universal testing machine to determine their ability to penetrate the stratum corneum (Supplementary Figure 1, https://links.lww.com/MEDMAT/A8). The stress-strain curves (Figure 5B) demonstrated GelMA, synthesized by modifying gelatin macromolecular chains with MA and crosslinked via UV curing, exhibited improved mechanical properties due to the dense network structure formed by the crosslinked molecular chains. Although the GelMA-CS-β-CD and GelMA-CS-β-CD-CSMA complexes, obtained through further crosslinking with CS-β-CD NPs and GelMA, could withstand increased pressure, they were prone to fracture under large strains. The addition of a Gel-PDA gel matrix significantly enhanced the strain capacity and compressive strength of the MN, greatly improving their mechanical properties. Consequently, the MN could endure substantial strain without fracturing under higher pressure (Supplementary Figure 2, https://links.lww.com/MEDMAT/A8). As shown in Supplementary Figure 3, https://links.lww.com/MEDMAT/A8, the maximum stress achieved was 4.48 N/needle, which fully meets the stress requirements for skin puncture^[49,50]. To evaluate the mechanical properties of the MN under humid conditions, compression tests were conducted in a hydrated environment. As shown in Figure 5C, the compressive force of the MNs increased with strain in both dry and wet environments; however, the mechanical performance under dry conditions was significantly superior to that under wet conditions. This discrepancy can be attributed to the swelling of the MN material and the disruption of hydrogen bonding networks caused by moisture in the wet environment, leading to a reduction in compressive modules and mechanical strength. Nevertheless, even under wet conditions, the compressive force continued to rise with increasing strain, and the maximum compressive force sustained by the MNs reached 100 N (equivalent to 1 N/needle) (Supplementary Figure 4, https://links.lww.com/MEDMAT/A8). This demonstrates that the MNs can provide effective penetration force within the strain range required for stratum corneum puncture.

According to the contact angle results (Figure 5D and Supplementary Figure 5, https://links.lww.com/MEDMAT/A8), the droplets in each material showed good penetration attributable to the large number of carboxyl and amino groups in the Gel, making the materials highly hydrophilic (ie, good wettability and liquid absorption). Notably, the MN’s dissolution speed will directly affect the drug diffusion rate from the MN to the surrounding area. According to the dissolution characterization results (Figure 5E), the dissolution rates increased with time. However, its swelling rate was relatively slow, which led to slow dissolution during tissue fluid infiltration, delaying DOX release and preventing sudden release. Furthermore, to assess the swelling state of the MN in vivo, the skin tissue was simulated using agarose gel and MN was injected into the agarose. Compared to the initial state of the MN, after it was injected into the agar, the aqueous solution therein infiltrated the MN hydrogel network, increasing the MN’s volume. Although the MN’s increased volume resulted in an apparent expansion change, the MN retained its intact conical structure (Supplementary Figure 6, https://links.lww.com/MEDMAT/A8), which would facilitate the continuous release of the drug in the MN into the tissue fluid.

To systematically evaluate the degradation behavior of the MN tip materials developed in this study, we prepared Gel, GelMA, GelMA-CS-β-CD-CSMA, and MN samples and conducted a 28-day in vitro degradation study under both simulated physiological conditions (PBS, pH 7.4) and a simulated post-tumor resection complex microenvironment (pH 6.8+enzymes). As shown in Figure 5F, all 4 materials exhibited expected degradation trends in PBS (pH 7.4). Pure gelatin MNs, lacking a crosslinked network, degraded rapidly primarily through a dissolution mechanism, with the mass remaining dropping to (42.8% ± 3.8%) within 7 days, indicating their unsuitability for long-term drug delivery. In contrast, GelMA, due to its stable 3D network formed by photo-initiated crosslinking, demonstrated excellent resistance to dissolution, retaining a high mass remaining of (24.7% ± 3.5%) after 28 days (Supplementary Figure 7, https://links.lww.com/MEDMAT/A8). However, under the composite environment (pH 6.8+enzymes), the degradation behaviors of the 4 materials diverged significantly. Gel was rapidly disintegrated by the enzymatic action of collagenase, degrading (97.3% ± 4%) within just 3 days. Although GelMA could withstand acidic dissolution alone, it remained susceptible to enzymatic attack, degrading significantly faster than in PBS and being almost completely degraded by day 28 (Supplementary Figure 8, https://links.lww.com/MEDMAT/A8). Notably, while the MN, due to its crosslinked network, exhibited relatively good degradation resistance, it still degraded by approximately 90% over 28 days (Supplementary Figure 7, https://links.lww.com/MEDMAT/A8). This is attributed to the protonation of amine groups on the chitosan chains under acidic conditions, leading to their dissolution and leaching from the gel network, which disrupts the material’s microstructure and creates numerous pores and channels. Consequently, collagenase can penetrate more deeply into the compromised network, accelerating the enzymatic degradation of the GelMA backbone. This synergistic effect of acid and enzyme enables the MN composite to respond actively to the pathological microenvironment, establishing a solid foundation for targeted and sustained drug release. Figure 5G systematically reveals the self-healing properties of gelatin dopamine hydrogels from the macro-morphology and micro-molecular action levels: macroscopically, the broken interface fused and recovered the mechanical load-bearing capacity (Cut) within 24h after physically cutting the Gel-PDA hydrogel (Original). Whether it is Gel-PDA’s own material (“Cut” column), or on the surface of biological tissues such as skin, heart, spleen, and liver, after cutting treatment, Gel-PDA can achieve effective recovery of structural integrity in the “Healed” stage, reflecting the universality of its self-healing properties. Microscopically, the molecular structure of the cut state (Healed) showed that the external force destroys the catechol moiety of dopamine and gelatin. The molecular structure of the cut state (Cut) reveals that external forces break the hydrogen bonds and weak covalent bonds—such as those in the Schiff base precursor—between the dopamine catechol moiety and gelatin molecules. In the healed state, the catechol structure reconstructs the crosslinking network through the reorganization of reversible hydrogen bonds, redox reactions, and the regeneration of dynamic covalent bonds with gelatin amino groups, thereby achieving interfacial fusion at the molecular level.

To evaluate the bioadhesive performance of Gel-PDA, this study conducted adhesion tests across multiple body sites (hand, elbow, knee, and finger) and various biological tissues (skin, heart, spleen, and liver). By comparing pre- and post-adhesion conditions, we systematically analyzed its adhesive adaptability and universality (Figure 5H). The results demonstrated that Gel-PDA maintained effective adhesion during dynamic processes such as hand extension and fist clenching, elbow flexion, knee morphological changes, and finger movements. This indicates its excellent adhesive stability on human soft tissues under dynamic mechanical stimuli, accommodating mechanical deformations and stresses induced by body motion. Furthermore, adhesion tests on biological tissues revealed that Gel-PDA could tightly conform to the surfaces of various tissue types, reflecting its universal adhesive capability across diverse biological substrates. The adhesion mechanism is mainly through the formation of a large number of hydrogen bonds, π–π stacking and other noncovalent interactions between the catechol group of polydopamine and the surface group of the tissue, as well as the formation of Schiff base covalent bonds with gelatin and other molecules under oxidative conditions to achieve strong wet adhesion. These findings provide an experimental basis for its potential applications in areas such as organ repair and tissue engineering scaffolds.

2.5 Biocompatibility evaluation of MN

Given that bacterial infection is a major contributor to delayed wound healing, we assessed the antimicrobial efficacy of MN patches. Notably, antimicrobial experiments were conducted using MNs composed of different materials. The results showed that bacteria grew normally around the Gel patch, as it lacked antibacterial components. In contrast, GelMA, GelMA-CSMA, GelMA-CS-β-CD-CSMA, and MN patches exhibited varying degrees of antimicrobial zones around them (Figure 6B), the inhibition zone of MN against Staphylococcus aureus reached 3.3 cm, which was significantly higher than that of the control group and the gelatin group (P < 0.001), confirming the antimicrobial properties of each material. Furthermore, the MN patch containing polymyxin effectively killed over 95% of S aureus and Escherichia coli in the cocultured bacterial suspension (Figure 6C), significantly higher than the control group (P < 0.001), demonstrating its exceptional antibacterial activity. This is attributed to the synergistic effect of CS in the tip part and polymyxin in the back part of the MN in this study. The puncture performance of the tip part physically penetrates the skin barrier and directly acts on the infected site. Polymyxin adsorbs and destroys the bacterial cell membrane by electrostatic adsorption, resulting in leakage of cell contents and rapid sterilization (Figure 6A). The results showed that MN could enhance the “breakthrough” and “resident” ability of drugs to the infection microenvironment in the infection model, showed a good preliminary bacteriostatic effect^[51].

This efficacy is attributed to the synergistic coordination between polymyxin and CS, which endows the MNs with strong antibacterial effects, effectively inhibiting bacterial reproduction and growth.

Figure 6E, F illustrates that after incubation for the same duration, cell viability was comparable between the experimental and control groups. On the 5th day, the cell activity in the MN group was significantly higher than that of the control group and exceeded the activity observed before treatment. This indicates that MN administration enhanced cellular activity and promoted cell growth and proliferation. Results from the Tetrazolium salt colorimetric assay (MTT) demonstrated that none of the experimental groups exhibited significant cytotoxicity (Figure 6D), as evidenced by robust cell growth and distribution on day 3. Notably, there were fewer, red-stained dead cells, and the number of green-stained cells in the MN group increased after 5 days, indicating that MN is biocompatible and promotes cell proliferation. These findings underscore the clinical potential of MN.

2.6 Evaluation of whole skin defect repair in animals of MN

Researchers have shown that skin lesions caused by BC have 2 main characteristics; difficulty in healing and long-term susceptibility to cancer^[51], which necessitates the search for effective preventive and therapeutic measures. To evaluate the impact of MN on skin wound healing following BC surgery, we conducted in vivo testing to monitor the therapeutic efficacy of MN. This involved tracking changes in defective skin wounds and analyzing the associated histopathological changes in mice (Figure 7A). Rats were treated with 3 sets of MN patches, and Figure 7B displays the changes in skin wounds in mice after different treatments. When the treatment effects were applied to infected wound tissue, wound healing responses in the treatment group were less favorable from days 0 to 3. This was likely due to hemostatic occlusion caused by the MNs in the early stages, as well as minor cytotoxicity from the CS-β-CD NPs. At day 7, the skin wound area was significantly reduced in all treatment groups, and administration of GelMA, CSMA, and bFGF promoted angiogenesis and cell proliferation, enhancing the expression of wound healing. On day 14, the blank group showed the lowest degree of healing with 50.0% of the original wound area. Compared with the blank group, the GelMA-treated group showed a significant reduction in skin wound area. Notably, The healing rate was: MN group > GelMA-CS-β-CD-CSMA group > GelMA group > Control group (Figure 7C, D). In addition, at any time point, the wound area of rats in the MN group was the smallest among all groups, and the wound closure rate was higher than 95% after 14 days of treatment. The Gel-PDA backing part can reshape the typical inflammatory wound microenvironment into a repair-oriented immune homeostasis during wound healing. Its core is to actively regulate the immune response. bFGF fundamentally reverses the immune state that hinders healing by guiding macrophages to polarize from pro-inflammatory M1 to anti-inflammatory and pro-repairing M2, and reducing the level of local pro-inflammatory M2 and. At the same time, the hydrogel, as an “intelligent” delivery system, can respond to the wound environment, continuously and stably release bFGF, and accurately promote angiogenesis and cell proliferation; the dopamine component effectively scavenges reactive oxygen species, alleviates oxidative stress damage, and its potential antibacterial properties also prevent infection.

This is due to the fact that bEGF can stimulate the endogenous expression of fibroblasts and significantly promote cell proliferation, and the elevation of fibroblasts can directly promote the formation of collagen fibers and increase the connective tissues, which can enhance the antitensile strength of the wound and accelerate wound healing. Gel, CS, and growth factors synergistically accelerate vascularization in the wound, enabling the reconstruction of 90% new skin in a significantly shorter time. This indicated significant improvement in skin damage, demonstrating its effectiveness in repairing BC wounds.

To provide a more detailed comparison of microscopic changes within the wound, tissue sections were analyzed using immunofluorescence and pathological staining techniques. These included hematoxylin and eosin (H&E) staining, Masson’s trichrome staining, and CD31 staining for platelet endothelial cell adhesion molecules. Figure 7E exhibits the micrographs of wound tissue sections of different treatments on day 14. Wound healing is observed by H&E staining. Masson staining reflects the growth of collagen fibers in the wound.CD-31 specifically stains platelet endothelial cell adhesion molecules and plays an important role in the interaction with neutrophils. Key elements in the photographic results, such as blood vessels, neutrophils, hair follicles, and collagen deposits within the wound area, were highlighted to facilitate observation and analysis. Compared with the control group, more mature collagen and sustained epithelial tissue production was observed in the MN group (Figure 7F, G). H&E staining showed the lowest number of neutrophils in the MN treatment group, indicating minimal wound inflammation. Masson staining showed more collagen fiber deposition in the treatment groups, especially in the MN treatment group, which had the densest collagen fiber deposition, and the deposition of collagen fibers was almost the same as that of intact skin. In addition, the expression of CD31 was higher in the treated group, and a higher number of fibroblasts, keratinocytes, granulation and re-epithelialization processes were observed in the area, in agreement with the results of H&E staining and Masson staining. Optimal wound repair was achieved through the combined effects of Gel, CS, and bFGF. This further confirms that MN demonstrates excellent biosafety and effectively promotes cell regeneration. The new smart MN can accelerate wound healing providing a new idea for clinical BC wounds that are difficult to heal.

2.7 Drug release and in vitro anticancer characterization of MN

Given the strong absorption peak of DOX at 481 nm (Figure 8C), this wavelength was selected for establishing its standard calibration curve. The linear relationship between the absorbance and concentration of DOX standard solutions is presented in Figure 8D. As shown in Figure 8E, the initial DOX solution exhibited significantly higher absorbance compared to the supernatant. Substituting the measured supernatant absorbance (0.140) into the standard curve yielded a supernatant DOX concentration (c1) of 6.5 µg/mL. Subsequently, the DOX loading capacity in the MN was calculated as 68.8% using Equation. This high loading efficiency is primarily attributed to the hydrophilic cyclodextrin structures on the surface of the CS-β-CD NPs. These cyclodextrins are closely packed, creating a hollow internal cavity that provides efficient space for DOX encapsulation.

Subsequently, the drug release profiles of DOX-CS-β-CD NPs and MN were examined separately in PBS at different pH values (Figure 8F, G). In pH 7.4 PBS, DOX-CS-β-CD NPs exhibited a sustained release profile, with cumulative release rates of 42% at 24 hour and 52% at 48 hour. In contrast, release was significantly accelerated in pH 5.5 PBS, reaching cumulative rates of 65.2% at 24 hour and 85.5% at 48 hour. This accelerated release is attributed to the acid-triggered destabilization of CS-β-CD NPs and the responsive disassembly of their crosslinked CS/TPP core regions (Figure 8A). Similarly, MN displayed pH-responsive release behavior, with DOX release significantly faster in pH 5.5 PBS compared to pH 7.4 (Figure 8G, H). However, drug release from MN was slightly delayed relative to that from the DOX-CS-β-CD NPs. This delay is likely due to the requirement for DOX to first be released from the NPs, then diffuse through the MN’s 3D network structure driven by osmotic pressure before entering the release medium (Figure 8B). These results indicate that both DOX-CS-β-CD NPs and MN possess favorable pH-responsive drug release properties. MN can achieve programmed administration through pH response, demonstrating preliminary intelligent intervention in the TME. This “adaptive” treatment strategy is highly compatible with the cutting-edge nanomedicine concept^[52].

The antitumor efficacy of different DOX formulations was evaluated using HeLa cells (Figure 8I). After 72-hour incubation, all treatment groups demonstrated significant proliferation inhibition compared to the control. Notably, GelMA/DOX-CS-β-CD NPs showed reduced cytotoxicity versus DOX-CS-β-CD NPs solution, with significantly enhanced inhibition at pH 5.5 versus pH 7.4. This pH-dependent effect correlates with accelerated drug release kinetics and higher cumulative release under acidic conditions, resulting in prolonged tumor suppression. The attenuated efficacy of GelMA/DOX-CS-β-CD NPs likely stems from: (1) slower drug release from the hydrogel matrix, and (2) potential cytoprotective effects of gelatin. These observations align with previous release profiles and biocompatibility data. Flow cytometry analysis (Figure 8J) revealed significantly increased apoptotic populations (Q2 + Q3 quadrants) at pH 5.5 versus pH 7.4, confirming enhanced pH-responsive cytotoxicity. These results collectively demonstrate the system’s intelligent drug release capability and potent anticancer activity. To verify the DOX-specific inhibitory mechanism, we analyzed cellular uptake of DOX-CS-β-CD NPs and GelMA/DOX-CS-β-CD NPs in HeLa cells (Supplementary Figure 10, https://links.lww.com/MEDMAT/A8). Fluorescence imaging revealed distinct distribution patterns: free DOX showed nuclear and cytoplasmic localization, while nanocarrier-loaded DOX exhibited predominant cytoplasmic accumulation. This differential localization suggests distinct cellular internalization mechanisms – passive diffusion for free DOX versus likely receptor-mediated endocytosis for the nanocarrier systems^[53]. The observed intracellular DOX accumulation confirms effective cellular entry and subsequent growth inhibition, supporting the DOX-dependent nature of the cytotoxic effects.

2.8 In vivo assessment of anticancer effects of MN

Next, we established a mouse model of BC to investigate the in vivo therapeutic effect of MN (Figure 9A). BC cells were inoculated subcutaneously on the back of the mice, and after 7 days of tumor formation, treatment was initiated, and the tumors were observed for 14 days. To confirm the protective effect of MN transdermal drug delivery on tumor recurrence in vivo, MNs without a tip portion (G1) and intact MNs with a tip portion (G2) were used to cover the wound for treatment. Figure 9B, C reveals that the tumor volume of mice in the blank group increased rapidly in a time-dependent manner, and the tumor growth was inhibited to some extent in the G1 group to a certain extent after the treatment, but the tumor growth resumed afterward. The present study employed DOX, a broad-spectrum anthracycline chemotherapeutic agent, In the acidic TME, DOX is rapidly released from the MN tips via a pH-triggered mechanism. While DOX directly eliminates rapidly proliferating tumor cells primarily by intercalating into DNA and inhibiting topoisomerase II, it can also induce immunogenic cell death. During this process, DOX prompts the release of damage-associated molecular patterns and tumor antigens from the dying cells, which in turn activates a systemic antitumor immune response—promoting the maturation of dendritic cells and recruiting cytotoxic T lymphocytes. Ultimately, this leads to the clearance of tumor cells by the activated immune system^{[54–55–56–57]}. However, extensive evidence indicates that free DOX lacks selectivity in vivo due to its small molecular properties, enabling nonspecific systemic distribution via the enhanced permeability and retention effect and passive diffusion. This nontargeted distribution pattern not only results in rapid systemic dissemination but also leads to limited accumulation efficiency and short duration of therapeutic efficacy in tumor tissue. Consequently, the free DOX group failed to demonstrate significant tumor volume reduction in murine models^{[58–59–60]}. Tumor size in the G2 group was significantly smaller relative to that of the control group and free DOX, which demonstrated that transdermal administration of MN prevented BC recurrence by ensuring the sustained release of the drug in the TME. In addition, the relative tumor volume and weight were recorded after 14 days (Figure 9D, E), and the tumor in the G2 group was well decreased, confirming that the drug-carrying tip of the MN specifically released DOX in the TME, effectively eliminating residual tumor and thereby preventing tumor recurrence. The spleen is an important site of extramedullary hematopoiesis, producing suppressor myeloid cells in tumor-bearing mice^[61]. Studies have demonstrated a positive correlation between spleen weight and liver index and tumor weight^[62]. Figure 9F shows that tumor recurrence was effectively controlled in the G2 group.

All collected tumor tissues and major organs were sectioned and stained with HE for histological analysis. In addition, GPX4 analysis and TUNEL analysis were performed on tumor tissues to assess tumor cell apoptosis^[63]. The G1 and G2 groups induced more cancer cell damage, including vacuolization, necrosis, atrophy and cytoplasmic nuclear separation (Figure 10A). Furthermore, the degree of tumor necrosis was significantly higher in the G2 group compared to the G1 group. Immunofluorescence staining for GPX4 and TUNEL revealed a marked increase in the percentage of apoptotic tumor cells and greater cell death in the G1 group (Figure 10B, C). These findings collectively indicate that the growth of both primary and recurrent tumors was significantly inhibited.

DOX has been shown to have high antitumor efficacy in the treatment of various cancers, but its side effects, especially toxicity to major organs in circulation, should not be overlooked^[62]. Studies have revealed that both intravenous and locally injected free DOX exhibit significant systemic toxicity due to its nonspecific distribution in vivo^[64,65]. In contrast, in this experiment, the spleens of the treated and control mice were maintained at normal levels without significant damage. In addition, no significant toxicity was detected in major organs such as heart, liver, and kidney in the MN group based on histological section staining observations (Figure 10D). This further suggests that when DOX is delivered via MN, the drug is not diluted by entering the bloodstream and metabolized by the liver, but rather it enters the cancer tissues, which makes it safer by reducing the systemic toxicity of the drug. In conclusion, this MN is shown to be a promising local drug delivery system for the prevention of recurrence and distant metastasis after mastectomy of breast tumors.

4.1 Data analysis

Materials and methods are described in Supplementary Information, https://links.lww.com/MEDMAT/A8. All data are depicted as mean ± standard deviation (n = 3), and significant differences were analyzed using OriginPro 2018 via one-way analysis of variance (ANOVA). The value of P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).