Mesoporous polydopamine delivery system for intelligent drug release and photothermal-enhanced chemodynamic therapy using MnO2 as gatekeeper

Abstract The non-specific leakage of drugs from nanocarriers seriously weakened the safety and efficacy of chemotherapy, and it was very critical of constructing tumor microenvironment (TME)-responsive delivery nanocarriers, achieving the modulation release of drugs. Herein, using manganese dioxide (MnO2) as gatekeeper, an intelligent nanoplatform based on mesoporous polydopamine (MPDA) was developed to deliver doxorubicin (DOX), by which the DOX release was precisely controlled, and simultaneously the photothermal therapy (PTT) and chemodynamic therapy (CDT) were realized. In normal physiological environment, the stable MnO2 shell effectively avoided the leakage of DOX. However, in TME, the overexpressed glutathione (GSH) degraded MnO2 shell, which caused the DOX release. Moreover, the photothermal effect of MPDA and the Fenton-like reaction of the generated Mn2+ further accelerated the cell death. Thus, the developed MPDA-DOX@MnO2 nanoplatform can intelligently modulate the release of DOX, and the combined CDT/PTT/chemotherapy possessed high-safety and high-efficacy against tumors.


Introduction
Cancers possessed high mutation rate and metastasis, and caused great threatening to the life of human beings [1][2][3].In clinical, chemical drugs were widely applied, but conventional chemotherapy still faced with great challenges.Especially, many patients were seriously suffering from the side effects of chemotherapy, and the drug resistance was also a key factor of leading to the failure of chemotherapy [4,5].Therefore, it was very urgent of developing new chemotherapy drugs with high safety and high efficacy.
In the past decades, functional nanomaterials were investigated as drug carrier, which provided new strategies for improving chemotherapy performance [6,7].By constructing delivery nanosystems, the stability of drugs was improved, and the drug accumulation at tumor sites was significantly increased [8,9].Different nanocarrier systems have been investigated, including metal-organic frameworks [10,11], liposomes [12,13], inorganic oxide nanoparticles [14,15], graphene and mesoporous carbon nanospheres [16][17][18].Owing to the good photothermal performance and rich channels, Prussian blue and polydopamine with mesoporous structure (MPDA) had received much attention as nanocarriers [19][20][21].Especially, the abundant aromatic rings of MPDA made it possible to load chemical drugs by electrostatic adsorption, p-p stacking, and hydrophobic-hydrophobic interactions [22,23].However, before reaching the tumor sites, the premature leakage of drugs could reduce the therapeutic efficacy, and also bring toxicity to normal tissues [24].Therefore, it was very important of constructing intelligent delivery nanoplatform, avoiding the non-specific leakage of drugs in normal physiological environment, but achieving the controllable release of drugs in tumor microenvironment (TME).
Recently, exogenous/endogenous-responsive delivery nanoplatforms were reported to realize the modulation release of drugs.When they were delivered into tumor sites, the protective layer can be destroyed, which accelerated the release of drugs [25,26].TME-responsive liposomes [27,28], polypeptides [29] and inorganic nanomaterials with disulfide bond [30] could cleave into small nanocomponents by endogenous substances, by which the drugs release was caused.Thermosensitive polymer, a kind of temperature-responsive phase-transition material, can transition from solid into liquid under the triggering of photo-thermal conversion, by which the accelerated drug release and photothermal therapy (PTT) were realized [31,32].It was well known that glutathione (GSH) and hydrogen peroxide (H 2 O 2 ) with highconcentration were present in TME, and it was very meaningful of designing TME-responsive nanoplatform to modulate the release of drugs [33][34][35].Importantly, based on the Fenton/Fentonlike reactions of metal ions, TME-responsive chemodynamic therapy (CDT) showed promising application in cancer treatment [36][37][38].Among many TME-responsive nanoplatforms, manganese dioxide (MnO 2 ) was considered as the most promising protective layer, owing to its GSH-responsive degradation characteristics [39,40].Moreover, the reduced Mn 2þ can convert endogenous H 2 O 2 to highly toxic hydroxyl radical (ÁOH) for CDT [41][42][43][44].Therefore, using MnO 2 as protective layer, the release of drugs can effectively be modulated, and simultaneously the CDT amplified the anti-tumor efficacy.
In this work, by designing MnO 2 as 'gatekeeper', an intelligent delivery nanoplatform (MPDA-DOX@MnO 2 ) was constructed for simultaneous CDT/PTT/chemotherapy, in which MPDA nanoparticles were used as nanocarrier to load doxorubicin (DOX), and GSH-responsive MnO 2 was used as protective player to modulate the DOX release.In Scheme 1, MPDA nanospheres were firstly prepared, and DOX was loaded onto MPDA (MPDA-DOX) using electrostatic adsorption.Finally, MnO 2 shell was in situ grown on MPDA-DOX to form MPDA-DOX@MnO 2 .In normal physiological environment, the stable MnO 2 shell avoided the premature release of DOX.However, in TME, the overexpressed GSH reduced MnO 2 to generate Mn 2þ , which led to the degradation of MnO 2 shell and allowed the release of DOX.Furthermore, the photothermal property of MPDA accelerated the ÁOH generation of Mn 2þ .Thus, the MPDA-DOX@MnO 2 nanoplatform effectively modulated the DOX release, and achieved the combination of chemotherapy/PTT/CDT, opening new avenues for the development of intelligent delivery nanoplatforms.

Characterization
The morphological changes of the nanomaterials were observed with a Tecnai F30 transmission electron microscope (FEI, USA) Scheme 1. Schematic diagram of MPDA-DOX@MnO 2 for intelligent drug release and photothermal-enhanced CDT. and a Nova NanoSEM450 scanning electron microscope (FEI, USA).The size and potentials were recorded on a Nano-ZS Zetasizer (Malvern, UK).The Brunauer-Emmett-Teller (BET) surface area and pore size distribution were measured using an ASAP2020M automatic specific surface area and t porosity analyzer (Micromeritics, USA).A K-a X-ray photoelectron spectrometer (Thermo Fisher, USA) was used to analyze the X-ray photoelectron spectroscopy (XPS).A Lambda 365 ultravioletvisible (UV-vis) spectrophotometer (PerkinElmer, Korea) and a Nicolet iS10 Fourier transform infrared (FT-IR) spectrometer (Thermo Fisher, USA) were used to measure the UV-vis absorption and FT-IR spectra.

DOX loading
Five milliliters of DOX with different concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 mg/ml) were added to 5 ml of MPDA (1 mg/ml), and stirred at room temperature in the dark for 24 h.By centrifugation, the MPDA-DOX was obtained, and the absorbance of the obtained supernatants was recorded.In addition, by measuring the absorbance of free DOX, the standard curve was drawn and the DOX loading was calculated.

DOX release
Three milliliters of MPDA-DOX and MPDA-DOX@MnO 2 (5 mg/ml) in dialysis bag were placed in 57 ml of PBS (pH ¼ 7.4, 6.5 and 5.5).At different time points, 1 ml of PBS was removed, and 1 ml of newly PBS was added.Finally, the absorbance of removed PBS was measured, and the cumulative release of DOX was calculated.Alternatively, by adding GSH (2.5, 5.0, 7.5 and 10 mM) in PBS (pH ¼ 5.5), the cumulative DOX release in MPDA-DOX@MnO 2 was also measured.

Photothermal stability
One milliliter of MPDA-DOX@MnO 2 (180 lg/ml) was irradiated (1.0 W/cm 2 ) for 10 min.Then, the laser was turned off, and the temperature was recorded.Finally, the photothermal conversion efficiency was calculated.Moreover, by repeating on/off laser for five times, the photothermal stability was measured.

Cellular uptake
One milliliter of MPDA-DOX and MPDA-DOX@MnO 2 (90 lg/ml) was incubated with MDA-MB-435 cells for 2, 4 and 6 h.Then, they were fixed, and stained with Hoechst.Finally, the cells were observed on an LSM-880 laser scanning confocal microscope (Zeiss, Germany).

Cellular ÁOH generation
One milliliter of MPDA@MnO 2 (150 lg/ml) containing H 2 O 2 (100 lM) was incubated with cells for 4 h.Then, 1 ml of medium containing DCFH-DA (10 lM) was added and incubated for 30 min.Finally, the cells were stained with Hoechst, and the fluorescence of 2,7-dichlorofluorescein (DCF) was observed.Alternatively, the cells were irradiated (1.0 W/cm 2 ) for 5 min, and the DCF fluorescence was also observed.

In vivo biocompatibility and CDT/PTT/ chemotherapy
The animal experiments were done in Experiment Animal Center of Hebei University (SYXK (Ji) 2022-009), and the protocols were approved by Experimental Animal Welfare Ethics Committee of Hebei University.

In vivo biocompatibility
Healthy nude mice were injected intravenously with 100 ll of saline and MPDA-DOX@MnO 2 (500 lg/ml).After 3 weeks, the mice were executed, and the blood indexes were analyzed.Moreover, the major organs were collected to perform H&E staining.

In vivo CDT/PTT/chemotherapy
The in vivo experiment included seven groups: Saline, Saline (Lþ), MPDA-DOX, MPDA@MnO 2 , MPDA-DOX@MnO 2 , MPDA@MnO 2 (Lþ), and MPDA-DOX@MnO 2 (Lþ).Briefly, 100 ll of different samples (500 lg/ml) was injected through the tail vein, respectively.After 24 h, the tumors of mice in laser groups were irradiated (1.0 W/cm 2 ) for 5 min, and the temperature change was detected.By measuring the body weight and tumor volume, the in vivo CDT/PTT/chemotherapy was evaluated, and the survival rates were also recorded.In addition, the main organs in the groups of Saline and MPDA-DOX@MnO 2 (Lþ) were removed for H&E staining.

Results and discussion
Characterization of MPDA-DOX@MnO 2 Transmission electron microscopy (TEM) images were obtained to characterize the synthesis process and GSH-responsive degradation of MPDA-DOX@MnO 2 .The bare MPDA nanoparticles were roughly spherical with channel structure, and showed uniform size distribution with a diameter of 150 nm (Figure 1A).The channel of MnO 2 -DOX became blurry, owing to the occupation of DOX (Figure 1B).By coating MnO 2 , the core-shell structure was observed, and the diameter of MPDA-DOX@MnO 2 was 220 nm (Figure 1C).By adding GSH, it was clearly found that the MnO 2 layer disappeared, indicating the GSH-responsive degradation (Figure 1D).Further, the dynamic light scattering (DLS) and the potential were also measured.In Figure 1E, the hydrodynamic size of MPDA, MPDA-DOX, MPDA-DOX@MnO 2 and MPDA-DOX@MnO 2 (GSH) was 160, 167, 220 and 169 nm, and the corresponding zeta potential was À10.1, 8.5, À7.3 and 8.7 mV, in which the positive potential of MPDA-DOX can be attributed to the cationic DOX molecule (Supplementary Figure S1).It was observed that the morphology, size and potential of MPDA-DOX@MnO 2 (GSH) were similar to those of MPDA-DOX, demonstrating the synthesis of MPDA-DOX@MnO 2 and the degradation of MnO 2 .
In order to demonstrate the mesoporous structure of MPDA, the N 2 adsorption-desorption isotherms and pore size were measured.The BET surface area of MPDA was 29 m 2 /g (Supplementary Figure S3A), and the pore size distribution ranged from 2.5 to 20 nm, in which the two main peaks of pore size were located at 3.4 and 10.8 nm (Supplementary Figure S3B).By varying the concentration of DOX, the optimal rates of loading and encapsulation were calculated to be 52% and 92% (Figure 1L; Supplementary Figure S4), in which the high loading/encapsulation efficiency was related to the mesoporous structure and negative surface potential of MPDA.

Coating and GSH-responsive degradation of MnO 2
By changing KMnO 4 concentration, MPDA@MnO 2 nanoparticles with different thickness of MnO 2 shell were prepared.As shown in Figures 1C and 2A-D and Supplementary Figure S5, when the concentration of KMnO 4 increased from 40 to 140 lg/ml, the mesoporous structure of MPDA gradually disappeared, and the overall size of MPDA@MnO 2 increased from 150 to 225 nm.The absorbance of MnO 2 at 400 nm became larger by adding KMnO 4 concentration (Figure 2I), indicating the increased thickness of MnO 2 shell.
Fixing the GSH concentration (2 mM), the degradation behavior of MPDA@MnO 2 with different thickness was investigated.As shown in Supplementary Figure S6, the MnO 2 shell synthesized with a KMnO 4 concentration below 120 lg/ml disappeared completely, but was still present, when the KMnO 4 concentration was 140 lg/ml.By increasing GSH concentration from 0 to 2 mM, the MnO 2 shell synthesized with KMnO 4 at different concentration got thinner (Figure 2E-H; Supplementary Figures S7-S9).In addition, with the presence of GSH at different concentrations, the UV-vis absorption spectra exhibited that the absorbance of MnO 2 gradually decreased, in which the complete degradation of MnO 2 synthesized with KMnO 4 at the concentrations of 40-140 lg/ml corresponded to a GSH concentration of 0.15, 0.4, 0.8, 1.0, 2.0 and 3.0 mM, respectively (Figure 2J and K; Supplementary Figure S10).

GSH-responsive release of DOX
The release behavior of DOX in MPDA-DOX and MPDA-DOX@MnO 2 was examined, respectively.when the pH of PBS was 7.4, 6.5 and 5.5, the cumulative release rates of DOX were 25.3%, 42.9% and 59.9% in MPDA-DOX (Figure 3A), but only 4.9%, 8.6% and 12.1% in MPDA-DOX@MnO 2 (Figure 3B), in which the low release rates can be attributed to the protection of MnO 2 .In contrast, the DOX release of MPDA-DOX@MnO 2 in PBS (pH ¼ 5.5) was significantly accelerated by adding GSH concentration, in which the release rate reached 56.3%, when the GSH concentration was 10 mM (Figure 3B), which suggesting the GSH-responsive controllable release of DOX.

Photothermal conversion, ÁOH generation and GSH consumption
Under 808 nm laser irradiation, the temperature of MPDA-DOX@MnO 2 increased by 25 C (Figure 3C; Supplementary Figure S11), and the temperature change was nearly constant by repeating on/off laser (Supplementary Figure S12).Moreover, the photothermal conversion efficiency (g) was calculated was 40.1% (Supplementary Figure S13), indicating the good photothermal conversion and stability of MPDA-DOX@MnO 2 .In TME, the overexpressed GSH can scavenge the produced ÁOH to reduce CDT performance, and MnO 2 can be a candidate as an effective depleting agent of GSH, in which DTNB is used as a probe to detect the depletion of GSH.As shown in Figure 3D, by increasing the concentration of MPDA@MnO 2 , the absorbance of TNB at 412 nm significantly decreased, indicating the good GSH depletion performance of MnO 2 .Moreover, it was found that the absorbance of MB first decreased, and then increased by adding GSH concentration to above 2 mM (Figure 3E), demonstrating the ÁOH elimination of excess GSH.
With the increase of exogenous H 2 O 2 , the ÁOH production of MPDA@MnO 2 was measured.In Figure 3F, when the concentration of H 2 O 2 was 600 lM, the absorbance of MB greatly decreased by 74.4%,Moreover, it was found that the ÁOH production of MPDA@MnO 2 can be enhanced by improving incubation temperature.It was firstly confirmed that single MB showed good temperature stability (Supplementary Figure S14).When the incubation temperature was 25, 37 and 49 C, the absorbance of MB incubated with MPDA@MnO 2 decreased by 52%, 59% and 63% (Figure 3G; Supplementary Figures S15-S17), respectively.By comparing the ÁOH production MPDA@MnO 2 with different shell Regenerative Biomaterials, 2023, Vol. 10, rbad087 | 7 thickness, it was observed that the thicker MnO 2 shell consumed more MB (Supplementary Figures S18-S20), which was relevant with the different Mn 2þ amounts.In addition, the GSH depletion of MPDA@MnO 2 can also be accelerated by improving incubation temperature.As shown in Figure 3H and Supplementary Figures S21-S23, the absorbance of TNB at 412 nm decreased by 64.8%, 81.3% and 88.2% under the incubation temperature of 25, 37 and 49 C.All the above results indicated that the photothermal performance of MPDA can enhance the ÁOH production.

Biocompatibility and in vitro CDT/PTT/ chemotherapy
In order to demonstrate the controllable release capability of DOX, the cellular uptake behavior of MPDA-DOX and MPDA-DOX@MnO 2 was firstly compared.In Figure 4A, the laser scanning confocal microscopy (LSCM) images of MPDA-DOX can rapidly enter cells, and release in the nucleus.However, the cellular uptake of MPDA-DOX@MnO 2 was slowly, and only a portion of DOX entered into the nucleus after 6 h incubation, which suggested that GSH-responsive MnO 2 degradation can modulate the release behavior of DOX.Then, the cytotoxicity of MPDA and MPDA@MnO 2 was determined.After 24 h incubation, the viability incubated with MPDA and MPDA@MnO 2 was still above 95% even at the highest concentration of 180 mg/ml (Figure 4B).By comparing the mice injected with saline and MPDA-DOX@MnO 2 , no significant difference was found in the indexes of blood routine and biochemistry (Figure 4C).Furthermore, using H&E staining, no significant necrosis and fibrosis were observed in the organs (Figure 4D).All the results suggested that the MPDA-DOX@MnO 2 nanoprobe possessed good biocompatibility.
To detect intracellular ÁOH production, DCFH-DA was applied as a fluorescent indicator.In Figure 5A, green fluorescence was clearly observed in the sample of MPDA@MnO 2 , and the fluorescence got stronger after laser irradiation, indicating the photothermal-enhanced ÁOH production.Moreover, the intracellular GSH content can reduce to 53%, when the concentration of MPDA@MnO 2 was 180 lg/ml (Figure 5B), demonstrating the effective depletion of intracellular GSH level.
The performances of in vitro chemotherapy, PTT and CDT were evaluated, respectively.In Figure 5C, the chemotherapy performance of MPDA-DOX@MnO 2 was worse than MPDA-DOX with the incubation time of below 12 h, but the viability was almost equivalent after 24 h incubation owing to the slow-release effect of MPDA-DOX@MnO 2 , in which the viability can reduce to 26%.Under the laser irradiation with different power density (0.75 and 1.50 W/cm 2 ), the single PTT performance of MPDA@MnO 2 decreased the viability to 63% and 31% (Figure 5D).In addition, by adding H 2 O 2 at the concentrations of 50 and 100 lM, the single CDT performance of MPDA@MnO 2 decrease the cell viability to 76% and 57%, respectively (Supplementary Figure S24).
Further, the combination therapy performance of MPDA-DOX@MnO 2 was measured.As shown in Figure 5E, the viability decreased to 15% by CDT/chemotherapy, but can decrease to 6% by CDT/PTT/chemotherapy.As shown in Figure 5F, the live/dead cells staining indicated that only weak red fluorescence was observed in the group of MPDA@MnO 2 , but the red fluorescence was strong under laser irradiation.Importantly, in the group of MPDA-DOX@MnO 2 (Lþ), the green fluorescence was nearly disappeared, which was also consistent with the MTT assay.All the above results demonstrated the good in vitro CDT/PTT/chemotherapy performances of MPDA-DOX@MnO 2 .

In vivo CDT/PTT/chemotherapy
By building MDA-MB-435 tumor-bearing nude mice, the in vivo anti-cancer efficacy of MPDA-DOX@MnO 2 was evaluated.After the tumor sites injected with MPDA@MnO 2 and MPDA-DOX@MnO 2 were irradiated, the temperature increased to 56.4 C and 57.3 C, but the temperature was almost constant in the control group, indicating the good tumor accumulation (Figure 6A  and B).In 14 days, the tumors in Saline, Saline (Lþ), MPDA-DOX, MPDA@MnO 2 , and MPDA-DOX@MnO 2 grew by 12.7, 11.3, 8.2, 7.4 and 3.8 times.In the group of MPDA@MnO 2 (Lþ), the tumor size firstly decreased and then increased to $0.67 times.However, in the group of MPDA-DOX@MnO 2 (Lþ), the tumors completely disappeared (Figure 6C and D), demonstrating the good CDT/PTT/ chemotherapy.Compared with the mice in other groups, no body weight loss was found (Figure 6E).Importantly, in the group of MPDA-DOX@MnO 2 (Lþ), the survival rate was 100% (Figure 6F).By comparing the H&E staining of different organs, no metastasis was observed in the group of MPDA-DOX@MnO 2 (Lþ), but tiny liver metastasis appeared in the group of Saline (Figure 6G).All the results demonstrated the safety and high efficacy of CDT/PTT/chemotherapy.

Conclusion
In summary, using MnO 2 as gatekeeper, the TME-responsive MPDA-DOX@MnO 2 nanoplatform was constructed for modulating DOX release, by which the non-specific leakage of DOX was avoided, and the synergistic PTT, CDT, and chemotherapy were achieved.The results indicated MnO 2 shell protected DOX from leakage in normal physiological environment, while caused the DOX release in TME, owing to the GSH-responsive MnO 2 degradation.By adding GSH concentration, the DOX release of MPDA-DOX@MnO 2 (pH ¼ 5.5) increased from 12.1% to 56.3%.Moreover, the MnO 2 shell also acted as a GSH depleting agent, significantly reducing the ÁOH clearance.Using the endogenous H 2 O 2 and the photothermal conversion of MPDA, the ÁOH generation was promoted.Using the synergistic CDT/PTT/chemotherapy of MPDA-DOX@MnO 2 , the cell viability decreased to 6%, and the tumors were eliminated.Thus, this work designed TME-responsive delivery nanoplatform, and achieved the synergistic CDT/PTT/chemotherapy with high-safety and high-efficacy against tumors.