FeS2@COF primarily based nanocarrier for photothermal-enhanced chemodynamic/thermodynamic tumor remedy and immunotherapy through reprograming tumor-associated macrophages | Journal of Nanobiotechnology

November 15, 2024

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Synthesis and characterization of FeS₂@COF-HA/AIPH

The synthesis technique of FeS₂@COF-HA NPs is proven in Fig. 1a. In the meantime, efficient preparation of the FeS₂, COF, and floor functionalization had been confirmed by Transmission electron microscopy (TEM) and scanning electron microscopy (SEM). As depicted in Fig. 1b, the FeS₂ NPs had a uniform spherical form with a imply diameter of roughly 8 nm. The FeS₂ NPs incorporating in COF NPs through a normal encapsulation methodology. TEM photos (Fig. 1c, d) and SEM photos (Fig. S1 and S2) exhibited that each the synthesized TAPB-DMTP-COF and FeS₂@COF had been uniformly spherical with a mean dimension of 170 nm, demonstrating that FeS₂ NPs had no impact the expansion of COF NPs. Dynamic gentle scattering (DLS) evaluation additionally validated that the particle dimension of the ready FeS₂@COF was largely concentrated at 180 nm (Fig. 1e). Notably, the sizes of FeS₂@COF measured by TEM had been smaller than that measured by DLS, which was attributed to the hydration impact. To additional substantiate the profitable synthesis of FeS₂@COF NPs through FeS₂ encapsulation, the high-angle annular dark-field scanning transmission electron microscopy (HADDF-STEM) picture and corresponding power dispersive spectrometer (EDS) elemental mapping photos manifested that the Fe and S aspect had been clearly concentrated contained in the COF, which indicated the profitable formation of FeS₂@COF NPs (Fig. 1f). Particularly, the C, N, and O aspect had been derived from COF, whereas the Fe and S aspect corresponded to FeS₂.

UV-vis-NIR spectrum of FeS₂ NPs confirmed broad absorbance within the NIR area, demonstrating its superior photothermal results (Fig. 1g). The construction of the nanocomposites was additional characterised by XRD. Following the encapsulation of FeS₂ NPs, the XRD sample of FeS₂@COF displayed the distinct diffraction peaks of each FeS₂ and TAPB-DMTP-COF. This confirms the profitable preparation of the FeS₂@COF NPs and demonstrates that the incorporation of FeS₂ NPs doesn’t alter the construction of TAPB-DMTP-COF (Fig. 1h). In the meantime, the outcomes of X-ray photoelectron spectroscopy (XPS) additional indicated that there was a big Fe and S sign peak, demonstrating the presence of FeS₂ NPs (Fig. 1i). To reinforce the biocompatibility and tumor cell focusing on means, the floor of FeS₂@COF NPs was additional coated with FA modification. The alteration of floor zeta potentials of nanocomposites proved the profitable modification in every step (Fig. 1j). As well as, the particle dimension of FeS₂@COF-HA dispersed in three varieties of physiologically related media, together with water, PBS and cell tradition medium, was monitored over 7 days utilizing DLS and TEM. As proven in Fig. S3, the particle dimension and morphology of FeS₂@COF-HA didn’t considerably change, demonstrating superior colloidal stability in physiologically related environments.

In vitro photothermal efficiency

After proving the profitable synthesis of FeS₂@COF-HA, we additional investigated the photothermal results of FeS₂@COF-HA nanocomposites by recording the temperature change of FeS₂@COF-HA dispersion at totally different concentrations upon irradiation of 808 nm laser (1.0 W/cm², 10 min). Noteworthy, a big temperature elevation was noticed with an elevated focus of FeS₂@COF-HA dispersion (Fig. 2a and b), substantiating that the photothermal impact of FeS₂@COF-HA was dose-dependent. In the meantime, the photothermal heating curves of FeS₂@COF-HA options exhibited a laser power-dependent photothermal efficiency (Fig. 2c and S4). Past that, the temperature variation of various samples throughout 808 nm irradiation had been recorded (Fig. 2d). Particularly, the temperature is positively related to the irradiation time within the FeS₂@COF-HA and FeS₂ group in comparison with the water and COF group (Fig. 2e), which demonstrated that the photothermal results of the FeS₂@COF-HA originated from the sturdy NIR absorption of FeS₂ NPs. Furthermore, the photothermal conversion effectivity (η) of FeS₂@COF-HA NPs was calculated to be 28.7% based on the earlier methodology [49] (Fig. 2f and g), which prompt that FeS₂@COF-HA was able to changing laser power into native hyperthermia. Concurrently, the photothermal efficiency of FeS₂@COF-HA nonetheless didn’t deteriorate after 5 irradiation/cooling cycles, suggesting the superb photothermal stability (Fig. 2h). Thus, these outcomes validated that FeS₂@COF-HA might be used as a potent photothermal agent for NIR-triggered alkyl radical technology of AIPH.

AIPH loading and pH/NIR triggered launch of the brokers

The potential of FeS₂@COF-HA as drug supply system to encapsulate the alkyl radical initiator (AIPH) was additional investigated. Based mostly on spectrophotometry, the drug loading content material (LC) was measured to be 54.3 ± 2.1% from the stand curve of AIPH (Fig. S5), originating from the big pore floor space of FeS₂@COF. To substantiate the controllability of twin clever stimuli responsive “gatekeepers”, underneath low pH stimulation and NIR laser irradiation, the discharge profiles of AIPH from FeS₂@COF-HA had been detected. Notably, the HA coating might be dissociated for the discharge of Fe²⁺ and AIPH in a pH-responsive and NIR-enhanced method. As illustrated in Fig. 2i, negligible drug launch was noticed at pH = 7.4 with NIR irradiation or with out NIR irradiation. Quite the opposite, the AIPH launch at pH 5.0 reached 39.6% after 24 h and additional considerably elevated to 57.2% upon NIR laser irradiation. As well as, we investigated the discharge habits of the Fe²⁺ from the nanoplatform. As might be seen from Fig. S6, underneath the impartial situation of pH 7.4, the discharge of Fe²⁺ was insignificant. Nonetheless, as soon as the rise of H⁺ occurred (pH 5.0), an explosive launch of Fe²⁺ was famous after the identical incubation time. Specifically, it was seen that the discharge fee of the Fe²⁺ might be remarkably enhanced by the involvement of NIR irradiation. Subsequently, the in vitro drug launch outcomes offered clear proof that FeS₂@COF-HA possess the power to reply to acidic TME situation and NIR laser irradiation, which might successfully stop regular cell injury from the instantaneous technology of extreme free radical and permit for an on-demand managed therapeutic brokers launch for steady tumor remedy.

Free radical technology capability

Impressed by the superior photothermal conversion results of FeS₂@COF-HA NPs, we additional investigated the alkyl free radical manufacturing means of FeS₂@COF-HA/AIPH upon laser irradiation by using 2,2′-azobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a chemical probe to seize free radicals underneath totally different circumstances (Fig. 3a and b). Noticed from Fig. 3c, solely small quantity of alkyl radical was monitored when FeS₂@COF-HA/AIPH positioned at 37 °C, whereas a variety of alkyl radicals had been generated constantly at 44 °C, demonstrating that the excessive temperature is useful for the formation of alkyl radicals. In the meantime, laser-activated alkyl radical manufacturing by FeS₂@COF-HA/AIPH was additional detected (Fig. 3d). Sometimes, the power to generate alkyl radicals might be successfully strengthened with the extended irradiation time, suggesting a robust correlation between the technology of alkyl radicals and the period of laser publicity. Nonetheless, alkyl radicals weren’t detected within the AIPH and FeS₂@COF-HA group underneath the identical circumstances (Fig. 3e). Moreover, the absorption depth of ABTS^+• exhibited an approximate linear relationship with the prolongation of irradiation time (lower than 5 min). Nonetheless, the absorbance stabilized within the subsequent 5 min, which is attributed to the entire photo-induced degradation of AIPH (Fig. 3f). These outcomes indicated a promising potential for using FeS₂@COF-HA/AIPH in TDT, because it was able to producing alkyl radical underneath the situation of laser-triggered hyperthermia.

Mobile uptake and intracellular ·OH detection

Throughout the technique of most cancers remedy, delivering medication successfully to tumor cells stays a formidable problem [51]. Thus, previous to investigating the in vivo therapeutic outcomes, the tumor-targeting means of FeS₂@COF-HA was first evaluated. Clearly, it might be seen that the sturdy pink fluorescence sign was discovered within the FeS₂@COF-HA handled cells group (Fig. S6), whereas no vital fluorescent sign was famous in FeS₂@COF group. Thus, these findings indicated that the functionalization of HA on the floor of FeS₂@COF-HA might remarkably enhanced the general accumulation of nanocomposites inside the focused tumor cells, enabling the FeS₂@COF-HA appropriate candidates for particular tumor remedy.

Temperature is a crucial issue that impacts the speed of Fenton-like response, the place a rising native temperature can remarkably improve the CDT effectivity [52, 53]. Therefore, ·OH radical manufacturing activated by FeS₂@COF-HA within the presence or absence NIR laser irradiation are comprehensively detected by using 3,3′,5,5′-Tetramethylbenzidine dihydrochloride (TMB) (Fig. 3b). Notably, FeS₂@COF-HA exerted a robust ·OH sign within the presence of H₂O₂, whereas the opposite teams exhibited no apparent change, indicating that the ·OH radical technology was generated primarily based on the response of FeS₂@COF-HA and H₂O₂ (Fig. 3g). As well as, because the NIR irradiation added to the FeS₂@COF-HA group, the attribute absorption peaks of TMB at 665 m, had been remarkably enhanced, which demonstrated that the domestically excessive temperatures produced by NIR laser irradiation remarkably enhanced the catalytic means of the Fenton response. As well as, electron spin resonance (ESR) spectroscopy was carried out to detect the technology of •OH through the use of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) because the trapping agent. Compared to the FeS₂@COF-HA and AIPH + H₂O₂ teams, a gaggle of typical radical peaks was noticed within the FeS₂@COF-HA + H₂O₂ group, whereas the everyday peak depth was considerably elevated after irradiating with the 808 nm laser irradiation (Fig. Sx), confirming that the thermal impact of laser irradiation for the promotion for CDT. For extra particular evaluation, methylene blue (MB) was explored to detect the technology of ·OH radicals underneath the situation of assorted pH values (pH = 5.5, 6.5, and seven.4). As displayed in Fig. 3h, the absorption peak at 660 nm was considerably decrease than that at excessive pH values, suggesting {that a} low pH worth might promote the effectivity of the Fenton-like impact. Taken collectively, all of the above outcomes revealed that the Fe²⁺ might successfully set off the Fenton response of FeS₂@COF-HA to provide ·OH radicals.

To additional verify the technology of ROS inside cells, the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was employed for detect the full technology of intracellular ROS (Fig. S7). Against this, a robust inexperienced fluorescence indicators had been famous within the FeS₂@COF-HA and FeS₂@COF-HA + laser teams, reflecting that the endogenous H₂O₂ is transformed to dangerous ·OH radicals through Fe²⁺-mediated Fenton-like reactions. Extra importantly, after MNNG/HOS cells had been handled with FeS₂@COF-HA/AIPH and irradiated with 808 nm laser underneath each normoxia and hypoxia circumstances, the strongest inexperienced fluorescence might be noticed, demonstrating the very best intracellular ROS stage. This enchancment was attributed to the technology of each ·OH radicals and alkyl radicals induced by hyperthermia. Moreover, these findings additionally demonstrated that the numerous technology of intracellular ROS by FeS₂@COF-HA/AIPH is unbiased of ranges of oxygenation, holding nice potential for enhanced antitumor results.

In vitro anti-tumor impact

Inspired by the managed drug launch habits, wonderful photothermal impact and potential of booming ·OH technology means, we additional explored the combinational therapeutic outcomes of FeS₂@COF-HA/AIPH in vitro (Fig. 4a). Clearly, the outcomes of calcein-AM/PI staining verified that FeS₂@COF-HA/AIPH + laser induced obvious cell demise to MNNG/HOS cells when in comparison with different teams (Fig. 4b), additional demonstrating the fascinating mixed PTT/CDT/TDT results upon NIR laser irradiation. Furthermore, the cytotoxicity of FeS₂@COF-HA/AIPH on MNNG/HOS cells was additional assessed by cell-counting kit-8 (CCK-8) assay. As illustrated in Fig. 4c, no evident toxicity was famous with or with out laser irradiation when AIPH was used. Nonetheless, after therapy with FeS₂@COF-HA, cell viability was reasonably decreased because of the CDT impact. In the meantime, 808 nm laser irradiation additional enhanced the detrimental impact on tumor cells, benefitting from the improved therapeutic modality synergized by PTT. As anticipated, probably the most substantial detrimental impact on cell viability was noticed following the administration of FeS₂@COF-HA/AIPH + laser group, leading to a discount of roughly 10% in cell viability underneath each normoxia and hypoxia circumstances. Moreover, the outcomes of colony formation additionally exerted that FeS₂@COF-HA/AIPH + laser group exhibited the strongest lethality for MNNG/HOS cells underneath each normoxia and hypoxia circumstances (Fig. 4d). The mix index (CI) of PTT/CDT/TDT is calculated to be 0.47, verifying the synergistic impact. General, this distinctive cell injury might be ascribed to the combinatorial impact of PTT/CDT/TDT, with therapy efficacy unbiased of oxygenation stage, making it extremely favorable for eliminating deep-seated tumors.

Quite a few research have proved that mitochondrial injury was intently related to ROS-induced oxidative stress [54,55,56], and the mitochondrial membrane potential (MMP) served as an essential and consultant indicator for assessing mitochondrial perform (Fig. 4e). Subsequently, 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolocarbocyanine iodide (JC-1) probe was then utilized to analyze the impression of nanoplatform on mitochondrial perform. Typically, the JC-1 probe displayed pink fluorescence underneath regular MMP circumstances, whereas underneath irregular circumstances, it confirmed inexperienced fluorescence. As proven in Fig. 4f, the outcomes exerted that FeS₂@COF-HA/AIPH + laser handled group confirmed the strongest inexperienced fluorescence underneath the situation of normoxia and hypoxia, which indicated that FeS₂@COF-HA/AIPH + laser might successfully induce abnormalities within the MMP of MMNHOS cells by producing an extreme technology of ROS [54]. Furthermore, the fluorescence depth ratio of inexperienced/pink fluorescence in MNNG/HOS cells was remarkably greater than that noticed within the different teams following therapy with FeS₂@COF-HA/AIPH + laser (Fig. S8), demonstrating that its antitumor impact was extra vital. Concurrently, there was no vital distinction in fluorescence depth ratio between teams (6) and (7), indicating that the therapeutic outcomes of FeS₂@COF-HA/AIPH + laser was unbiased of the native oxygenation stage. Moreover, the method of cell apoptosis was additional investigated utilizing the Annexin V-FITC/PI staining assay by circulate cytometry. In distinction to the opposite teams, therapy with FeS₂@COF-HA/AIPH + laser resulted within the highest fee of apoptosis in each normoxia and hypoxia (Fig. 4g and h), which might be attributed to the excellent therapeutic outcomes ensuing from the synergistic results of PTT/CDT/TDT.

Immune activation impact of nanocomposites

Immunotherapy act a key function in inhibiting tumor proliferation, and the effectiveness of immunotherapy correlated considerably with macrophage polarization [57, 58]. Furthermore, it’s identified that intracellular oxidative stress generated by Fe²⁺ play an essential function within the activation of immune response, which might successfully promote the polarization of M1 macrophages [59]. Subsequently, we then explored the impression of FeS₂@COF-HA on reprogramming M2 macrophages by investigating its affect on macrophage polarization in vitro (Fig. 5a). As illustrated in Fig. 5b-d, FeS₂@COF-HA/AIPH + laser group might considerably improve the CD86 expression by 11.5% and 50.1%. In the meantime, the CD206 expression dramatically decreased from 62.0 to 24.7%. Importantly, immunofluorescence staining was utilized to additional examine the polarization of the RAW 264.7 cell after corresponding therapy. As displayed in Fig. 5e and f, the fluorescent depth of M1 macrophages (pink fluorescence) elevated, whereas M2 macrophages (inexperienced fluorescence) decreased following FeS₂@COF-HA/AIPH + laser therapy. Collectively, these findings verified that FeS₂@COF-HA mixed with AIPH encapsulation underneath laser irradiation might successfully repolarize M2-like macrophages by upregulating M1-related CD86 markers and downregulating M2-related CD206 markers.

In vivo biodistribution and photothermal imaging

To additional validate the tumor-targeting accumulation of the nanocomposites, the tumor particular imaging and biodistribution had been explored by a small animal in vivo fluorescence imaging system. As demonstrated by the in vivo bioimaging, the FeS₂@COF-HA group displayed sturdy fluorescent indicators on the tumor web site 6 h after intravenous injection (Fig. 6a), whereas the management group solely reveals delicate fluorescence sign. Additionally, the ex vivo imaging additionally exhibited that FeS₂@COF-HA had been largely collected within the tumor area (Fig. 6b). In the meantime, the fluorescence photos of the tumor slice additional confirmed this conclusion (Fig. 6c). Taken collectively, these outcomes revealed that FeS₂@COF-HA can particularly goal into the tumor websites as a perfect nanocarrier for the extremely environment friendly and protected supply of medication to tumor web site.

Based mostly on the outcomes of above imaging observations, the NIR laser irradiation ought to be carried out at 6 h post-injection, and this time level was adopted for the following in vivo photothermal remedy experiment. The thermal imaging images exhibited the temperature of FeS₂@COF-HA group rising clearly, whereas the temperature of management group exhibited a average temperature improve in tumor websites following injection for six h underneath laser irradiation (Fig. 6d and e). These observations prompt that the FeS₂@COF-HA possessed superior photothermal property, which might successfully induce native hyperthermia for tumor remedy.

In vivo anti-tumor effectiveness analysis and M1 macrophage polarization

Impressed by the above outcomes, the therapeutic outcomes of subcutaneous MNNG/HOS induced tumor mannequin had been proven in Fig. 7a. When the tumor quantity grew to roughly 100 mm³, the mice had been randomly divided into the next six group: (1) management, (2) laser, (3) AIPH + laser, (4) FeS₂@COF-HA, (5) FeS₂@COF-HA + laser, (6) FeS₂@COF-HA/AIPH + laser. As displayed in Fig. 7c-e, it’s famous that the fast tumor development was present in management group and littler therapeutic impact for laser and AIPH + laser group. Nonetheless, each FeS₂@COF-HA and FeS₂@COF-HA + laser therapy exhibited sure inhibitory impact, owing to the power of CDT and PTT/CDT to induce tumor cell demise. Extra importantly, the mice handled with FeS₂@COF-HA/AIPH + laser confirmed minimal tumor quantity, arising from that the synergistic results of PTT/CDT/TDT and immunotherapy. As well as, there was no vital weight fluctuations of the mice throughout the entire therapy interval (Fig. S10), suggesting that the as-synthesized nanocomposites had no systemic unintended effects.

Following 14 days of therapy, Hematoxylin and eosin (H&E) and TdT-mediated dUTP Nick-Finish Labeling (TUNEL) staining had been additional carried out to additional elucidate the mechanisms of tumor apoptosis (Fig. 7f). It’s discovered that H&E-staining photos exhibited that FeS₂@COF-HA/AIPH + laser handled mice suffered extra extreme tumor cell demise than different teams. In the meantime, TUNEL staining photos revealed that tumors in FeS₂@COF-HA/AIPH + laser group exerted a excessive stage of cell apoptosis than different teams. Moreover, Ki-67 antibody staining of tumors indicated that there was a big suppression on tumor cell proliferation within the FeS₂@COF-HA/AIPH group in comparison with different therapy teams. To additional detect the ROS manufacturing within the tumor, ex vivo ROS staining was additionally performed. In contrast with different experimental teams with comparatively low ROS indicators, the FeS₂@COF-HA/AIPH + laser group exhibited stronger pink fluorescence (Fig. 7f), demonstrating a lot better ROS manufacturing within the tumor web site. Collectively, the above outcomes indicated that combining multimodal therapies enhanced the antitumor therapeutic effectivity.

To additional validate TAM reprogramming in vivo, the tumor tissues had been collected and stained with CD206 and iNOS. The immunofluorescence staining outcomes exhibited that the iNOS expression in FeS₂@COF-HA/AIPH + laser group was considerably up-regulated, whereas the CD206 expression decreased apparently (>Fig. S9). These outcomes additional prompt that the FeS₂@COF-HA/AIPH NPs + laser might successfully activate the antitumor immune response and certainly enhance the tumor immunotherapeutic efficacy.

Organic security analysis

Biocompatibility and low toxicity of the nanocomposites is extremely crucial for tumor remedy. As illustrated in Fig. S11a, FeS₂@COF-HA NPs exhibited a superior cytocompatibility with the excessive survival fee even at excessive focus. As well as, hemolysis testing confirmed that each one samples exhibited negligible hemolytic impact even uncovered to a excessive focus of 800 µg/mL, demonstrating the superb hemocompatibility of as-synthesized FeS₂@COF-HA NPs (Fig. S11b). On the similar time, no considerable organ injury was noticed within the H&E staining of tissue slices collected kind the NPs-treated mice, indicating the superb biosafety of the as-synthesized nanoplatform (Fig. S11c). Furthermore, the outcomes of blood biochemical indexes additional validated favorable biosafety of FeS₂@COF-HA NPs (Fig. S12). Accordingly, the above outcomes prompt that FeS₂@COF-HA is a perfect candidate for tumor remedy owing to its excellent biosafety.

Mechanism of FeS2@COF-HA/AIPH NPs synergistic most cancers remedy analyzed by RNA-sequencing

To additional work out the potential therapeutic mechanism of FeS₂@COF-HA/AIPH-mediated mixed antitumor efficiency, the RNA-sequencing was carried out (Fig. 8a). As proven in Fig. 8b, the volcano plot indicated that 767 genes had been considerably up-regulated and 214 genes had been considerably down-regulated. Amongst them, oxidative stress associated genes, together with GPX8, GSR, and LOX, and so on. are regulated, demonstrating that the generated ROS acts an essential function in tumor development inhibition. In the meantime, the expression of tumor necrosis issue (TNF) signaling pathway associated genes (CAPSE-3, BTRAF1, and BCL3, and so on.) and apoptosis associated genes (FOS, CAPN2, and CASP8, and so on.) was additionally regulated within the experimental group. As well as, the experimental group considerably upregulated the expression of immune-related genes, corresponding to LTA, HPX, and LTF, and so on. (Fig. 8c). Afterwards, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway evaluation had been additional carried out to research the genes set enrichment. GO evaluation revealed that many of the DEGs had been primarily concerned in tumor necrosis and immune-related pathways (Fig. 8d). KEGG evaluation reveals that DEGs are primarily concentrated in TNF signaling pathway, NF-kappa B signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and p53 signaling pathway, and so on. Gene set enrichment evaluation (GSEA) evaluation additional revelated that the experimental group elevated the expression of genes had been centered on the IL-17 signaling pathway, the MAPK signaling pathway, the TNF signaling pathway, and the reactive oxygen species pathway (Fig. 8e). Subsequently, these outcomes indicated that FeS₂@COF-HA/AIPH + laser therapy might evoke an inflammatory storm and antitumor immune responses, thereby inflicting tumor cell demise.

FeS2@COF primarily based nanocarrier for photothermal-enhanced chemodynamic/thermodynamic tumor remedy and immunotherapy through reprograming tumor-associated macrophages | Journal of Nanobiotechnology