Cinobufagin loaded Prussian blue-like nanoparticles for chemo/gasoline remedy of multidrug resistant most cancers | Journal of Nanobiotechnology

November 9, 2025

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CS-1 induces pyroptosis-mediated cytotoxicity throughout various MDR cells

CS-1, the construction of which is depicted in Fig. 1A, demonstrated a wide-ranging anti-tumor exercise [32]. By investigating the cytotoxicity of CS-1 on a number of MDR cell traces (MCF-7/ADR, A2780/DDP, and HepG2/DDP) in addition to their respective parental counterparts, the three resistant cell traces demonstrated important resistance to their corresponding chemotherapeutic brokers (Fig. 1B-C), as beforehand reported [33,34,35]. Quite the opposite, CS-1 exhibited potent cytotoxicity in a dose-dependent method towards these resistant strains and their parental counterparts with IC₅₀ values starting from 1 to five µM (Fig. 1D). The resistance index (RI) of those cell traces was computed to be lower than 5 to CS-1 (Fig. 1E), in line with the components of resistance index (RI) = IC₅₀ of resistant cells/IC₅₀ of parental cells [36]. Microscopic pictures demonstrated typical pyroptotic morphology in CS-1 handled cells, together with cell swelling and the formation of enormous bubbles [37] (Fig. 1F). DCFH-DA probe staining assay visually demonstrated a major enhance in inexperienced fluorescence of three MDR cell traces after therapy with CS-1 for 12 h (Fig. 1G), reflecting the flexibility of CS-1 to induce excessive ranges of ROS in MDR tumor cells.

Contemplating the upper RI and sensitivity to CS-1 (RI = 1.425 ± 0.024), we performed RNA-seq assay to completely examine the impact of CS-1 on the gene transcription in MCF-7/ADR cells. The volcano plot confirmed 4520 upregulated genes and 2256 downregulated genes in MCF-7/ADR cells relative to the MCF-7. In distinction, CS-1 therapy induced the upregulation of 1386 genes and the downregulation of 2175 genes (p ≤ 0.05, fold change ≥ 2) (Fig. S1A-B). The Venn diagram additionally confirmed 1943 intersecting genes with frequent modifications amongst MCF-7, MCF-7/ADR and CS-1 group (Fig.S1C). Amongst these 1943 genes, 978 genes upregulated in MCF-7/ADR group have been inhibited by CS-1, which have been just like these of MCF-7 group. Conversely, 248 genes downregulated in MCF-7/ADR group have been upregulated by CS-1, which have been just like that of the MCF-7 group. KEGG assay indicated the excessive enrichment of drug resistance protein regulatory pathway of PI3K-AKT and apoptotic pathways of MAPK, NF-κB and JAK-STAT in MCF-7/ADR cells (Fig. 1H). In distinction, CS-1 therapy induced excessive enrichment of pyroptosis-related inflammatory pathways equivalent to MAPK, NF-κB and TNF (Fig. 1I). GSEA evaluation additionally demonstrated that CS-1 therapy induced the activation of pyroptosis-related inflammatory pathways equivalent to TNF, MAPK, and NF-κB in MCF-7/ADR cells (Fig. 1J-L). Heatmap evaluation of DEGs confirmed downregulation of Multidrug resistance-associated protein ABC transporters (ABCG2) and Bcl-2, an antiapoptotic protein in MCF-7/ADR cells with CS-1 therapy (Fig. 1M). PPI evaluation indicated that TNF, a grasp regulator of irritation, linked NF-κB with Caspase-3 (Fig. 1N). Subsequently, we additional indicated the associated protein expression stage of pyroptosis by western blot combining with our reported end result [38]. Then, we detected the modifications of pyroptosis-associated proteins in MCF-7/ADR cells after therapy with totally different concentrations of CS-1. As we anticipated, CS-1 considerably activated Caspase-3 and GSDME-N ranges (Fig. 1O). In conclusion, CS-1 can successfully induce TNF associated Caspase-3-dependent pyroptosis in MCF-7/ADR cells (Fig. 1P), whereas could inhibiting ABCG2 to reinforce drug focus, which is pressing for successfully killing tumor cells.

Preparation and characterization of HA@Lip-CS-1@PBCO NPs

Our research revealed that CS-1 has manifested important anti-tumor potential in MDR tumors, nonetheless, its scientific implementation is restricted by the cardiotoxic potential of high-dose administration [39]. To protect the antitumor efficacy of CS-1 towards MDR tumors whereas minimizing cardiotoxicity, we adopted a combinational technique of CS-1/CO. MnCO was used as a precursor for producing CO. MTT assay demonstrated MCF-7/ADR cell viability of 48.2% after the combinational therapy of 1 µM CS-1 and 40 µM MnCO (Fig.S2A-B). Nonetheless, the low concentrating on and bioavailability of CS-1 and MnCO restrict their scientific software. Thus, we engineered a tumor-targeted “chemo-gas” nanocomplexe (HA@Lip-CS-1@PBCO NPs) to handle these questions. Determine 2A demonstrated that subsequent to lipid encapsulation and hyaluronic acid modification, a 9.5 nm thick movie coated the floor of PB NPs. DLS evaluation disclosed an increment in particle measurement subsequent to the encapsulation of MnCO by PB NPs (leading to a measurement of 102.7 nm), lipid encapsulation (yielding a measurement of 157.1 nm), and HA modification (leading to a measurement of 160.1 nm), as illustrated in Fig. 2B. Moreover, alterations in potential have been noticed. Ranging from PB NPs with a worth of −28.6 ± 1.6 mV, it modified to PBCO NPs at −25.1 ± 1.2 mV, then to Lip-CS-1@PBCO NPs at −36.9 ± 2.1 mV, and at last to HA@Lip-CS-1@PBCO NPs with a worth of −37.56 ± 1.3 mV (Fig. 2C). Component mapping characterization outcomes indicated that the manganese ingredient in MnCO shows a excessive diploma of overlap with the iron ingredient in PB NPs, suggesting the efficient loading of MnCO into PB NPs. Moreover, the detected phosphorus ingredient sign additional confirmed profitable liposome encapsulation (Fig. 2D and S3A). The UV-vis absorption spectrum demonstrated attribute peaks of MnCO and CS-1 at 340 nm and 290 nm, respectively, as proven in Fig. 2E. The FT-IR spectra of HA@Lip-CS-1@PBCO NPs exhibited attribute absorption peaks comparable to lipid elements (C = O at 1740 cm⁻¹, P = O at 1230 cm⁻¹, and CH₂/CH₃ at 2850 cm⁻¹), MnCO (C ≡ O at ~ 2080 cm⁻¹) and the cyanide bridges of PB NPs (C ≡ N at 2080 cm⁻¹) [40], additional confirming the profitable integration of hyaluronic acid-modified Lip-CS-1 with PBCO NPs (Fig. 2F). It’s noteworthy that ultrasonic therapy hardly impacts the discharge of CO from PBCO NPs (Fig.S3B). UV spectroscopy assay information indicated that the encapsulation effectivity of CS-1 and MnCO have been 21.45 ± 4.3% and 69.03 ± 1.13%, respectively (Fig. 2G). Moreover, the fabric remained secure for about three days in water, PBS, and DMEM supplemented with 1% FBS, which is conducive to its in-vivo drug motion (Fig. 2H). Collectively, these outcomes counsel the profitable institution of HA@Lip-CS-1@PBCO NPs.

Purposeful characterization of HA@Lip-CS-1@PBCO NPs

In keeping with the design options of HA@Lip-CS-1@PBCO NPs, we first investigated the discharge traits of CS-1 and CO (Fig. 3A). As proven in Fig. 3B, the discharge charge of CS-1 from HA@Lip-CS-1@PBCO NPs attained 88.28 ± 1% in 72 h in an acidic atmosphere (pH6.8), which was attributed to the insertion of pH-responsive molecule DSPE-PEOz2K into the lipofilm. Then, utilizing the FL-CO-1/PdCl₂ probe (Fig. 3C) [28], we evaluated the CO launch functionality underneath microenvironment situation (excessive ranges of H₂O₂). As proven in Fig. 3D, the CO launch profiles of Lip-CS-1@PBCO NPs exhibited important pH-dependent kinetics. Below physiological situations (pH7.4 + 1 mM H₂O₂), the NPs demonstrated sustained however restricted CO launch, accumulating 18.5 ± 0.5 µM at 24 h with a near-linear development. In stark distinction, an acidic microenvironment (pH5.4 + 1 mM H₂O₂) triggers fast CO liberation, culminating in 70.5 ± 0.5 µM by 24 h – representing a 3.8-fold enhancement in comparison with impartial pH. Subsequently, the FL-CO-1 probe was utilized to additional detect intracellular CO in cells subjected to therapy with HA@Lip-CS-1@PBCO NPs, with intracellular H₂O₂ serving because the substrate. Whereas cells handled with PBS and CS-1 (1 µM) exhibited no enhance in fluorescence, a major enhancement of inexperienced fluorescence was noticed in cells handled with HA@Lip-CS-1@PBCO NPs (1 µM CS-1, 20 µg/mL PBCO NPs). This enhancement was notably greater than that in cells handled with PBCO NPs (20 µg/mL), as illustrated in Fig. 3E-F. This end result steered that ROS induced by CS-1 can promote CO launch habits. As well as, the CO gasoline launched by MnCO has the potential to kind microbubbles inside the organism. Owing to their comparatively excessive acoustic impedance, these microbubbles can create a definite distinction with the encircling tissues and fluids [41], which supplies the likelihood for in vivo ultrasound imaging. As anticipated, an ultrasonic sign was detected within the pattern containing 2 mM H₂O₂ and HA@Lip-CS-1@PBCO NPs answer (Fig. 3G). Conversely, no ultrasonic sign was detected within the H₂O₂ free pattern. This end result indicated that MnCO may very well be effectively launched within the high- H₂O₂ atmosphere, which is analogous to the tumor microenvironment (normally containing 100 µM-1 mM H₂O₂) [42].

Earlier research indicated that HA floor modification enhances nanodrug concentrating on effectivity by way of CD44 interplay, a receptor extremely expressed on tumor cells [43, 44]. To judge mobile uptake, Ce6-labeled HA@Lip-Ce6@PBCO NPs have been employed. Outcomes revealed a progressive enhance in attribute pink fluorescence inside tumor cells over time, peaking at 6 h (Fig. 3H-I). In comparison with free Ce6 or Lip-Ce6@PBCO NPs, HA@Lip-Ce6@PBCO-treated MCF-7/ADR cells exhibited stronger fluorescence depth. Conversely, pretreatment with free HA markedly diminished this fluorescence sign (Fig. 3J), confirming CD44-mediated uptake inhibition. Utilizing MCF-7/ADR multicellular spheroids to imitate strong tumors, the pink fluorescence at depths of 0 ~ 60 μm was considerably greater in HA@Lip-Ce6@PBCO-treated samples than in free Ce6 or Lip-Ce6@PBCO teams (Fig. 3Okay). According to earlier experiences [45], these outcomes additionally demonstrated that HA could facilitate the entry of HA@Lip-Ce6@PBCO NPs into MCF-7/ADR cells through interplay with CD44. In conclusion, HA@Lip-Ce6@PBCO NPs display glorious concentrating on and penetration skills, which maintain nice promise for tumor remedy.

HA@Lip-CS-1@PBCO NPs successfully kill tumor cells in vitro

The cytotoxicity of HA@Lip-CS-1@PBCO NPs was initially assessed towards MCF-7/ADR cells. As depicted in Fig. 4A, therapy with HA@Lip-CS-1@PBCO NPs diminished cell viability to 50.5 ± 10%, considerably decrease than that achieved with PBCO NPs (95.5 ± 0.69%) or CS-1 alone (68.3 ± 5.5%). In the meantime, this type of NPs can considerably inhibit the formation of cell colony (Fig.S4A). Moreover, Stay/lifeless staining equally demonstrated essentially the most intense pink fluorescence in MCF-7/ADR cells handled with HA@Lip-CS-1@PBCO NPs, which mirrored the excessive cell demise charge. In distinction, tumor cells handled with PBCO NPs and CS-1 exhibited average pink fluorescence in comparison with the group handled with HA@Lip-CS-1@PBCO NPs. As a management group, nearly all cells confirmed inexperienced fluorescence after PBS therapy (Fig. 4B). According to these findings, FACS evaluation demonstrated that the cell demise charge in HA@Lip-CS-1@PBCO NPs handled cells was 80.46%, which is considerably greater than that of CS-1 handled cells (60.92%) (Fig. 4C-D). To increase these observations to a extra physiologically related mannequin, 3D spheroids have been employed. Following 5 days of therapy with HA@Lip-CS-1@PBCO NPs, spheroids exhibited unfastened and disintegrated morphology (Fig. 4E), and reside/lifeless staining confirmed predominant pink fluorescence, confirming low cell viability (Fig. 4F). Collectively, these outcomes spotlight the excellent tumor-killing capability of HA@Lip-CS-1@PBCO NPs in each two-dimensional and three-dimensional fashions.

Contemplating the development of efficacy of the nanodrug formulation, we additional explored the modifications within the genes of MCF-7/ADR earlier than and after therapy. RNA-seq evaluation revealed profound transcriptomic alterations in MCF-7/ADR cells following HA@Lip-CS-1@PBCO NPs therapy. Venn diagram in Fig.S4B recognized modifications of 12,208 mRNAs following therapy with HA@Lip-CS-1@PBCO NPs. In keeping with the usual of fold change ≥ 2 fold (p ≤ 0.05), HA@Lip-CS-1@PBCO NPs induced 1689 gene upregulation and 2298 gene downregulation in MCF-7/ADR cells (Fig.S4C). In the meantime, KEGG evaluation of those genes with reverse modifications revealed that HA@Lip-CS-1@PBCO NPs performs a major position within the regulation of mitochondrial function-related PI3K-Akt pathway [46], the inflammation-related TNF, NF-κB, and MAPK pathways and oxidative stress-related HIF-1 and FoxO pathways, in addition to the oxidative phosphorylation (OXPHOS) pathway (Fig. 4G). Amongst them, downregulation of OXPHOS-related genes (e.g., COX7C, ATP5F1D, MT-ND1, MT-ND4, and MT-ND6) strongly correlates with mitochondrial electron transport chain impairment, suggesting HA@Lip-CS-1@PBCO NPs induce oxidative stress through mitochondrial harm (Fig. 4H). Concurrent activation of HIF-1 (Fig. 4I) and FoxO pathways (Fig. 4J) additional helps this speculation, as each pathways are redox-sensitive and amplify ROS accumulation underneath mitochondrial dysfunction. Heatmap evaluation indicated the downregulation of antioxidant genes (NQO1, HSPA8, HSPD1, CAT) and mitochondrial fusion protein MFN1/MFN2, whereas upregulation of mitochondrial oxidative stress-related proteins (TNF, IL-18, IL-1β) in handled MCF-7/ADR cells (Fig. 4Okay). PPI evaluation signifies the central position of HIF-1α, connecting MFN1/MFN2 with TNF, which is related to Caspase-3-dependent pyroptosis (Fig. 4L). These findings collectively indicated that HA@Lip-CS-1@PBCO NPs can successfully kill MDR breast most cancers cells by inducing mitochondrial oxidative stress.

HA@Lip-CS-1@PBCO NPs activate pyroptosis through oxidative stress-induced mitochondrial harm

On condition that CS-1 induces pyroptosis in vitro, together with our RNA-seq information following HA@Lip-CS-1@PBCO NPs therapy, we investigated whether or not HA@Lip-CS-1@PBCO NPs may induce pyroptosis in MCF-7/ADR cells. The intense discipline pictures of cells handled with HA@Lip-CS-1@PBCO NPs revealed the cell swelling attribute of pyroptotic cells (Fig. 5A). Moreover, the staining results of T11 dyes, which may discriminate ruptured cell membrane from regular cell membrane, exhibited apparent membrane harm, cell swelling, and content material leakage in MCF-7/ADR cells handled with HA@Lip-CS-1@PBCO NPs (Fig. 5A). TEM corroborated these findings, demonstrating important plasma membrane harm within the HA@Lip-CS-1@PBCO NPs group (Fig. 5B). According to membrane disruption, PI staining—which selectively labels cells with compromised membranes—confirmed markedly elevated fluorescence depth in HA@Lip-CS-1@PBCO NPs-treated cells in comparison with PBS group (Fig. 5C). As plasma membrane integrity loss facilitates the discharge of cytosolic contents, we quantified lactate dehydrogenase (LDH) launch, an indicator of pyroptotic cytotoxicity. HA@Lip-CS-1@PBCO NPs therapy resulted in a major elevation of LDH launch (10.3 ± 2.3-fold enhance relative to PBS group), confirming profitable pyroptosis induction (Fig. 5D). Subsequently, we examined whether or not HA@Lip-CS-1@PBCO NPs-induced pyroptosis was depending on the activation of caspase-3/GSDME pathway. This end result was just like the Fig. 1 and RNA-seq information displaying that HA@Lip-CS-1@PBCO NPs-activated caspase-3 triggers pyroptosis by cleaving GSDME (Fig. 5E).

Subsequent, we endeavored to disclose the underlying course of by way of which HA@Lip-CS-1@PBCO NPs induces GSDME-mediated pyroptosis. Mixed with the RNA-seq outcomes, we investigated whether or not pyroptosis triggered by HA@Lip-CS-1@PBCO NPs necessitates mitochondrial dysfunction. Notably, TEM pictures revealed that HA@Lip-CS-1@PBCO NPs considerably enhanced mitochondrial swelling compared to the management cells (Fig. 5F). Moreover, mitochondrial fluorescence staining after therapy with HA@Lip-CS-1@PBCO NPs additionally confirmed extreme mitochondrial harm within the cells (Fig. 5G). Mitochondrial membrane potential (ΔΨm) collapse, typically related to mitochondrial harm, triggers cytochrome c (Cyt C) launch into the cytosol. Western blot evaluation confirmed a major enhance in Cyt C launch from mitochondria in HA@Lip-CS-1@PBCO NPs-treated cells (Fig. 5H). Evaluation of ΔΨm utilizing the JC-1 probe demonstrated essentially the most intense inexperienced fluorescence (indicative of depolarization) within the HA@Lip-CS-1@PBCO NPs group in comparison with different remedies (Fig. 5I). Given the established position of mitochondrial dysfunction in ROS technology and the reported affiliation between ROS and pyroptosis induction in most cancers inhibition [47], we hypothesized that HA@Lip-CS-1@PBCO NPs may elevate mobile ROS ranges. Certainly, each fluorescence imaging and movement cytometry evaluation confirmed a major upregulation of intracellular ROS in HA@Lip-CS-1@PBCO NPs-treated cells, exceeding ranges noticed with CS-1 or PBCO NPs alone (Fig. 5J).

Contemplating that mitochondria are a serious supply of ROS, we proceeded to analyze whether or not HA@Lip-CS-1@PBCO NPs induces the technology of mitochondrial ROS in MCF-7/ADR cells. As anticipated, HA@Lip-CS-1@PBCO NPs therapy triggered a major upregulation of mitochondrial ROS (Fig. 5Okay). To evaluate the ensuing oxidative stress and its influence on antioxidant defenses, we measured key biomarkers. First, mobile glutathione (GSH) ranges, a important antioxidant important for sustaining mitochondrial structural/useful integrity and defending mitochondrial DNA from oxidative harm, plummeted by 54.9 ± 0.9% in handled cells (Fig. 5L). This extreme depletion signifies a profound disruption of the mobile antioxidant system in MCF-7/ADR cells. Additional proof of mitochondrial harm was noticed: 1) Extracellular ATP launch, a marker of mitochondrial permeability transition, was considerably elevated in HA@Lip-CS-1@PBCO NP-treated cells in comparison with PBCO NP-treated controls (Fig. 5M); 2) Malondialdehyde (MDA) ranges, a well-established biomarker of lipid peroxidation and oxidative stress [48], elevated dramatically (8.7 ± 1.8-fold) within the NPs-treated MCF-7/ADR cells (Fig. 5N), confirming in depth oxidative membrane harm. Given the established hyperlink between oxidative stress sensing and the p62/Nrf2 pathway [49], we investigated their involvement. Western blot evaluation revealed that HA@Lip-CS-1@PBCO NPs considerably suppressed each p62 and Nrf2 protein ranges in MCF-7/ADR cells (Fig. 5O), suggesting impaired activation of the endogenous antioxidant response. Importantly, and critically for overcoming MDR [50, 51], HA@Lip-CS-1@PBCO NPs considerably downregulated key MDR-associated efflux pumps, together with P-glycoprotein (P-gp) and ABCG2 (Fig. 5P). This discount functionally impairs drug efflux, a major MDR mechanism. Collectively, these findings underscore the exceptional capacity of HA@Lip-CS-1@PBCO NPs to induce pyroptosis and circumvent drug efflux, thereby facilitating the elimination of multidrug resistant tumor cells (Fig. 5Q).

Pharmacokinetics and biodistribution of HA@Lip-Ce6@PBCO NPs in vivo

The pharmacokinetic profile of HA@Lip-Ce6@PBCO NPs was evaluated by monitoring Ce6 fluorescence depth in blood samples post-injection. As proven in Fig. 6A, blood samples confirmed a gradual lower in fluorescence depth following intravenous injection. The NPs exhibited considerably extended circulation, with a half-life (t₁/₂) of three.4 h in BALB/c nude mice – 2.3-fold longer than free Ce6 (1.5 h) (Fig. 6B). Actual-time fluorescence imaging revealed distinct biodistribution patterns. HA@Lip-Ce6@PBCO NPs gathered preferentially at tumor websites, with fluorescence depth of Ce6 and Lip-Ce6@PBCO NPs rising progressively and plateauing at 6 h post-injection. Tumor fluorescence depth within the HA-modified NP group constantly exceeded that of Lip-Ce6@PBCO NPs and free Ce6 at 6, 8, and 12 h (Fig. 6C), demonstrating HA-mediated lively concentrating on. In distinction, free Ce6 exhibited weak, quickly declining fluorescence as a result of non-specific distribution whereas the fluorescent depth of Lip-Ce6@PBCO NPs was greater than that within the free Ce6 group because of the EPR impact, and HA@Lip-Ce6@PBCO NPs exhibited the strongest tumor fluorescent depth. Ex vivo evaluation at 12 h post-injection confirmed fluorescence indicators primarily localized within the liver and lungs (Fig. 6D), attributable to hepatic metabolism and residual circulating NPs [52]. Notably, minimal cardiac fluorescence indicated diminished cardiotoxicity in comparison with free Ce6. Tumor fluorescence depth within the HA@Lip-Ce6@PBCO NPs group was 2.6 ± 0.6-fold greater than within the free Ce6 group, confirming enhanced tumor concentrating on.

We subsequent evaluated the ultrasound imaging potential of HA@Lip-Ce6@PBCO NPs in vivo. Following intravenous injection into tumor-bearing mice, ultrasound imaging revealed a detectable acoustic sign on the tumor web site, which was because of the stimulation of endogenous H₂O₂ within the tumor microenvironment (TME) triggering the discharge of CO from the NPs. This gasoline technology considerably amplified the intratumoral acoustic sign in comparison with pre-injection ranges (Fig. 6E). The pronounced sign enhancement demonstrates each profitable tumor-targeted supply of HA@Lip-CS-1@PBCO NPs and H₂O₂-responsive CO technology inside the TME. General, these findings spotlight the nanocomplexes’ sturdy concentrating on capacity and extended blood half-life, important for its therapeutic efficacy in animals.

HA@Lip-CS-1@PBCO NPs exhibit potent antitumor exercise towards multidrug-resistant most cancers in vivo

Constructing on their in vitro cytotoxicity and in vivo tumor-targeting capabilities, we evaluated the therapeutic efficacy of HA@Lip-CS-1@PBCO NPs in nude mice bearing MCF-7/ADR xenografts in line with the routine in Fig. 7A. In comparison with PBS group, all therapy teams (CS-1, PBCO NPs, HA@Lip-CS-1@PBCO NPs) confirmed considerably diminished tumor volumes (Fig. 7B). Ex vivo evaluation confirmed placing tumor development inhibition by HA@Lip-CS-1@PBCO NPs. Imaging and gravimetric quantification revealed a ultimate tumor weight of 20.2 ± 9.4 mg within the HA@Lip-CS-1@PBCO NP group versus 112.8 ± 58.6 mg in PBS group (Fig. 7C-D), translating to a tumor inhibition charge (TIR) of 82.2% ± 5.4%. This efficacy considerably surpassed each free CS-1 (TIR: 54.3% ± 5.2%) and PBCO NPs (TIR: 49.5% ± 15.2%) (Fig. 7E). Critically, no important physique weight reduction was noticed throughout any group, supporting the biocompatibility of the nanomaterials (Fig. 7F). Furthermore, H&E staining demonstrated in depth, demarcated tissue necrosis in all therapy teams (CS-1, PBCO NPs, HA@Lip-CS-1@PBCO NPs), contrasting sharply with the intact morphology and nuclear density of PBS-treated tumors (Fig. 7G). HA@Lip-CS-1@PBCO NPs therapy induced essentially the most pronounced discount in mobile proliferation, evidenced by the bottom Ki67 expression (Fig. 7G). Moreover, important upregulation of the pyroptosis executioner GSDME and the apoptosis-to-pyroptosis change marker cleaved caspase-3 was detected through immunofluorescence in HA@Lip-CS-1@PBCO NP-treated tumors (Fig. 7H-I). These outcomes display that HA@Lip-CS-1@PBCO NPs exhibit potent antitumor exercise towards MDR breast most cancers in vivo.

We additional assessed therapeutic exercise in an A2780/DDP (cisplatin-resistant) xenograft mannequin utilizing the identical routine (Fig. 7J). HA@Lip-CS-1@PBCO NPs achieved a ultimate tumor quantity of 38.0 ± 24.5 mm³, markedly smaller than these of PBS group (236.8 ± 59.5 mm³; Fig. 7Okay). Ex vivo tumor imaging and weight quantification corroborated this potent efficacy: HA@Lip-CS-1@PBCO NP-treated tumors weighed 42.3 ± 12.5 mg, representing a 67.1% discount in comparison with PBS group (128.7 ± 57.9 mg) (Fig. 7L-M). The corresponding TIR was 67.2% ± 6.0% (Fig. 7N). Physique weight remained secure (Fig. 7O), and histological analysis recapitulated the MCF-7/ADR findings: HA@Lip-CS-1@PBCO NP-treated A2780/DDP tumors exhibited important necrosis (Fig. 7P), minimal Ki67 staining (Fig. 7P), and robust induction of cleaved caspase-3 and GSDME attribute of pyroptosis (Fig. 7Q). These outcomes robustly display that HA@Lip-CS-1@PBCO NPs effectively inhibit the expansion of MDR breast tumors and exhibit potent, broad-spectrum exercise towards various MDR cancers in vivo.

Biocompatibility and biosafety analysis of HA@Lip-CS-1@PBCO NPs

Given the important significance of biocompatibility for scientific translation, we rigorously evaluated the security profile of HA@Lip-CS-1@PBCO NPs by way of a number of assays: hemolysis, platelet aggregation, cytotoxicity in regular cells, zebrafish embryo toxicity, and systemic toxicity in tumor-bearing mice. As proven in Fig.S5A-B, all examined formulations (PBS, CS-1, PBCO NPs, HA@Lip-CS-1@PBCO NPs) exhibited glorious blood compatibility, with hemolysis charges under the 5% security threshold after 6 h incubation with pink blood cells (RBCs). Moreover, HA@Lip-CS-1@PBCO NPs considerably suppressed platelet aggregation, as evidenced by an optical density (OD650 nm) worth of 94.8% in comparison with 48.6% for thrombin (Fig.S5C). MTT assays confirmed low cytotoxicity of HA@Lip-CS-1@PBCO NPs. Cell viability in VSMC, NIH/3T3, and H9c2 cells remained above 95% after 24 h publicity, considerably greater than free CS-1 and PBCO NPs in NIH/3T3 and H9c2 cells (Fig.S5D). As well as, zebrafish embryotoxicity testing revealed no important variations in physique size throughout therapy teams (Fig. 8A-B). Notably, HA@Lip-CS-1@PBCO NPs (122 beats per minute) mitigated the cardiotoxic impact noticed with free CS-1 (130 beats per minute), demonstrating a considerably decrease coronary heart charge (Fig. 8C). This means the nanoformulation successfully reduces inherent cardiac antagonistic results.

In vivo biosafety evaluation was important for evaluating the scientific feasibility of nanomedicines. We investigated the impact of HA@Lip-CS-1@PBCO NPs by performing complete blood cell rely and liver and renal perform evaluation. In MCF-7/ADR-bearing mice, HA@Lip-CS-1@PBCO NPs confirmed good systemic security profile. Full blood counts revealed no antagonistic results on RBC, PLT, or HGB ranges. Importantly, handled mice exhibited a major lower in white blood cell (WBC) rely in contrast with PBS, suggesting attenuation of the tumor-associated inflammatory response (Fig. 8D). Liver and kidney perform markers (ALT, AST, CRE, URE) remained inside regular ranges, confirming no hepatorenal toxicity (Fig. 8E). H&E staining of key organs (coronary heart, liver, spleen, lung, kidney) confirmed no notable lesions or morphological modifications (Fig. 8F). Collectively, these outcomes display that HA@Lip-CS-1@PBCO NPs possess glorious biocompatibility, successfully diminished cardiotoxicity in comparison with the free drug, and low systemic toxicity, supporting their potential for additional scientific improvement.

Cinobufagin loaded Prussian blue-like nanoparticles for chemo/gasoline remedy of multidrug resistant most cancers | Journal of Nanobiotechnology

CS-1 induces pyroptosis-mediated cytotoxicity throughout various MDR cells

Preparation and characterization of HA@Lip-CS-1@PBCO NPs

Purposeful characterization of HA@Lip-CS-1@PBCO NPs

HA@Lip-CS-1@PBCO NPs successfully kill tumor cells in vitro

HA@Lip-CS-1@PBCO NPs activate pyroptosis through oxidative stress-induced mitochondrial harm

Pharmacokinetics and biodistribution of HA@Lip-Ce6@PBCO NPs in vivo

HA@Lip-CS-1@PBCO NPs exhibit potent antitumor exercise towards multidrug-resistant most cancers in vivo

Biocompatibility and biosafety analysis of HA@Lip-CS-1@PBCO NPs

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