Abstract

   There is an important clinical need and social significance, especially
   for young patients, to explore a new breast-conserving strategy that is
   not dependent on biomarkers for anti-triple-negative breast cancer.
   Disulfiram, historically employed for the treatment of chronic
   alcoholism, has recently emerged as a promising antitumor agent in
   combination with Cu^2+. However, reported disulfiram–Cu^2+ codelivery
   regimens often suffer from instability as well as inadequate drug
   metabolism, which is detrimental to the production and action of the
   antitumor active ingredient copper(II) bis(diethyldithiocarbamate). To
   address this obstacle, this study tested nanosystems
   ICG-CuET@PLGA-CS-HA (IC@PCH) nanoparticles (NPs) carrying the
   chemotherapeutic agent copper(II) bis(diethyldithiocarbamate) and
   photosensitizer indocyanine green for the efficient delivery of
   antitumor drugs. Benefiting from the involvement of hyaluronic acid,
   the prepared IC@PCH NPs not only targeted CD44 on the surface of tumor
   cells but also showed a longer in vivo circulation time. The in vitro
   and in vivo results demonstrated that IC@PCH NP-mediated
   photothermal-chemotherapy treatment led to pyroptosis via the
   NLRP3/caspase-1 classical pathway, which had a significant therapeutic
   effect on triple-negative breast cancer. In addition, targeting IC@PCH
   NPs allows photoacoustic–magnetic resonance–fluorescence trimodal
   imaging, which is capable of detecting more insidious cancer foci and
   opens up new avenues for precise cancer diagnosis and treatment.

Introduction

   The latest World Health Organization statistics show that breast cancer
   (BC) has surpassed lung cancer globally for both incidence and
   mortality rates in the female population [[38]1]. Actors like genetics,
   environment, and lifestyle are driving BC prevalence up annually and at
   younger ages [[39]2,[40]3]. Triple-negative breast cancer (TNBC) is a
   subtype of BC that lacks the expression of the estrogen receptor, the
   progesterone receptor, and human epidermal growth factor receptor 2
   (HER2). Due to the lack of therapeutic targets, TNBC is insensitive to
   endocrine therapy as well as anti-HER2 targeted therapy treatment, and
   the prognosis is often poor [[41]4,[42]5]. This necessitates an
   emphasis on the diagnosis and treatment of middle-aged and young female
   TNBC patients. Current treatments involve surgery, radiation,
   chemotherapy, and neoadjuvant chemotherapy [[43]6,[44]7]. However,
   these typically lack specificity, causing emotional trauma of surgery
   and severe adverse effects from radiotherapy/chemotherapy
   [[45]8,[46]9]. Thus, to explore a new breast-conserving strategy,
   without being dependent on biomarkers, anti-TNBC has a marked clinical
   need and social significance for young patients.

   Disulfiram (DSF) has been approved by the Food and Drug Administration
   for the treatment of alcohol dependence since 1951 and was first found
   to have anticancer effects in 1977 [[47]10]. Since then, several
   studies have demonstrated that DSF has excellent tumor toxicity, with
   this efficacy being strongly contingent upon the presence of Cu^2+
   [[48]11]. In vivo, DSF is metabolized to dithiocarbamate, and its
   antitumor activity is greatly enhanced after liganding with Cu^2+ to
   form a dithiocarbamate–copper complex (copper(II)
   bis(diethyldithiocarbamate) [CuET]), considering the determining
   metabolite for the tumor-suppressing effect [[49]12,[50]13]. Several
   studies have demonstrated that DSF has excellent tumor toxicity and
   plays an important role in mitigating drug resistance, inhibiting
   angiogenesis, suppressing tumor metastasis, and modulating the immune
   microenvironment [[51]14,[52]15]. Despite the proven efficacy of DSF
   combined with Cu^2+, its in vivo transportation faces many challenges
   in practical use. Clinical trials of oral as well as intravenous
   DSF/Cu^2+ supplements have shown disappointing results due to the fact
   that DSF is highly unstable under the biological conditions of gastric
   acid and blood [[53]16]. What is more, to make matters worse, single
   treatments are often shown to be less effective [[54]17]. It is
   essential to probe into other effective and efficient strategies
   against TNBC.

   Nanomedicine is a new field of medicine that utilizes nanomaterials for
   disease diagnosis and treatment at the molecular level, with little
   invasiveness and side effects [[55]18–[56]21]. Using nanodrug delivery
   systems to deliver DSF-based drugs is an exciting choice. However,
   DSF/Cu^2+ codelivery nanosystems cannot ensure the efficient generation
   of the antitumor active ingredient CuET in vivo. This is due to the
   inconsistent pharmacokinetic profiles of Cu^2+ and DSF, and the
   formation of CuET in tumors is not efficient and selective, greatly
   impairing therapeutic efficacy [[57]22]. Meanwhile, most DSF-based
   delivery nanosystems are passively targeted to reach the tumor site
   just through the enhanced permeability and retention effect, which
   cannot achieve tumor site-specific accumulation ideally
   [[58]23,[59]24]. To overcome the above difficulties, we developed a
   nanoparticle (NP)-based targeted delivery system to increase the
   stability of CuET during systemic circulation by
   poly(lactic-co-glycolic acid) (PLGA) enclosure wrapped CuET. Based on
   previous studies, hyaluronic acid (HA) was loaded around the outermost
   layer of NPs, which specifically targeted CD44-overexpressing TNBC
   cells, without the systemic toxicity and solubility issues of
   chemotherapeutic drugs [[60]25,[61]26]. After precise targeting,
   combination therapy is carried out at the same time, which is expected
   to be anti-TNBC.

   Here, we report an ICG-CuET@PLGA-CS-HA (named IC@PCH) NP nanodrug
   delivery system, incorporating the chemotherapeutic drug CuET and the
   photosensitizer indocyanine green (ICG) (Fig. [62]1A). Studies suggest
   that chitosan (CS) is adhesive and positively charged, while HA
   specifically targets CD44-overexpressing 4T1 cells with high
   biocompatibility and nonimmunogenicity [[63]27,[64]28]. Thus,
   ICG-CuET@PLGA-CS NPs can attract HA to their surface via charge
   interaction, enhancing the particles’ tumor-targeting ability and
   extending their circulation time. After intravenous injection, the NPs
   bind to the CD44 receptor via HA and target aggregation at the tumor
   site. They disintegrate in the acidic microenvironment of the tumor,
   releasing CuET and ICG, and realize combined chemotherapy-photothermal
   therapy under laser irradiation for minimally noninvasive treatment of
   BC (Fig. [65]1B). Meanwhile, ICG and CuET endow NPs with photoacoustic
   (PA)–magnetic resonance (MR)–fluorescence imaging capabilities
   [[66]29–[67]31]. The NPs are targeted to reach the tumor cells, provide
   rich imaging information, and specifically suggest tumor lesions,
   promising to increase the cancer diagnosis rate and improve the
   prognosis. More importantly, we performed RNA sequencing (RNA-seq) and
   western blot to demonstrate that ICP@CH NP-mediated
   photothermal-chemotherapy treatment led to pyroptosis via the
   NLRP3/caspase-1 classical pathway. This demonstrates that this
   therapeutic modality is without dependent biological targets of TNBC
   and can effectively mediate cell death. In summary, this study reveals
   a superb strategy by creating an NP-based targeted delivery system for
   tumor microenvironment (TME)/laser-triggered photothermal-chemotherapy,
   effectively addressing the nonspecificity of endocrine/genetic
   treatment in TNBC.

Fig. 1.

   Fig. 1.
   [68]Open in a new tab

   (A) Diagram of the ICG-CuET@PLGA-CS-HA (IC@PCH) nanoparticle (NP)
   synthesis procedure. (B) Schematic illustration of the effect of IC@PCH
   NPs: photothermal-chemotherapy mediates tumor cell pyroptosis via the
   caspase-1 signaling pathway. ICG, indocyanine green; CuET, copper(II)
   bis(diethyldithiocarbamate); PLGA, poly(lactic-co-glycolic acid); PVA,
   polyvinyl alcohol; CS, chitosan; HA; hyaluronic acid; MRI, magnetic
   resonance imaging; PAI, photoacoustic imaging; NIR, near-infrared;
   DAMPs, damage-associated molecular patterns; GSDMD-N, N-terminal
   fragment of GSDMD; PTT, photothermal therapy; FLI, fluorescence
   imaging.

Materials and Methods

Materials

   ICG and polyvinyl alcohol (PVA) were obtained from Sigma-Aldrich (St.
   Louis, MO, USA). CuET was obtained from TCL (Shanghai, China).
   Carboxyl-PLGA (COOH–PLGA) (molecular weight 12,000 Da, 50:50) was
   obtained from Daigang Biomaterial (Jinan, China). CS and HA were
   purchased from Macklin (Shanghai, China). All other reagents and
   materials were directly obtained from the lab and were commercially
   available.

Preparation of IC@PCH NPs

   ICG-CuET@PLGA (IC@P) NPs and IC@PCH NPs were prepared using the
   double-emulsification method. In a 50-ml centrifuge tube, 50 mg of
   PLGA, 5 mg of CuET, and 2 ml of dichloromethane were added and
   processed using an ultrasonic oscillator for 30 s to dissolve them
   completely. Subsequently, 4 mg of ICG was dissolved in 200 μl of
   ultrapure water and added to the above mixture and emulsified using a
   sonicator (VCX130; Sonics and Materials, USA) at 100 W for 3 min to
   obtain the primary emulsion. An 8-ml volume of 5% (w:v) PVA was
   introduced into the primary emulsion and emulsified for 2 min to obtain
   a complex emulsion. To obtain the IC@P NP solution, isopropanol was
   added to the complex emulsion and evaporated with stirring to evaporate
   the dichloromethane. Finally, the NP solution was washed 3 times by
   high-speed centrifugation, resuspended in pure water, and stored in a 4
   °C refrigerator protected from light.

   To coat HA, we first prepared ICG-CuET@PLGA-CS NPs. The preparation
   procedure was the same as for IC@P NPs except that 8 ml of 5% PVA
   solution was replaced by 3 ml of 5% PVA solution and 3 ml of 2% CS,
   which had been dissolved in 3% acetic acid. Then, 10 mg of HA was
   added, shaken on a shaker for 1 h, washed 3 times, and resuspended in
   pure water to obtain an aqueous solution of IC@PCH NPs.

Characterization of IC@PCH NPs

   The morphological features of NPs were observed by scanning electron
   microscopy (Hitachi SU8010, Japan) and low-pressure transmission
   electron microscopy (TEM; FEI Tecnai G2 12, USA). The composition of
   the NPs was analyzed by an ultraviolet (UV)–visible–near-infrared (NIR)
   spectrometer (Shimadzu U4000, Japan), a Fourier transform infrared
   spectrometer (Nicolet iS10, USA), an x-ray diffractometer (Bruker D8
   Advance, Germany), and an x-ray photoelectron spectrometer (Thermo
   Escalab 250XI, USA) to analyze the composition. The particle size and
   potential were analyzed by a particle size potentiostat, and the 1-week
   stability was analyzed.

CuET release

   Equal amounts of IC@PCH NP solutions were separately packed into
   dialysis bags (molecular weight cutoff 3,500, soluble) and randomly
   divided into 2 groups (n = 3). The samples of the 2 groups were placed
   in phosphate-buffered saline (PBS) buffer medium (30% ethanol, 0.02%
   sodium azide, and 0.01% Tween-80) at pH = 7.4 and pH = 6.5,
   respectively. A blue-necked bottle containing NPs and buffer medium was
   placed in a thermostatic oscillator, and 1 ml of buffer medium was
   collected at the time points of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
   and 12 h. After each sample collection, another 1 ml of fresh buffer
   medium was added to the blue-necked bottle to ensure a constant volume
   of buffer medium. The samples obtained above were analyzed by
   high-performance liquid chromatography, and the cumulative release rate
   of CuET was deduced from the standard curve.

In vitro photothermal effect of IC@PCH NPs

   PBS and different concentrations of IC@PCH NPs (0.5, 1, 2, 4, and 6
   mg/ml) were irradiated under an 808-nm laser (1.5 W/cm^2) for 6 min to
   explore the effect of concentration on the warming effect (n = 3); 2
   mg/ml IC@PCH NPs were irradiated under different intensities of an
   808-nm laser (0.5, 1.0, 1.5, and 2 W/cm^2) for 6 min to explore the
   effect of laser intensity on the warming effect (n = 3). A 10 μg/ml
   solution of ICG, CuET, and PLGA was exposed to a 1.5 W/cm^2 laser to
   explore the source of the photothermal conversion performance of the
   NPs (n = 3). The IC@PCH NP solution and free ICG solution were exposed
   to a 1.5 W/cm^2 laser 4 times to observe the effect of the laser
   irradiation and cooling process on the temperature. The warming process
   was recorded by infrared thermography.

Cell lines

   4T1 mouse BC cells and MCF-10A cells were purchased from Procell
   (Wuhan, China). Human umbilical vein endothelial cells (HUVECs) were
   obtained from the Department of Ultrasound and Chongqing Key Laboratory
   of Ultrasound Molecular Imaging. 4T1 cells and HUVECs were cultured in
   complete RPMI 1640 (Procell). MCF-10A cells were cultured in MCF-10A
   cell-specific medium (Procell). All cells were cultured in a 37 °C
   incubator with 5% CO[2].

Intracellular endocytosis of IC@PCH NPs

   4T1 cells and MCF-10A cells were inoculated in laser copolymerization
   dishes for 24 h. Then, 2 mg/ml of IC@P-DiI NPs, and IC@PCH-DiI NPs were
   added to the 4T1 cell group and MCF-10A cell group, respectively,
   followed by coincubation for 1, 2, and 3 h. Each group was fixed by
   adding paraformaldehyde and then washed with PBS, and finally the cells
   (blue) were observed to phagocytose the NPs (red) under a laser
   confocal microscope (Nikon, Japan). Each group was incubated with
   different NPs, and then cells were washed, harvested, centrifuged, and
   resuspended. The cells were analyzed for phagocytosis in each group by
   flow cytometry analysis.

In vitro safety evaluation and efficacy evaluation

   HUVECs and MCF-10A and 4T1 cells were inoculated in 96-well plates and
   were incubated with different concentrations of IC@PCH NPs (0, 0.25,
   0.5, 1, 2, and 5 mg/ml) for 24 h. The cellular activity was then
   determined by Cell Counting Kit-8 (CCK-8) assay.

   4T1 cells were inoculated in 96-well plates, and they were divided into
   9 groups, namely, the control (PBS) group, laser-only (PBS + laser)
   group, free CuET group, DSF@PLGA-CS-HA (D@PCH) NPs + CuCl[2]@PLGA-CS-HA
   (Cu^2+@PCH) NPs group, IC@PCH NPs group, CuET@PLGA-CS-HA (C@PCH) NPs
   group, ICG@PLGA-CS-HA (I@PCH) NPs + laser group, IC@P NPs + laser
   group, and IC@PCH NPs + laser group. All drugs were used in 3
   concentrations (0.5, 1, and 2 mg/ml), and the laser intensity was
   controlled at 1.5 W/cm^2. After the treatment, the cell viability of
   each group was determined using the CCK-8 method.

   4T1 cells were plated in a 12-well plate and incubated for 24 h before
   9 treatments were applied. Following 3 washes with PBS, each well
   received 500 μl of calcein-AM and propidium iodide (PI) working
   solution to stain live (green) and dead (red) cells. After 30 min, 3
   more washes were performed followed by observation under fluorescence
   microscopy (Olympus CKX53, Japan). 4T1 cells were plated in a 6-well
   plate and incubated for 24 h before 7 treatments. Posttreatment, cells
   were washed thrice with PBS, harvested via trypsin digestion,
   centrifuged at 2,000 rpm for 5 min, and resuspended in PBS containing
   annexin V–fluorescein isothiocyanate (5 μl) and PI (10 μl). Cells were
   then analyzed for apoptosis through flow cytometry (CytoFLEX,
   California, USA).

Mechanistic investigation of IC@PCH NPs in inducing photothermal-chemotherapy

   The 4T1 cells were treated with 2 mg/ml of IC@PCH NP solution as well
   as 1.5 W/cm^2 of an 808-nm laser, and then the morphology of cells was
   observed under a light microscope. Next, 4T1 cells were divided into
   the control and IC@PCH NPs + laser groups (n = 3) and were subjected to
   transcriptome sequencing and bioinformatic analysis after treatment.
   Differentially expressed genes (DEGs) with fold change >2 and P value
   <0.05 were screened out. Functional enrichment analysis of DEGs was
   performed utilizing the Gene Ontology framework. In addition, the Kyoto
   Encyclopedia of Genes and Genomes (KEGG) was employed to pinpoint
   pathways that exhibited significant associations with the identified
   DEGs.

Western blot validation of the screened NLRP3 signaling pathway

   4T1 cells were inoculated into 6-well plates and grouped for treatment.
   The groupings were control, laser-only, C@PCH NPs, I@PCH NPs + laser,
   and IC@PCH NPs + laser groups. Proteins were extracted from the cells
   and separated by sodium dodecyl sulfate–polyacrylamide gel
   electrophoresis. Then, they were transferred to a polyvinylidene
   fluoride membrane (Bio-Rad, USA) and blocked. Target proteins were
   detected using specific antibodies, including NLRP3, caspase-1, cleaved
   caspase-1, GSDMD, N-terminal fragments of GSDMD (GSDMD-N), and β-actin
   (Proteintech). Stained bands were observed by a chemiluminescent
   detection system and analyzed in terms of grayscale intensity utilizing
   the ImageJ software. The scheme illustrating the mechanism was drawn
   using BioRender.

Animals

   All animal experiment protocols were approved by Institutional Animal
   Care and Use of Chongqing Medical University (approval number:
   IACUC-CQMU-2024-0594). Female BALB/c mice (8 weeks) were obtained from
   the Laboratory Animal Center of Chongqing Medical University; 4 × 10^6
   4T1 cells were dispersed in a 100-μl solution and injected into the
   right dorsal side of BALB/c mice to establish a mouse hormonal tumor
   model. Treatment was started when the tumor grew to 45 to 75 mm^3. The
   tumor volume was calculated using the formula [1/2 × length ×
   (width)^2].

In vivo biosafety

   Eight-week-old female BALB/c mice (n = 15) were selected and grouped
   according to 20 d–15 d–10 d–5 d–pre time points (n = 3 per time). Each
   group was injected with 8 mg/ml NPs through the tail vein at a dose of
   200 μl each. Animal blood collection operation was carried out after 20
   d for routine blood (white blood cells, lymphocytes, red blood cells,
   hemoglobin, mean corpuscular volume, and platelets) and blood
   biochemistry examination (alanine aminotransferase, aspartate
   aminotransferase, urea, creatine kinase, creatine kinase-MB, and
   lactate dehydrogenase 1). The major organs of mice were collected for
   hematoxylin–eosin (H&E) staining.

In vivo photothermal capacity and photothermal-chemotherapy

   The BALB/c mice were divided into the laser-only (PBS + laser) group,
   IC@P NPs + laser group, I@PCH NPs + laser group, and IC@PCH (ICG-CuET
   @PLGA-CS-HA) NPs + laser group. After the mice in each group were
   injected with NPs (5 mg/ml, 200 μl) for 2 h, the tumors were irradiated
   with a 1.5 W/cm^2 laser for 6 min, and the warming process of the tumor
   site was recorded by using an infrared thermography camera.

   Twenty-eight mice were randomly divided into 7 groups: the control
   (PBS) group, laser-only (PBS + laser) group, D@PCH NPs + Cu^2+@PCH NPs
   group, IC@PCH NPs group, C@PCH NPs group, I@PCH NPs + laser group, and
   IC@PCH NPs + laser group, with 4 mice per group. Two hours after tail
   vein injection (5 mg/ml, 200 μl), the tumors were irradiated with an
   808-nm laser (1.5 W/cm^2) for 6 min, and the entire course of treatment
   was performed only once. The tumor size and body weight of the mice
   were measured every other day throughout the treatment cycle. The mice
   were executed after 20 d of treatment, and the tumors were collected
   for H&E staining, terminal deoxynucleotidyl transferase dUTP nick end
   labeling (TUNEL) fluorescence staining, and proliferating cell nuclear
   antigen (PCNA) fluorescence staining.

In vitro and in vivo multimodality imaging

   CuET, ICG, and IC@PCH NP solutions (1.2 mg/ml) were injected into the
   agarose gel model, respectively, at 200 μl per well. The optimal
   excitation wavelength was explored by irradiating with a 690- to 960-nm
   laser using a PA imager (Visual Sonics Inc., Toronto, Canada).
   Different concentrations (0.4, 0.6, 0.8, 1.0, and 1.2 mg/ml) of IC@PCH
   NP solutions were injected into the agarose gel model and were
   irradiated by the laser at the optimal excitation wavelength to observe
   the PA signal values. Different concentrations (0.4, 0.6, 0.8, 1.0, and
   1.2 mg/ml) of Cu^2+ and IC@PCH NP solutions were injected into the
   agarose gel model, and the MR signal values were recorded. Different
   concentrations (0.4, 0.6, 0.8, 1.0, and 1.2 mg/ml) of IC@PCH NP
   solutions were injected into 48-well plates, and the fluorescence
   signal values were observed. IC@P NP, IC@PCH NP, ICG@PCH NP, and
   CuET@PCH NP solutions (5 mg/ml, 200 μl) were injected into mice through
   the tail vein, and PA imaging of the tumor site was performed before
   and 1 h–2 h–3 h–6 h–12 h after the injection, and the signal values of
   the tumor area were recorded. IC@P NP and IC@PCH NP solutions (5 mg/ml,
   200 μl) were injected into mice through the tail vein, and magnetic
   resonance imaging (MRI) of mice was performed before and 1 h–2 h–3 h–6
   h–12 h after the injection, and the signal values of the tumor area
   were recorded. IC@P NP and IC@PCH NP solutions (5 mg/ml, 200 μl) were
   injected into mice, and PA imaging of the tumor site was performed
   before and 0.5 h–1 h–2 h–3 h–6 h after the injection, and the signal
   values of the tumor area were recorded.

Biodistribution in vivo

   IC@P NPs and IC@PCH NPs (5 mg/ml, 200 μl) were injected into 4 T1
   hormonal mice through the tail vein. Major organs (including the heart,
   liver, spleen, lungs, and kidneys) and tumors were collected 24 h after
   injection. The biodistribution of IC@P NPs and IC@PCH NPs was measured
   ex vivo using a Xenogen IVIS spectral imaging system (IVIS Lumina III,
   PerkinElmer, Waltham, MA).

Statistical analysis

   All data were analyzed using GraphPad Prism 9.0, presented as mean ±
   standard deviation (SD). Calculations were performed using a 2-tailed
   Student t test. P values less than 0.05 represent statistically
   significant differences.

Results and Discussion

Preparation and characterization of IC@PCH NPs

   IC@PCH NPs were successfully synthesized using a simple 2-emulsion
   method, and the synthesis process is shown in Fig. [69]2A. These IC@PCH
   NPs exhibit spherical morphology with a smooth surface and a uniform
   particle size as depicted by scanning electron microscopy (Fig.
   [70]2B). A more obvious halo ring surrounding the surface of IC@PCH NPs
   is observed under TEM compared to IC@P NPs (Fig. [71]2C and D),
   verifying successful HA/CS decoration. Figure [72]2E reveals that the
   I@P NPs appear green, C@P NPs are brownish, and IC@PCH NPs and IC@P NPs
   exist somewhere between these 2 color schemes, signifying the
   successful incorporation of both IC@PCH and IC@P with both ICG and
   CuET. IC@PCH NPs exhibit superior greenness compared to IC@P NPs,
   suggesting an increase in ICG loading after HA/CS incorporation,
   possibly due to the negative charge of ICG being attracted by the
   positive CS. The particle size potentiostat results showed that the
   particle size of the IC@P NPs was about 219.8 nm (polydispersity index
   [PDI] = 0.048), and the size was increased to 248.4 nm (PDI = 0.108)
   and 259.2 nm (PDI = 0.119) sequentially after loading of CS and HA,
   which was in agreement with the TEM results (Fig. [73]2F and Table
   [74]S1). In pure aqueous solution at pH 7.4, the ζ charge of IC@P NPs
   was −17.67 mV, which changed to 21.57 mV after loading CS and reversed
   to −16.17 mV after continued loading of HA, indicating that HA was
   loaded by positive and negative charge attraction (Fig. [75]2F).

Fig. 2.

   Fig. 2.
   [76]Open in a new tab

   Synthesis and characteristics of IC@PCH NPs. (A) A schematic
   illustration depicting the synthetic pathway of IC@PCH NPs. Scanning
   electron microscopy (SEM) images of (B) IC@PCH NPs. Transmission
   electron microscopy (TEM) images of (C) ICG-CuET@PLGA (IC@P) NPs and
   (D) IC@PCH NPs. (E) Digital images of I@P NP, C@P NP, IC@P NP, and
   ICP@CH NP solutions. (F) Size distribution and ζ potential (inset) of
   IC@P NPs, IC@PC NPs, and IC@PCH NPs. (G)
   Ultraviolet–visible–near-infrared (UV–Vis–NIR) absorption spectra of
   ICG solution, CuET solution, IC@P NPs, and IC@PCH NPs. (H) Fourier
   transform infrared (FTIR) spectra of IC@PCH NPs. (I) X-ray diffraction
   (XRD) of IC@PCH NPs and CuET. (J and K) High-resolution Cu 2p and S 2p
   XPS spectra of IC@PCH NPs.

   Subsequently, we analyzed the drug loading performance of IC@PCH NPs
   using UV spectroscopy. The results showed that the IC@PCH NPs had
   obvious absorption peaks at 263, 545, and 699 nm, which proved the
   successful loading of ICG and CuET (Fig. [77]2G). The UV absorption
   peaks of ICG were shifted from 713 and 780 nm to 545 and 699 nm, and
   the UV absorption peaks of CuET were shifted from 437 to 263 nm, which
   may be due to the spatial site resistance effect [[78]32]. Based on the
   UV spectroscopy and standard curves of ICG and CuET (Fig. [79]S1A to
   D), it was determined that the encapsulation rate of ICG in IC@PCH NPs
   was about 52.40% with a loading rate of about 7.33%, and the
   encapsulation rate of CuET in IC@PCH NPs was about 34.40% with a
   loading rate of about 6.01%. In addition, the successful incorporation
   of CuET was confirmed through Fourier transform infrared spectroscopy,
   which exhibited characteristic peaks in the range of 1,020 to 1,200
   cm^−1 (C=S), indicative of the stretching vibrations associated with
   CuET (Fig. [80]2H). This analysis provides additional verification of
   the successful loading process. The absorption peak of the unsaturated
   group C=O double bond at 1,747 cm^−1 was from PLGA. Notably, no
   markedly changes were detected between the x-ray diffraction patterns
   of IC@PCH and CuET, suggesting that the crystal structure of CuET
   remained unchanged after being loaded into NPs (Fig. [81]2I). Through
   x-ray photoelectron spectroscopy (XPS), we assessed the chemical state
   of Cu and S. The binding energy centers at 932.3 and 952.8 eV
   correspond to high-resolution XPS peaks of Cu 2p[3/2] and Cu 2p[1/2],
   respectively (Fig. [82]2J). The binding energy centers of S 2p[3/2] and
   S 2p[1/2] were located at 161.5 and 162.5 eV, respectively, which
   indicated the presence of sulfides and disulfides (Fig. [83]2K). These
   findings align with the XPS data of CuET nanocrystals.

   The stability and responsiveness of IC@PCH NPs to the release of CuET
   were particularly to be verified in subsequent experiments. After
   storing the aqueous solution of IC@PCH NPs at 4 °C for 1 week, no
   significant change in average particle size or potential was observed,
   and no aggregation or precipitation occurred within 7 d (Fig. [84]S2A),
   highlighting the exceptional long-term stability of the NPs obtained.
   NPs’ release of CuET is significantly faster in pH 6.5 buffer compared
   to its release in a typical neutral pH environment (pH = 7.4) (Fig.
   [85]S2B). This is owing to PLGA’s solubility under acidic conditions
   [[86]33]. Therefore, NPs can release drugs triggered by the TME and
   thus exhibit antitumor activity. Meanwhile, this slow-release property
   facilitates the continuation of chemotherapy at the tumor site.

In vitro photothermal effect of IC@PCH NPs

   Given ICG’s high absorption in the NIR region, this study examined the
   photothermal conversion performance of IC@PCH NPs in an aqueous
   solution. The infrared thermograms at varying IC@PCH NP concentrations
   and laser powers are shown in Fig. [87]3A. Then, Fig. [88]3B shows that
   the temperature of the IC@PCH NP aqueous solution significantly
   increases upon 808-nm laser irradiation (1.5 W/cm^2, 6 min), exhibiting
   a concentration-dependent pattern. Furthermore, evident laser power
   dependence appeared during NIR irradiation for 6 min, suggesting easy
   control of sample temperature by adjusting laser power (Fig. [89]3C).
   The heat curve analysis suggested that IC@PCH NP’s photothermal
   properties are primarily driven by ICG, with limited heating effect
   from CuET (Fig. [90]3D). Furthermore, compared to free ICG, IC@PCH NPs
   exhibited superior photothermal stability during 4 heating and cooling
   cycles, possibly due to the enhanced photostability provided by coating
   modifications of ICG and CuET in polymer materials (Fig. [91]3E).
   Through calculations, the photothermal conversion efficiency of IC@PCH
   NPs was calculated at 20.23% (Fig. [92]3F). These findings suggest that
   as a photothermal agent, IC@PCH NPs efficiently convert light energy
   into thermal energy.

Fig. 3.

   Fig. 3.
   [93]Open in a new tab

   In vitro photothermal performance of IC@PCH NPs. (A) Infrared
   thermograms of IC@PCH NPs under 808-nm laser irradiation at different
   concentrations, powers, and times. (B) Heating curves of varying
   concentrations of IC@PCH NPs by 1.5 W/cm^2. (C) Heating curves of
   IC@PCH NP aqueous dispersion (2 mg/ml) exposure at varying power
   densities. (D) ICG, CuET, and PLGA’s photoheating curves at 10 μg/ml
   concentration. (E) Heating curve of IC@PCH NPs and ICG aqueous
   dispersion under 4 cycles of heating and subsequent cooling processes.
   (F) The time constant was derived from the duration of the cooling
   phase within these cycles. PBS, phosphate-buffered saline.

Intracellular endocytosis of IC@PCH NPs

   Utilizing confocal laser scanning microscopy, we evaluated the cellular
   uptake efficiency of IC@PCH and ICP NPs, which were decorated with
   1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate
   (DiI, red), and cell nuclei were identified using
   4′,6-diamidino-2-phenylindole (blue). Despite ICG’s inherent
   fluorescence, DiI was utilized because it is within the range of
   confocal laser scanning microscopy and flow cytometry
   excitation/emission wavelengths (the ICG emission wavelength is 780 nm,
   the excitation wavelength is 820 nm, the DiI emission wavelength is 565
   nm, and the excitation wavelength is 549 nm) [[94]34]. The CD44
   receptor is a cell surface glycoprotein receptor with dual roles and
   expression during cellular processes and is particularly important for
   the targeting properties of HA-coated NPs [[95]35]. Under normal
   physiological conditions, CD44s are predominantly expressed and
   involved in the maintenance of tissue homeostasis and repair functions
   (e.g., low levels of CD44 expression in HUVECs play a protective role
   by stabilizing the endothelial barrier), whereas under pathological
   conditions (e.g., TNBC), expression of CD44v6 is significantly
   up-regulated, promoting disease progression by driving
   epithelial–mesenchymal transition and tumor stem cell properties
   [[96]36,[97]37]. Molecularly, the Link module of the CD44 receptor
   binds specifically to the hexasaccharide unit of HA via a hydrogen
   bonding network [[98]28]. These molecular characteristics directly
   informed our NP design. Experiments included 4 groups: (1) MCF-10A
   cells + IC@P NPs, (2) MCF-10A cells + IC@PCH NPs, (3) 4T1 cells + IC@P
   NPs, and (4) 4T1 cells + IC@PCH NPs.

   As incubation time advanced, the red dots representing NPs were
   progressively engulfed by cells. After 3 h of culture, DiI-labeled
   IC@PCH NPs rapidly congregated around the membrane of the group 4 4T1
   cells and infiltrated within these. Only limited IC@P NPs were observed
   to accumulate on MCF-10A cells in group 3. Conversely, no significant
   aggregation of IC@PCH NPs was detected near MCF-10A cells in group 1
   and group 2 (Fig. [99]4A). This may be attributed to the
   ligand–receptor interaction between HA-coated NPs and
   CD44-receptor-overexpressing 4T1 cells. Meanwhile, there was no
   specific binding of receptor ligands in groups 1, 2, and 3, but NPs
   were endocytosed more in the IC@P NPs + 4T1 cell group than in the
   MCF-10A cells, probably due to the more vigorous metabolism and active
   phagocytosis process in tumor cells. Flow cytometry data (Fig. [100]4B
   and C) show that after 3 h, the fluorescence intensity of IC@PCH NPs in
   4T1 cells is 90.99%, compared to 57.08% for IC@P NPs. The fluorescence
   intensity of IC@P NPs and IC@PCH NPs in MCF-10A cells at the third hour
   was only 25.85% and 30.11%, respectively. These findings suggest the
   CD44 receptor’s critical role in the endocytosis of HA-functionalized
   NPs in 4T1 cancer cells. IC@PCH NPs can target cancer cells
   overexpressing CD44 receptors, potentially facilitating targeted cancer
   therapy.

Fig. 4.

   Fig. 4.
   [101]Open in a new tab

   Intracellular endocytosis of IC@PCH NPs. (A) CLSM analysis (scale bars:
   100 μm) and (B) FCM analysis of intracellular uptake of DiI-labeled
   IC@P and IC@PCH NPs in 4T1 and MCF-10A cells for 1, 2, and 3 h. (C)
   Quantitative analysis of (B) (n = 3). The results are presented as mean
   ± standard deviation (SD). ****P < 0.0001. ns, not significant; CLSM,
   confocal laser scanning microscopy; FCM, flow cytometry; DiI,
   1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate.

In vitro photothermal-chemotherapy treatment against 4T1 cells

   Cell viability was examined by standard CCK-8 assay to validate IC@PCH
   NPs for the safety and efficacy of photothermal-chemotherapy treatment
   in vitro (Fig. [102]5A). As shown in Fig. [103]5B, there was no
   significant decrease in the viability of HUVECs after 24 h of
   coincubation with 5 mg/ml IC@PCH NPs compared with that of the control
   group, indicating that the injection of 5 mg/ml IC@PCH NPs was almost
   nontoxic to the blood vessels and had good biocompatibility. Compared
   with the control group, MCF-10A cells showed no significant decrease in
   survival after 24 h of coincubation with 2 mg/ml IC@PCH NPs, indicating
   that 2 mg/ml ICP@CH NPs at the tumor treatment site had no significant
   toxicity to normal breast cells. Exposure to 4T1 cells at IC@PCH NP
   concentrations of 2 and 5 mg/ml reduced their viability to 69.14% and
   56.24% respectively, revealing potential tumor cell toxicity dependent
   on NP concentration. Meanwhile, as 4T1 cells show a higher level of
   CD44 expression compared to normal cells, the endocytosis of IC@PCH NPs
   by 4T1 cells is promoted, thereby resulting in a greater killing
   potency. Next, the anticancer effect of each treatment group on 4T1
   cells was evaluated. As illustrated in Fig. [104]5C, the cell viability
   in the IC@PCH NPs + laser group was lower than that in both the C@PCH
   NPs (no ICG) and I@PCH NPs (no CuET) + laser groups. This indicates a
   synergistic anticancer effect of photothermal therapy and chemotherapy.
   Under laser irradiation, the cytotoxicity of IC@PCH NPs is higher than
   that of IC@P NPs, indicating that targeting NPs enhances anticancer
   efficacy. This could be attributed to the CD44-receptor-mediated
   internalization of more IC@PCH NPs by 4T1 cells, thereby potentiating
   photothermal therapy and chemotherapy efficacy. Given the superior
   biosecurity of the 808-nm laser, only the laser group exhibited low
   cytotoxicity. Due to the direct use of CuET skipping the reaction loss
   between DSF and Cu^2+, the cell survival rate of the C@PCH group is
   lower than that of the D@PCH NPs + Cu^2+@PCH NPs group. The higher cell
   survival of the IC@PCH NPs group compared to that of the IC@PCH NPs +
   laser group demonstrates the good safety and controllability of IC@PCH
   NPs; i.e., the strong antitumor properties are triggered after laser
   irradiation. Compared to IC@PCH NPs, C@PCH NPs have higher cytotoxicity
   because their CuET loading rate is higher, resulting in more CuET
   entering tumor cells. Additionally, short-term tumor cell toxicity was
   reduced with C@PCH NPs compared to that with the free CuET group,
   likely due to PLGA nanoshell’s pH-dependent sustained release property,
   conferring a milder yet enduring antitumor capacity. Lastly, similar
   results were obtained using different concentrations of NPs (0.5, 1,
   and 2 mg/ml), with the efficacy escalating with concentration.

Fig. 5.

   Fig. 5.
   [105]Open in a new tab

   In vitro photothermal chemotherapy treatment against 4T1 cells. (A) A
   schematic illustration depicting the utilization of IC@PCH NPs for
   photothermal chemotherapy treatment against 4T1 cells. (B) Cytotoxicity
   against human umbilical vein endothelial cells (HUVECs) and MCF-10A and
   4T1 cells after incubation with IC@PCH NPs for 24 h; n = 3. (C) The
   anticancer efficacy of various therapies against 4T1 cells; n = 3. (D)
   Live and dead fluorescence images of 4T1 cells in different treatment
   groups (scale bar: 50 μm). (E) Flow cytometry apoptosis assay stained
   with annexin V–fluorescein isothiocyanate (FITC) and propidium iodide
   (PI) after varying treatments. The results are presented as mean ± SD.
   **P < 0.01; ***P < 0.001; ****P < 0.0001.

   As shown in the fluorescence microscope image (Fig. [106]5D), both the
   C@PCH NPs and I@PCH NPs + laser groups revealed modest red
   fluorescence, suggesting limited cytotoxicity via chemo + targeted
   treatment, photothermal + targeted treatment. The prominent red
   fluorescence signal was observed when 4T1 cells were co-cultured with
   IC@P NPs or IC@PCH NPs followed by laser irradiation. Comparable
   outcomes were noted in the annexin V–fluorescein isothiocyanate/PI
   staining assay (Fig. [107]5E). Laser-irradiated IC@P NPs and IC@PCH NPs
   evoke high levels of apoptosis, with the cytotoxic potency of IC@PCH
   NPs marginally higher than that of IC@P NPs. These findings validate
   the necessity of a photothermal + chemo + targeted strategy for tumor
   eradication. It should be noted that although IC@P NPs and IC@PCH NPs
   showed similar killing ability in tumor cells, considering the
   biocompatibility and targeting of HA, IC@PCH NPs may be more
   advantageous in in vivo circulation.

Mechanistic investigation of IC@PCH NPs in inducing photothermal-chemotherapy

   It is worth noting that the cells after treatment showed a variety of
   morphological damage of tumor cells mediated by IC@PCH NPs, and the
   “balloon-like” changes were observed under the light microscopy, and
   the cells were enlarged and burst (Fig. [108]6A). To further
   investigate the mechanism of action of IC@PCH NP-mediated
   photothermal-chemotherapy, RNA-seq and bioinformatic analysis of 4T1
   cells in the control group and IC@PCH NPs + laser group was performed.
   In the heat map, blue denoted genes that were down-regulated, while red
   indicated genes that were up-regulated (Fig. [109]6B). The volcano plot
   displayed that, in comparison to the control group, the IC@PCH NPs +
   laser group exhibited significant up-regulation of 547 genes and
   notable down-regulation of 668 genes (log[2]FC ≥ 1, P ≤ 0.05 for
   up-regulation; log[2]FC ≤ −1, P ≤ 0.05 for down-regulation; otherwise
   no differential expression) (Fig. [110]6C). To better understand the
   major metabolic and signaling pathways of DEGs in the IC@PCH NPs +laser
   group, we conducted KEGG pathway enrichment analysis. The results
   highlighted the involvement of insulin resistance, allograft rejection,
   cell-death-related pathways, and the NLRP3 signaling pathway in the
   therapeutic mechanism of IC@PCH NPs (Fig. [111]6D). Some studies have
   shown that the NLRP3 signaling pathway is closely related to a new mode
   of death, which is pyroptosis [[112]38,[113]39].

Fig. 6.

   Fig. 6.
   [114]Open in a new tab

   Mechanistic investigation of IC@PCH NPs in inducing photothermal
   chemotherapy. (A) Light micrograph of 4T1 cells treated with PBS and
   IC@PCH NPs + laser (scale bar: 20 μm). (B) Heat map of the
   differentially expressed genes between the control and IC@PCH NPs +
   laser groups. Red and blue indicate the up-regulated and down-regulated
   genes. (C) Volcano plot of genes that were differentially expressed
   between the control and IC@PCH NPs + laser groups. (D) Representative
   Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with
   genes that were significantly differentially expressed between the
   control and IC@PCH NPs + laser groups. (E) The expression levels of
   NLRP3, caspase-1, cleaved caspase-1, GSDMD, and N-terminal fragments of
   GSDMD (GSDMD-N) in 4T1 tumors after various treatments detected by
   western blot. (F) A scheme illustrating the mechanism of
   photothermal-chemotherapy-induced pyroptosis. (G) Quantitative analysis
   of (E); n = 3. The results are presented as mean ± SD. **P < 0.01; ***P
   < 0.001; ^##P < 0.01; ^###P < 0.001. MAP, mitogen-activated protein;
   ECM, extracellular matrix; cAMP, cyclic adenosine 3,5-monophosphate;
   PI3K, phosphoinositide 3-kinase.

   Pyroptosis is programmed cell death mediated by the pore-forming
   protein gasdermin and is closely associated with the inflammatory
   response [[115]40]. Pattern-recognition receptors such as toll-like
   receptors and nucleotide oligomerization domain-like receptors
   recognize intra- and extracellular signals and form inflammatory
   vesicles assembled with pro-caspase-1 and apoptosis-associated
   speck-like protein to activate caspase-1. Activated caspase-1 cleaved
   GSDMD into GSDMD-N. GSDMD-N nonselectively perforates membrane pores in
   the cell membrane, and the inflammatory factors were secreted,
   ultimately causing cell swelling, lysis, and death [[116]41,[117]42].
   The light microscopy and RNA-seq results suggested that IC@PCH NPs may
   mediate 4T1 cell death through the pyroptosis pathway (Fig. [118]6F).
   To validate the above results and understand the mechanism of IC@PCH
   NP-mediated pyroptosis, the NLRP3, caspase-1, cleaved caspase-1, GSDMD,
   and GSDMD-N protein expression levels in 4T1 cells with different
   treatments were evaluated by western blot analysis (groups: control
   group, laser-only group, C@PCH NPs group, I@PCH NPs + laser group, and
   IC@PCH NP + laser group) (Fig. [119]6E). As expected, the levels of key
   proteins for pyroptosis were significantly elevated in the I@PCH NPs +
   laser group and IC@PCH NP + laser group (Fig. [120]6G), which was
   attributed to the predominance of photothermal effects in the
   treatment. Meanwhile, the expression level of pyroptosis key proteins
   was significantly higher in the IC@PCH NP + laser group than in the
   I@PCH NPs + laser group, proving the chemotherapeutic agents’ effective
   synergism. Therefore, we concluded that IC@PCH NPs could inhibit tumor
   cell proliferation by initiating a classical caspase-1-dependent
   pathway leading to tumor cell pyroptosis. During treatment, TNBC often
   has no response to conventional therapy, while pyroptosis can bypass
   the “treatment blockade” and find new therapeutic targets. This makes
   IC@PCH NP-induced photothermal-chemotherapy a promising cancer
   treatment strategy.

In vivo biosafety

   Using a novel biomedical nanomaterial in vivo requires first
   verification of its biosafety. An evaluation of the biological safety
   of IC@PCH NPs was conducted comprehensively. Healthy BALB/c mice were
   administered IC@PCH NPs (8 mg/ml, 200 μl) via intravenous injection on
   days 5, 10, 15, and 20, with a control group receiving saline. Blood
   and major organs (including the heart, liver, spleen, lungs, and
   kidneys) from these mice were collected for analysis. H&E staining of
   major organs showed little histopathologic change (Fig. [121]7A).
   Furthermore, biochemical and hematological analyses indicated no
   significant differences between groups at each time point (Fig. [122]7B
   and C), indicating the high biosecurity of IC@PCH in subsequent
   treatments.

Fig. 7.

   Fig. 7.
   [123]Open in a new tab

   Biosafety of IC@PCH NPs in vivo. (A) Hematoxylin–eosin (H&E) staining
   of the main organs (heart, liver, spleen, lung, and kidney) in mice
   sacrificed at different time intervals (0, 5, 10, 15, and 20 d) after
   intravenous injection of IC@PCH NPs; the scale bars are 500 μm. (B)
   Blood routine and (C) blood biochemistry of mice at different time
   points after intravenous injection of IC@PCH NPs; n = 3. WBC, white
   blood cells; Lymph, lymphocytes; RBC, red blood cells; HGB, hemoglobin;
   MCV, mean corpuscular volume; PLT, platelets; MCH, mean corpuscular
   hemoglobin; RDW, red cell distribution width; ALT, alanine
   aminotransferase; AST, aspartate aminotransferase; CK, creatine kinase;
   CK-MB, creatine kinase-MB; LDH1, lactate dehydrogenase 1.

In vivo photothermal properties and in vivo treatment of hormonal mice

   The synergistic in vivo targeted anticancer efficacy of photothermal
   and chemotherapeutic treatments with IC@PCH NPs was evaluated.

   As shown in Fig. [124]8A and B, the laser-only group experienced a
   slight increase in temperature to 38.5 °C, below the heat therapy
   threshold of 42 °C. This finding suggests that the NIR laser is safe
   for normal tissues and suitable for future clinical applications. The
   temperature of IC@PCH NPs and I@PCH NPs rapidly increased to 53.5 and
   53.4 °C, respectively, upon laser irradiation, indicating that the
   efficacy of NP photothermal therapy is mainly derived from ICG.
   Moreover, compared to IC@PCH NPs, the temperature of the IC@P NPs +
   laser group was only 44.5 °C. This indicates that more ICG accumulated
   in the tumor region due to the superior targeting ability of the NPs
   coated with HA and higher ICG encapsulation rate, resulting in higher
   temperatures.

Fig. 8.

   Fig. 8.
   [125]Open in a new tab

   In vivo therapeutic efficacy of IC@PCH NPs. (A) Infrared thermographic
   images and (B) heating curves following tail vein injection of PBS,
   IC@P NPs, I@PCH NPs, and IC@PCH NPs under 808-nm laser irradiation (1.5
   W/cm^2). (C) Schematic of intratumor treatments. (D) Tumor volume
   growth trajectories for the control, laser-only, C@PCH NPs, I@PCH NPs +
   laser, and IC@PCH NP + laser groups posttreatment. (E) Pictures of the
   dissected tumors from each treatment group. (F) Tumor weight, (G) tumor
   inhibition rate, and (H) body weights of hormonal mice. (I) H&E
   staining, proliferating cell nuclear antigen (PCNA), and terminal
   deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
   immunohistochemistry staining in the tumor region in various treatment
   groups (scale bars: 50 μm). The data are shown as mean ± SD, n = 4 per
   group. *P < 0.05; ***P < 0.001; ****P < 0.0001.

   The protocol of intratumor treatments is sketched out in Fig. [126]8C.
   To further assess the synergistic anticancer effect, when the tumor
   volume grew to 45 to 75 mm^3, 4T1 hormonal mice were randomly divided
   into 7 groups (n = 4): the control group, laser-only group, D@PCH NPs +
   Cu^2+@PCH NPs group, IC@PCH NPs group, C@PCH NPs, I@PCH NPs + laser,
   and IC@PCH NP + laser groups. After the mice in each group were
   injected with the corresponding drug intravenously (5 mg/ml, 200 μl)
   for 2 h, the tumor area of the corresponding group was irradiated with
   NIR laser at 1.5 W/cm^2 (808 nm) for 6 min (Fig. [127]8A). Tumor
   volumes were measured every 2 d (Fig. [128]8D). The IC@PCH NPs + laser
   group significantly inhibited tumor growth, the I@PCH NPs + laser group
   and the C@PCH NPs group exhibited moderate inhibition, while the D@PCH
   NPs + Cu^2+@PCH NPs group and the IC@PCH NPs group slowed down the
   growth rate of the tumor only mildly. In contrast, the control and
   laser-only groups displayed notable tumor volume increases, suggesting
   no therapeutic effects. Tumor growth inhibition was more pronounced in
   the C@PCH NPs group than in the D@PCH NPs + Cu^2+@PCH NPs group,
   suggesting that direct delivery of CuET helps to overcome the
   limitation that the DSF–Cu^2+ delivery nano-delivery system is unable
   to efficiently form the antitumor active ingredients. The IC@PCH NPs +
   laser group had better treatment results compared to the IC@PCH NPs
   group, again demonstrating the good laser control properties of IC@PCH
   NPs. Harvested tumor weights and in vivo tumor suppression rates from
   each treatment group further confirmed these results (Fig. [129]8E to
   G). Additionally, there were no significant differences in body weight
   among the groups (Fig. [130]8H), demonstrating the high biosecurity of
   IC@PCH NPs for in vivo application.

   As illustrated in Fig. [131]S3, mice treated with IC@PCH NPs + laser
   demonstrated optimal anticancer efficacy. Before treatment, a tumor was
   evident on the right hind limb of the mouse. By day 4 posttreatment,
   the tumor region began scabbing. By day 12, the wound started healing.
   By day 16, a solitary small scab was observed, accompanied by adjacent
   scar tissue formation. By day 20, the skin had nearly completed its
   healing process, and no recurrence of the tumor was evident. At the
   same time, the control and laser-only groups showed a significant
   increase in tumor volume, while the I@PCH NPs + laser group and the
   C@PCH NPs group were able to alleviate the increase in tumor volume to
   a certain extent compared with the control group. The D@PCH NPs +
   Cu^2+@PCH NPs group and the IC@PCH NPs group also had a slight
   inhibitory effect on tumor growth. In conclusion, the HA-coated
   targeted drug delivery system, in conjunction with photothermal therapy
   and chemotherapy, exhibits potent synergistic antitumor activity,
   successfully preventing the recurrence of tumors. Also, the favorable
   recovery of the mice at the skin wound provides confidence in applying
   this treatment strategy to clinical breast-conserving therapy.

   On the second day after treatment, one mouse from each group was
   randomly selected to be executed and used for immunohistochemical
   staining and immunofluorescence staining (Fig. [132]8I). H&E staining
   showed that tumor cells in the control and laser-only groups were
   blue-stained, with a large proportion of nucleoplasm and nuclear
   fragmentation was visible. Some tumor cells in the D@PCH NPs +
   Cu^2+@PCH NPs group and the IC@PCH NPs group showed nuclear
   consolidation or fragmentation. Extensive nuclear consolidation of the
   tumor cells was seen in the I@PCH NPs + laser group, and extensive
   nuclear fragmentation of the tumor cells was seen in the C@PCH NPs
   group. Most of the tumor cells had nuclear fragmentation, and extensive
   nuclear lysis was seen in the IC@PCH NPs + laser group. This indicates
   that chemotherapy + targeting treatment, photothermal + targeting
   treatment, and photothermal + chemotherapy + targeting treatment can
   all induce tumor cell necrosis, and the degree of damage is deepened
   sequentially. The results of the TUNEL assay and PCNA staining have
   been widely recognized as indicators of apoptosis and tumor
   proliferation, respectively. The positive index of the TUNEL assay was
   highest in the IC@PCH NPs + laser group, with some in the D@PCH NPs +
   Cu^2+@PCH NPs group, IC@PCH NPs group, C@PCH NPs, and I@PCH NPs + laser
   group, and was almost invisible in the control and laser-only groups,
   indicating that the targeted chemo-phototherapy combination therapy
   effectively induced TNBC cells’ death. In addition, the PCNA staining
   level was the lowest in the IC@PCH NPs + laser group, suggesting that
   the targeted photothermal- chemotherapy significantly suppressed tumor
   cell proliferation. The control and laser-only groups showed low
   expression of TUNEL and high expression of PCNA, and the absence of a
   notable disparity between the 2 groups suggests that the utilization of
   a 808-nm laser poses no adverse effects on tissue at the molecular
   level. These exciting, superb results provided an experimental basis
   for minimally invasive treatment of TNBC.

In vitro and in vivo multimodal imaging and in vivo distribution

   Studies suggest that an ideal nanomedicine could accomplish effective
   tumor treatment while also providing precise imaging guidance and
   immediate therapeutic effect analysis, especially for highly malignant
   TNBC. Multimodal imaging techniques, such as combining ultrasound with
   computed tomography technology or fluorescence with MRI, have emerged
   as a recent research focus [[133]43,[134]44]. A specific imaging
   technique possesses unique advantages yet also presents inherent
   technical limitations. Incorporating multimodal imaging techniques on a
   single nanoplatform effectively addresses these shortcomings for
   achieving multistep-free theranostics [[135]45,[136]46]. Based on this,
   we prepared IC@PCH NPs as triple-mode contrast agents for
   PA/MR/fluorescence imaging, designed to integrate diagnosis and
   treatment.

   PA imaging is an innovative technique based on the PA effect. The
   full-wavelength scanning results showed that the IC@PCH NPs had a
   distinct absorption peak at 845 nm in the NIR region (Fig. [137]9A).
   The optoacoustic properties of the NPs were attributed to the combined
   effect of ICG as well as CuET; the latter 2 optoacoustic signal
   waveforms are shown in Fig. [138]S4A and B. A laser at a wavelength of
   845 nm was used to excite the PA signals of the NPs, and PA images
   showed that the in vitro PA signals were progressively enhanced with
   the concentration of IC@PCH NPs in a linear manner (Fig. [139]9B). To
   noninvasively and precisely monitor the accumulation of IC@P NPs and
   IC@PCH NPs at tumor sites in real time and acquire tumor morphology, we
   injected the NPs intravenously and conducted PA imaging at the tumor
   site, visualizing the therapeutic process (Fig. [140]9C). After the
   injection of IC@P NPs and IC@PCH NPs, the tumor site’s PA signal was
   found to enhance up to its peak intensity at 2 h postinjection. ICP@CH
   NPs were more concentrated at the tumor site than the IC@P NPs group at
   each time point, demonstrating their better tumor-targeting ability
   (Fig. [141]9D). Both the ICG@PCH NPs group and CuET@PCH NPs group
   showed weaker fluorescence at the tumor site than the IC@PCH NPs group,
   again indicating that the PA imaging ability of IC@PCH NPs originated
   from the combined effect of ICG and CuET (Fig. [142]S5A and B).

Fig. 9.

   Fig. 9.
   [143]Open in a new tab

   In vitro and in vivo multiple imaging. (A) The optimal excitation
   wavelength of IC@PCH NPs was identified by comprehensive spectral
   scanning. (B) Photoacoustic (PA) images (inset) and corresponding
   signal intensities of IC@PCH NPs at increasing concentrations. (C) PA
   images and (D) quantitative analysis of tumor sites at different times
   after intravenous injection of IC@P NPs and IC@PCH NPs; n = 3. (E)
   T1-weighted magnetic resonance (MR) images (inset) and intensities of
   Cu^2+ at increased concentration. (F) T1-weighted MR images and (G)
   quantitative analysis of tumors in 4T1 tumor-bearing mice at various
   times following intravenous injection of IC@P NPs and IC@PCH NPs; n =
   3. Nanoparticle aggregation zones are circled in dotted lines, and peak
   MRI times are marked with red pentagrams. (H) Fluorescence images
   (inset) and intensities of IC@PCH NPs at increased concentration. (I)
   In vivo fluorescence images of mice treated with IC@P NPs and IC@PCH
   NPs at different time points, and ex vivo fluorescence images of key
   organs and tumors excised from mice at 24 h posttreatment. (J) Tumor
   site fluorescence intensity quantification; n = 3. (K) Ex vivo
   fluorescence intensity analysis in major organs and tumors; n = 3. All
   results are expressed as mean ± SD, with statistical significance
   indicated as *P < 0.05 and **P < 0.01.

   Compared to PA imaging, MRI has superior soft tissue contrast and
   infinite penetration depth. T1-weighted MR images of the IC@PCH NPs’
   dispersed system in vitro showed no significant MR signal enhancement
   ability (Fig. [144]S4C), whereas T1-weighted images of Cu^2+ in vitro
   and IC@PCH NP solution in vivo showed MR enhancement, and there was a
   linear correlation between the concentration of Cu^2+ and MR signal
   (Fig. [145]9E). This suggests that IC@PCH NPs can metabolize to Cu^2+
   at the tumor site and have desirable contrast-enhanced MRI capabilities
   for subsequent imaging.

   Following injection of IC@P and IC@PCH NP suspension, with the gradual
   accumulation of NPs, the tumor demonstrates peak MRI signal strength at
   2 h postinjection (Fig. [146]9F). HA-coated IC@PCH had a stronger
   tumor-targeting ability than IC@P and prolonged the NP retention time
   (Fig. [147]9G), which was consistent with PA imaging results. PA
   imaging can provide high-spatial-resolution microstructural images of
   the tumor site, reflecting the accumulation of NPs at the tumor site,
   and it is relatively inexpensive and portable. However, its depth of
   penetration is severely limited by reflection, refraction, and
   absorption of acoustic waves, and its whole-body imaging capability is
   poor [[148]47]. MRI not only provides detailed tissue information that
   identifies fine (~3 mm) as well as precise anatomical information
   in vivo but also allows scanning of the entire body without tissue
   depth limitations, but it is expensive and has a long imaging time
   [[149]48]. Therefore, integrating MR/PA imaging modes can effectively
   guide and oversee the entire treatment process and provide valuable
   complementary information for clinical applications.

   Fluorescence imaging is also a frequently used imaging modality in
   clinical practice. It has the advantages of high sensitivity, high
   specificity, and multicolor imaging, but the limited depth of
   penetration of light into the tissue (micrometers to centimeters,
   depending on the wavelength) [[150]49]. Thus, fluorescence imaging can
   be used as a complement to the 2 imaging modalities mentioned above
   [[151]50]. We investigated the ex vivo and in vivo fluorescence imaging
   ability and biodistribution of IC@P NPs and IC@PCH NPs. The
   fluorescence intensity of IC@PCH NPs showed a linear enhancement
   relationship at concentrations less than 150 μg/ml (Fig. [152]9H).
   Notably, the maximum fluorescence intensity of tumor tissue was
   observed after 0.5 h of accumulation of IC@P NPs and IC@PCH NPs (Fig.
   [153]9I). The peak time was about 1.5 h earlier than that of PA imaging
   and MRI modalities, which may be mainly due to the short circulating
   half-life, high safety index, and rapid hepatic clearance of ICG.
   Quantitative analysis of fluorescence intensity at the tumor site
   similarly demonstrated that HA promotes the accumulation of IC@PCH NPs
   at the tumor site (Fig. [154]9J).

   To observe the biodistribution of IC@P, IC@PCH NPs, major organs
   (including the heart, liver, spleen, lungs, and kidneys) and tumors
   were collected for ex vivo fluorescence imaging at the end of the whole
   observation period (Fig. [155]9I). In ex vivo bioimaging, the
   fluorescence signal of IC@PCH NPs within the tumor increased about
   2.79-fold compared with that of IC@P NPs, demonstrating that the
   fluorescence imaging ability was greatly improved, which was attributed
   to its good targeting ability as well as the increase in ICG loading
   rate (Fig. [156]9K).

   Based on these findings, we conclude that each component of the
   triple-modality probe enhances the other modalities, ultimately
   providing more accurate information in biological systems. The superior
   tumor accumulation efficiency of IC@PCH NPs was attributed to the
   specific binding of CD44 overexpressed on tumor cell membranes to HA.
   Therefore, HA-modified IC@PCH NPs may offer highly sensitive and
   specific outcomes for nanomedicine TNBC monitoring.

Conclusion

   In conclusion, we have successfully constructed CD44-receptor-targeted
   IC@PCH NPs and proposed a breast-conserving therapeutic strategy
   combining chemotherapy and photothermal therapy for a population of
   young women with TNBC. NPs bind to tumor cells with high CD44 receptor
   expression through a HA shell and release the drug in an acidic TME.
   The IC@PCH NPs were effective in killing tumors by
   photothermal-chemotherapy, leading to pyroptosis. Pyroptosis breaks
   through the blockade of TNBC to conventional therapeutic modalities,
   finding new therapeutic targets and re-sensitizing TNBC to treatment.
   The repurposing of the old drug DSF overcomes the insensitivity of TNBC
   while reducing the drug development cycle, offering new possibilities
   for curing TNBC. In addition, copper ions mediate the generation of PA
   and MRI signals, while ICG mediates the generation of PA and FI
   signals, which together detect and guide the therapeutic process,
   better suggesting occult tumorigenic lesions. These findings present
   new insights into DSF–copper NP-based targeted delivery systems for
   pyroptosis-induced augmentation of photothermal-chemotherapy against
   TNBC. Given the Food and Drug Administration approval of all components
   within our strategy, coupled with the considerable attention garnered
   by combination therapy, our research holds potential for advancing
   toward clinical implementation.

Acknowledgments