Theranostic mesoporous platinum nanoplatform delivers halofuginone to remodel extracellular matrix of breast cancer without systematic toxicity

Abstract The enriched collagens in the extracellular matrix (ECM) of breast cancer substantially impede drug delivery. Halofuginone (HF), a potent antifibrotic agent, was effective to deplete the collagens and remodel the ECM by inhibiting the TGFβ pathway. However, the application of HF was hindered by its strong liver toxicity. Herein, mesoporous platinum (mPt) nanoparticles were constructed to load HF as theranostic nanoplatforms. mPt had a uniform spherical structure with a diameter of 79.83 ± 6.97 nm and an average pore diameter of 20 nm and exhibited good photothermal conversion efficiency of 62.4%. The obtained HF‐loaded nanoplatform (PEG@mPt‐HF) showed enhanced cytotoxicity through the combination of photothermal therapy and the anti‐TGFβ effect induced by HF. The animal imaging and histochemical assays confirmed the PEG@mPt‐HF could efficiently deliver HF to tumors (monitored by CT) and remodel the ECM by TGFβ pathway inhibition, which resulted in increased anti‐cancer efficacy. Importantly, the liver toxicity observed in HF‐treated mice was negligible in those treated by PEG@mPt‐HF. Overall, this study designed a theranostic nanoplatform to remodel the ECM with remarkably reduced systematic toxicity and enhance the therapeutic efficacy through combination treatment.


| INTRODUCTION
Breast cancer (BC) is the most common cancer for women, with an incidence of 5% worldwide. 1,2 Rich extracellular matrix (ECM) is one of the tumoral microenvironment hallmarks of BC. 3,4 It is composed of enriched collagens and serves as a dynamic regulator of numerous cellular processes including cell adhesion, migration, and signaling. [5][6][7] Recently, increasing findings suggested the ECM played a critical role in the therapy resistance of cancers, 8,9 because the dense structure gives rise to substantial barriers to the perfusion, diffusion, and convection of drug delivery. [10][11][12] Therefore, it is a great necessity to deplete the collagen in tumor ECM to improve the delivery of drugs and thus the therapeutic efficacy.
Halofuginone (HF) is an effective antifibrotic agent whose active principle (febrifugine) was isolated from the Chinese herb Chang Shan Jie Zhang, Ziqing Xv, and Yang Li contributed equally.
(Dichroa febrifuga Lour). 13,14 HF could not only inhibit the formation of fibrosis but also deplete the collagens in cancer stroma. 15 This antifibrotic effect was mainly from the inhibition of the transforming growth factor-β (TGFβ) signaling pathway. 16 TGFβ is one of the major cytokines implicated in the activation of myofibroblasts. 17 It induced the phosphorylation of Smad3 and transformed quiescent fibroblasts to activated myofibroblasts that overexpress α-smooth muscle actin (αSMA). 18,19 HF inhibited the TGFβ-Smad3 signaling pathway and thus deplete the collagens in the ECM of tumors. 20,21 Our previous studies have shown that HF could enhance the therapeutic efficacy of nanoparticles by disrupting the collagens in tumor stroma. 22 However, to visualize and optimize the dosage of HF, it is desired to load HF on nanoparticles with imaging ability, whose distribution can be readily monitored by imaging to reflect the depletion of collagens in tumor stroma. Moreover, HF provokes high toxicity to the liver and other human organs, 23,24 which further requires suitable nanocarriers to improve its biodistribution and reduce side effects.
The mesoporous platinum (mPt) nanoparticles have porous structure and high surface area, which could act as excellent drug delivery carriers. Previous reports showed that mPt would help to reduce the toxicity of chemotherapeutic drugs to normal tissue. 25 Because of the high atomic number of Pt, mPt could be also applied as computed tomography (CT) imaging contrast. 26 Moreover, the mPt could convert light energy to heat to exert photothermal therapy (PTT), which could be used to enhance the therapeutic efficacy of HF.
The dense collagen matrix in tumors significantly hampers the penetration and impacts the efficacy of nanotherapeutics. 21,27,28 The success of PTT is directly related to the delivery of nanoparticles to tumors. 29 Besides, the depletion of collagens would boost the immunogenic effect of PTT and thus further enhance the therapeutic efficacy. 30 Hence, we speculate that the reduction of tumor stroma would also benefit the penetration and distribution of mPt and thus enhance its therapeutic efficacy.
Herein, we built a multi-function nanoplatform by loading HF into polyethylene glycol (PEG) coated mPt (PEG@mPt-HF). This nanoplatform could deplete the collagens in the stroma of BC, improve the distribution of nanoparticles, reduce the liver toxicity of HF and enhance the therapeutic efficacy by combing HF with PTT. More importantly, the success of collagen depletion could be monitored by the enhanced CT contrast due to the imaging ability of PEG@mPt-HF.

| Bioinformatics analysis of BC patient
The expression profiles of collagen-I gene (COL1A1) in pan-cancer were displayed using Tumor Immune Estimation Resource (TIMER) database and TGFB3), were explored using BC data on the TIMER database.

| Synthesis and characterization of mPt and PEG@mPt
In the first step, mPt was synthesized. Typically, 450 mg of Pluronic

| Assessment of the photothermal conversion efficacy and CT imaging capability
The photothermal effect of mPt was characterized in different concentrations (0, 20, 40, and 80 μg/ml) under various power of laser irradiation (808 nm, Nanjing spectrum of optics-electrical Technology Co., Ltd.) (0.5, 1.0, and 1.5 W/cm 2 ) recorded by an IR thermal camera (FOTRIC's IR camera, 220s, China). The photothermal stability of mPt was determined through 5 rounds of irradiation (5 min) flowed cooling (10 min) process. To assess the photothermal conversion efficacy, the solution of mPt (100 μg/ml) was irradiated (1 W/cm 2 ) for 15 min, stable at the highest temperature, then cooled to room temperature. The following equation was used to calculate the photothermal conversion efficacy of mPt.

| Assessing the distribution of NP in vivo
Twenty-four hours after the mice received the last injection (n ≥ 4 mice/group), they were scanned by dual-source CT system. The CT value of each tumor was calculated. Parameter: the slice thickness of 0.6 mm, the voltage of 120 kVp, and the current of 23 mA.
The mice were euthanatized after the CT scan. Then tumors and major organs (heart, liver, spleen, lung, and kidney) of mice in PEG@mPt and PEG@mPt-HF groups were collected. All of the samples were weighed and digested to estimate the Pt concentration by ICP-OES.

| Statistical analysis
Data are presented as means ± standard deviation of ≥3 independent experiments performed. Student t-test was used for comparison between two groups. Kaplan-Meier plot was used to analyze the association of COL1A1 or COL1A2 levels with BC patient survival.

| ECM enrichment in BC patients
As collagen-I is the major component of ECM, its gene expression profiles across cancers were identified using TCGA/GTEx database. It is noted that collagen-I was overexpressed across cancers, especially in BC patients (Figure 1a,b). The IHC data also confirmed that collagen-I was highly expressed in BC tumors compared with normal breast tissue ( Figure 1c). Moreover, the high expression of collagen-I was significantly related to the poor prognosis of BC patients (Figure 1d). The expression of collagen-I was also positively related to the expression

| Theranostic characteristics of PEG@mPt-HF
To explore the theranostic potential of mPt, its CT imaging ability was compared to the clinical contrast agent, iodine. It is shown that the contrast of mPt was similar to that of iodine at the same concentration (Figure 3a,b). Furthermore, upon 808 nm laser irradiation, the temperature of mPt solutions increases smoothly, and heat production capacity was positively correlated with the concentration of mPt and the laser power (Figure 3c,d). No significant change in temperature was observed for H 2 O during 1.5 W/cm 2 laser irradiation ( Figure 3d). The photothermal conversion efficacy of mPt was calculated to be 62.4% (Figure 3e,f). Besides, the photothermal curve of the mPt was stable during five cycles of laser irradiation (Figure 3g).
In summary, these results showed that mPt possessed the potential to be used as a theranostic platform for CT imaging and PTT.
To investigate the anti-cancer effect of PEG@mPt-HF, the cell viability was tested by MTT assay. After incubation with different concentrations of PEG@mPt for 48 h, the cell viability remained above 90%, indicating the great biocompatibility of PEG@mPt ( Figure 4a). As shown in Figure 4b, the PEG@mPt-HF showed similar cytotoxicity with free HF at the same concentration, indicating the PEG@mPt could deliver the HF into the cancer cells and release it to exert TGFβ inhibition effect. It is noted that the therapeutic efficacy of PEG@mPt-HF with laser irradiation (IC50: 20.61 μg/ml) was greater than that of PEG@mPt with laser irradiation (IC50: 27.82 μg/ml) and PEG@mPt-HF in dark (IC50: 42.13 μg/ml). These results showed that the PEG@mPt-HF could enhance the therapeutic efficacy of HF through the combination of TGFβ pathway inhibition and PTT.

| ECM degradation effect
To investigate the influence of PEG@mPt-HF on the ECM of BC, the 4T1 tumors were collected for IHC staining after i.v. injection of PEG@mPt-HF every 3 days for 2 weeks. As shown in Figure  between PEG@mPt and PEG@mPt-HF treated tumors was lower than that of Pt concentration, which is probably because the CT values would be more easily affected by hemorrhage and necrosis, which were common in tumors. These results indicated the PEG@mPt-HF could improve the delivery of nanoparticles by depletion of collagens, and further enhance the CT image contrast of tumors, which is critical for the detection and assessment of BC.

| In vivo therapeutic efficiency
The therapeutic efficiencies of HF, PEG@mPt-HF, PEG@mPt + laser, and PEG@mPt-HF + laser were evaluated on 4T1 tumor-bearing mice.
The treatment workflow was depicted in Figure 7a.

| Biocompatibility
To evaluate the biosafety of PEG@mPt, various concentrations of PEG@mPt were mixed with RBCs. As shown in Figure  writingreview and editing (lead).

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.