Thermosensitive Nanogels with Cross-Linked Pd(II) Ions for Improving

Jun 18, 2019 - Figure 2A shows that light scattering intensities increased from 17.1 ± 3.3 to 342.9 ± 10.9 kcps as the coordination ratios (CRs) of ...
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Cite This: Chem. Mater. XXXX, XXX, XXX−XXX

Thermosensitive Nanogels with Cross-Linked Pd(II) Ions for Improving Therapeutic Effects on Platinum-Resistant Cancers via Intratumoral Formation of Hydrogels Hao Zhao,† Jiabao Xu,† Wenjing Huang, Yanbing Zhao,* and Xiangliang Yang* National Engineering Research Center for Nanomedicine, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China Downloaded via BUFFALO STATE on July 17, 2019 at 10:59:48 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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ABSTRACT: Although bivalent palladium [Pd(II)] compounds exhibit favorable antitumor efficacy against platinumresistant cancers, the mechanism remains unclear. Moreover, some drawbacks, such as structural lability, rapid elimination in vivo, and poor intratumoral retention, heavily limit their application in systemic tumor chemotherapy. Here, thermosensitive Pd-PNS nanogels are rationally designed by crosslinking coordination of Pd(II) ions with poly(styrenesulfonate-b-N-isopropylacrylamide-b-styrenesulfonate) (PNS), achieving high drug-loading ability (ca. 80% of entrapment efficiency and 40% of drug-loading efficiency), sustained release, and long-term retention of highly water-soluble K2PdCl4 in intratumoral administration. Cytotoxicity on sensitive/resistant MCF-7 cells and cancer stem cell (CSC) spheroids indicates that Pd-PNS nanogels could inhibit the palladium efflux of P-glycoprotein and multidrug resistance-associated protein-1 and thus enhance the DNA damage and toxicity of platinum-resistant MCF-7/Pt cells and their CSCs. Based on their temperature-sensitive sol−gel transition, Pd-PNS nanogels show sustained release profiles and long-term retention in tumors, further enhancing in vivo antitumor efficacy on MCF-7/Pt tumor-bearing BALB/c nude mice. Owing to these advantages of prolonged retention, efflux inhibition, and efficient CSCs killing ability, Pd-PNS nanogels are promising to be developed as a new temperature-sensitive delivery nanoplatform for injectable regional chemotherapy on platinum-resistant cancers.

1. INTRODUCTION Although chemotherapeutic drugs have been extensively used in cancer therapy over the last decades, the therapeutic efficacies of small molecular compounds have been heavily limited by their poor pharmacokinetics/pharmacodynamics (PK/PD), nonspecific biodistribution, insufficient cellular uptake, and serious side effects.1,2 As one of the first metalbased chemotherapeutic agent, for instance, cis-diammineplatinum dichloride (cisplatin) encounters worsening antitumor efficiency and serious toxicities (e.g., nephrotoxicity and neurotoxicity), owing to indiscriminate accumulation in normal and tumor tissues, cellular uptake inhibition, and the increase of cisplatin efflux and detoxification.3,4 Under longterm chemotherapies with cisplatin and other platinum drugs (carboplatin, nedaplatin, oxaliplatin, etc.), moreover, cancer cells are prone to acquire multiple drug resistance (MDR), being associated with a series of inter/intracellular events, such as abnormality of membrane transporters (e.g., Ctr1, Pglycoprotein (P-gp), multidrug resistance-associated protein-1 (MRP-1)) and DNA repair.5−7 Recently, some studies indicated that platinum drug resistance was highly dependent on the occurrence and progression of cancer stem cells (CSCs), playing significant roles in the recurrence and metastasis of tumors.8−10 To date, great efforts have been © XXXX American Chemical Society

made for overcoming platinum resistance via preparing new platinum compounds such as cisplatin analogues with different leaving groups and non-leaving groups, octahedral Pt(IV) complex prodrugs, macromolecular platinum complexes, or nanomedicines.11−16 However, it is still a great challenge for platinum-based drugs to overcome MDR, improve in vivo PK/ PD behavior, and reduce undesirable toxicity. Inspired by the success and the limitations of cisplatin, plenty of organometallic compounds with other transition metals (titanium, iron, ruthenium, rhodium, iridium, palladium, copper, gold, etc.) have been developed as emerging alternatives to platinum-based chemotherapeutics.17−20 Owing to the wide range of oxidation states, different coordination geometries, diversity of in vivo hydrolysis kinetics, and mechanism of action, these nonplatinum metal compounds exhibited better antitumor activity than cisplatin, especially against platinum-resistant cancer cells. Among these metal elements, bivalent palladium [Pd(II)] compounds attracted increasing attention owing to their coordination chemistry similar to platinum, exhibiting better anticancer activity as well Received: March 11, 2019 Revised: June 12, 2019 Published: June 18, 2019 A

DOI: 10.1021/acs.chemmater.9b00986 Chem. Mater. XXXX, XXX, XXX−XXX

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Chemistry of Materials Scheme 1. Antitumor Mechanism of Pd-PNS Nanogels on a Platinum-Resistant Tumora

a (A) The schematic of self-assembling behavior. (B) Thermosensitive sol−gel transition and resistance-reversal mechanism of platinum-resistant cells.

as lower toxicity.21−23 More interestingly, they also showed promising capacity in overcoming platinum resistance, although the mechanism remains unclear.24,25 Unfortunately, since these small molecular Pd(II) compounds have intrinsically more rapid hydrolysis rate in blood and stronger water solubility than platinum compounds, it is difficult to maintain in vivo structural integrity and realize long-term blood circulation and selective delivery to tumor tissue/cells after intravenous (i.v.) administration, resulting in severe toxicity and poor therapeutic efficacy.26,27 Plenty of nanoscale drug vehicles (micelles, liposomes, emulsions, organic/inorganic nanoparticles, endogenous exosomes, etc.) have been skillfully designed as promising delivery platforms of small molecular chemotherapeutics by i.v. administration, due to many advantages including improvement of drug stability, controlled release, prolonged blood circulation time, enhanced permeation and retention (EPR, also called passive targeting) effect, and enhancement of cellular uptake.28−33 In particular, several nanomedicines exhibited highly efficient performance to inhibit the resistance of cancer cells to cisplatin by improved cell internalization, reduced drug efflux, or co-delivery with other drugs (e.g., photosensitizers, photothermal agents, siRNA, etc.).5−7,12,28,29 However, these nanomedicines, most of which were intravenously delivered, must pass through a series of biological barriers (blood circulation, protein adsorption, extravasation, retention/penetration in tumor tissues, tumor cell internalization, etc.), resulting in reduced accessibility to the targeted site and susceptibility of cancer cells.34,35 For example, the EPR effect, which is highly dependent on tumor vascular permeability in the i.v. route, has been overestimated in targeted delivery efficiency and clinical outcome owing to tumor heterogeneity and tumor microenvironments (high interstitial fluid pressure, hypoxia, etc.).36,37

In contrast to the vasculature-dependent delivery strategies, intratumoral administration is increasingly considered as a promising delivery approach of nanomedicines, biomacromolecules, immunotherapeutics, viruses, and cells because of its unique advantages such as independence from blood supply, high regional drug concentration in tumor sites, sustained release, low systemic toxicities, and minimal invasiveness.38−45 In particular, several in situ forming hydrogel/nanogel platforms (e.g., OncoGel and Pluronic F127 hydrogels) were skillfully designed for increasing intratumoral drug concentration and retention time in tumor sites, thereby further enhancing their antitumor efficacy and reducing side effects.41−45 However, a major challenge still remains in exploring an efficient approach of intratumoral administration to improve the concentration and retention time of highly water-soluble drugs in tumor sites. Here, a triblock polymer poly(styrenesulfonate-b-N-isopropylacrylamide-b-styrenesulfonate) (pSS100-b-pNIPAM200-bpSS100, PNS) was first synthesized using atom transfer radical polymerization (ATRP) and used as a temperature-sensitive hydrogel ligand for coordination with potassium tetrachloropalladate [Pd(II)] owing to its tunable thermosensitivity,46 good biocompatibility,47,48 and potential as a carrier for antitumor drugs.49−51 By displacing four chloride ions with the sulfonate groups of PNS, the thermosensitive nanogels of Pd(II)-coordination-cross-linking PNS (Pd-PNS) were rationally developed as new intratumoral delivery nanomedicines for achieving sustained release and enhanced intratumoral retention of highly water-soluble Pd(II) ions, which was further improved by temperature-sensitive sol−gel transition of concentrated Pd-PNS nanogel dispersions. Based on their capacity to prolong retention time in tumor sites, Pd-PNS nanogels in intratumoral injection route might sufficiently exert their antitumor activities on platinum-resistant cancer cells, especially on the related CSCs. The design idea is schematiB

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Figure 1. Characterization of pNIPAM200 and PNS polymers. (A) Atom transfer radical polymerization (ATRP) synthetic route. (B) Proton nuclear magnetic resonance (1H NMR) spectra and (C) gel permeation chromatography (GPC) of pNIPAM200 and PNS polymers.

to 342.9 ± 10.9 kcps as the coordination ratios (CRs) of Pd(II) and sulfonate units increased from 0.1 to 100%, suggesting that linear PNS polymeric chains were gradually cross-linked into three-dimensional (3D) networks by the replacement of Pd−NO3 bonds with Pd−sulfonate bonds. Meanwhile, the hydrodynamic diameters of Pd-PNS nanogels decreased from 1097.1 ± 12.4 to 158.4 ± 6.5 nm and the ζ potentials increased from −41.9 ± 1.0 to −4.4 ± 1.2 mV with the increase of CRs (Figure 2B), indicating that the crosslinking degrees of Pd-PNS nanogels increased with the increase of CRs. Transmission electron microscopy (TEM) also showed that the sizes of Pd-PNS nanogels decreased with the increasing CR (Figure 2C), agreeing with the size data by DLS (Figure 2B). Moreover, Pd-PNS nanogels exhibited a distinct core−shell structure, composed of the complexing core between Pd(II) and sulfonate units of PNS and the hydrophilic shell of poly(N-isopropylacrylamide) chains. In their Fourier transform infrared (FTIR) spectra (Figure 2D), after coordination cross-linking between Pd(II) and sulfonate units, the characteristic peak of K2Pd(NO3)4 (1384.8 cm−1, N−O stretching vibration of −NO3 groups) disappeared, whereas the characteristic peak of PNS (1178.4 cm−1, SO stretching vibration of −SO3− groups) was weakened. The data of XPS characterization also indicated that after coordination cross-linking of Pd(II) the binding energy peaks of Pd 4d3/2 and Pd 4d5/2 shifted from 343.3 and 338.1 eV to 342.8 and 337.6 eV, respectively, whereas that of O 1s of PNS shifted from 531.3 to 530.9 eV (Figure 2E). These results suggested that Pd-PNS nanogels with a well-defined structure were efficiently fabricated using a simple mixing approach of Pd(II) and PNS polymers. 2.2. Temperature Sensitivity of Pd-PNS Nanogels. Temperature-sensitive sol−gel phase transition, facilitating prolonging of the retention time of drugs in tumor tissues, played a vital role in enhancing the therapeutic efficacy of injectable hydrogels in intratumoral administration. First, the temperature-dependent transmittance and hydrodynamic diameters of Pd-PNS nanogels were characterized, respectively, using a UV/vis spectrometer and DLS. As shown in Figure 3A, the transmittances of three Pd-PNS nanogels (CR values are 5, 50, and 100%) decreased rapidly from 80−100 to 10−20% at about 35 °C, and their hydrodynamic diameters also showed a

cally illustrated in Scheme 1. Ultimately, Pd-PNS nanogels will be demonstrated to be promising intratumoral injection hydrogel delivery platforms of water-soluble nonplatinum agents to overcome cisplatin resistance of cancer cells more efficiently, by in vitro and in vivo pharmacodynamic evaluations with MCF-7/Pt cells.

2. RESULTS AND DISCUSSION 2.1. Synthesis and Characterization of the PNS Polymer and Pd(II)-Cross-Linked Nanogels (Pd-PNS Nanogels). First, a PNS triblock polymer was synthesized via classic ATRP reaction as shown in Figure 1A. The molecular composition and relative molecular weight (MW) of the PNS polymer were characterized by 1H NMR spectra and gel permeation chromatography (GPC), indicating the welldefined structure of the triblock PNS polymer as theoretical design (Figure 1B,C and Table S1). That is, the central poly(N-isopropylacrylamide) (pNIPAM) block was measured by GPC as 23.4 kDa and triblock PNS polymer was measured as 64.1 kDa. The molar ratio of p-styrenesulfonate (SS) groups and NIPAM groups was 0.99, agreeing with the feeding molar ratio (SS/NIPAM = 1.0). The temperature sensitivity of PNS polymer solution (5 mg mL−1, pH 6.5) was characterized by measuring the transmittance with temperature using a UV−vis spectrophotometer, and its lower critical solution temperature (LCST) was calculated as 37.3 °C from the curve of transmittance versus temperature (Figure S1). To obtain Pd(II)-induced coordination cross-linking nanogels with PNS (Pd-PNS nanogels), K2PdCl4 was first mixed with AgNO3 (molar ratio = 1:4) to replace chloride ions with more labile nitrate ions (NO3−). The resulting K2Pd(NO3)4 was confirmed by X-ray photoelectron spectroscopy (XPS) owing to the disappearance of the 2p binding energy peak of chlorine after replacement (Figure S2). To fabricate Pd-PNS nanogels, K2Pd(NO3)4 solution was added dropwise into PNS dispersion, and the coordination cross-linkages between Pd(II) and sulfonate units were formed under moderate stirring (Scheme 1). To investigate the coordination-assembling kinetics of Pd-PNS nanogels, the mean count rates, hydrodynamic diameters, and ζ potentials of these Pd-PNS nanogels were measured by dynamic light scattering (DLS). Figure 2A shows that light scattering intensities increased from 17.1 ± 3.3 C

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Figure 2. Assembly behavior study and structural characterization of Pd-PNS nanogels at different CR values. (A) Titration curve of the PNS polymer with cisplatin by monitoring their scattering light intensity (mean count rate, kcps). The inset pictures show the typical Tyndall effect of Pt-PNS nanogels at coordination ratio (CR) values of 5, 25, 50, 75, and 100%. (B) Variation curves of size and ζ potential of Pd-PNS nanogels with the dropwise addition of Pd(II). The concentration of Pd-PNS nanogel dispersions was 2 mg mL−1 for (A) and (B). (C) Transmission electron microscopy (TEM) images of Pd-PNS at the CR values of 5, 50, and 100%. (D) Fourier transform infrared (FTIR) spectra and (E) X-ray photoelectron spectroscopy (XPS) analysis of Pd(II), PNS, and Pd-PNS nanogels at CR values of 5, 50, and 100%. Plots 1 and 2 show the highresolution XPS spectra of O 1s and Pd 3d, respectively.

monotonous decrease with temperature (Figures 3B and S3). The LCSTs and volume phase transition temperatures (VPTTs) of the three nanogels were measured, respectively, from the curves of transmittance and hydrodynamic diameters, and all were lower than 37 °C (ca. 32−36 °C). Moreover, PdPNS nanogels showed temperature-dependent transmittance similar to that of PNS polymers, indicating that the coordination cross-linkage of Pd(II) with PNS had a little influence on the temperature sensitivity of PNS. The temperature-sensitive sol−gel transition of concentrated Pd-PNS nanogel dispersions was further investigated using the inverting-vial method. As shown in Figure 3C,D, a so-called temperature-sensitive free-flowing sol to free-standing gel transition occurred at the critical gelation temperature (CGT, ca. 31−42 °C), depending on the nanogel concentration and CR values of Pd-PNS nanogels. For instance, the CGTs of Pd-PNS nanogels decreased from 35.8, 34.7, 33.1, and 32.4 to 30.8 °C as the CRs increased from 5, 25, 50, and 75 up to 100%, respectively, attributed to the reduced

hydrophilicity of Pd-PNS nanogels due to the coordination cross-linkage of Pd(II) with sulfonate units. In rheological studies, the storage modulus (G′) and loss modulus (G″) of the Pd-PNS nanogel dispersions (10 wt % of PNS, 5% of CR) increased from 1.27 and 0.31 Pa at 25 °C to 1270 and 65.8 Pa at 50 °C, respectively, whereas those of the Pd-PNS nanogel dispersions (10 wt % of PNS, 100% of CR) increased from 1.29 and 0.24 Pa at 25 °C to 1190 and 15.89 Pa at 50 °C (Figure 3E,F), respectively. This indicated that PdPNS nanogel dispersions had good flowability at 25 °C owing to lower G′ and G″ values and high gelation strength at 37 °C because of temperature-sensitive sol−gel transition, facilitating in overcoming the dilemma between injection and gelation of Pd-PNS nanogel dispersions in intratumoral administration. In addition, although both Pd-PNS nanogel dispersions (5 and 100% of CR) showed the similar temperature dependence of rheological behavior, their CGTs, defined as the temperature at loss tangent (tan α) = 1.0, decreased as CR increased (e.g., 35.9 °C at 5% of CR and 32.6 °C at 100% of CR). D

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Figure 3. Study of the temperature sensitivity of Pd-PNS nanogels. (A) Transmittance vs temperature curves and (B) size vs temperature curves of Pd-PNS nanogels at CR values of 5, 50, and 100%. The pH of the Pd-PNS nanogel dispersions was 6.5 for (A) and (B). The concentration of PdPNS nanogel dispersions was 5 mg mL−1 for (A) and 2 mg mL−1 for (B). The influence of (C) nanogel concentration and (D) CR value on the gelation temperature of Pd-PNS nanogels. Inset photos are the representative pictures of Pd-PNS nanogels (CR = 5%) in the sol phase and gel phase. The minimum concentration at which the temperature-sensitive sol−gel transition occurred was 6 wt %. Rheological properties (G′, G″, and tan α) of Pd-PNS nanogels at the CR values of (E) 5% and (F) 100%, and the concentration of Pd-PNS nanogels was 10 wt %.

Figure 4. Pd(II) loading capacity and Pd(II) release behavior of Pd-PNS nanogels at different CR values. (A) Evaluation of the drug-loading (DL) amount and entrapment efficiency (EE) of Pd-PNS. (B) Influence of CR values on Pd(II) release behavior of Pd-PNS nanogels at 37 °C and pH 6.5. (C) Influence of pH value on Pd(II) release behavior of Pd-PNS nanogels at 37 °C.

2.3. Loading Capacity and in Vitro Release Profiles of Pd-PNS Nanogels. To prepare Pd-PNS nanogels, the coordination interaction between sulfonate units of PNS and Pd(II) ions was used as an efficient driving force to load Pd(II) ions using a simple one-step method. Figure 4A shows that the entrapment efficiency (EE) of Pd-PNS nanogels reached nearly 100% at 5% of CR and also 80% at 100% of CR. Moreover, PdPNS nanogel dispersions exhibited good colloidal stability in the CR range of 5−100%. Therefore, the drug-loading (DL) capacity of Pd-PNS nanogels could be easily tailored in the range of 0−40% by the simple mixing of K2Pd(NO3)4 and PNS solutions, facilitating the realization of clinical dosage

regimen individualization of Pd-PNS nanogels in intratumoral administration. Sustained release of water-soluble drugs is always a main limiting factor in enhancing antitumor efficacy and alleviating side effects of intratumoral chemotherapy. Therefore, in vitro Pd(II)-releasing behavior from various Pd-PNS nanogels was investigated at various pH values of media using a drug-eluting device, which was used for simulating intratumorally releasing properties.42,43 Pd-PNS nanogels showed favorable sustained release profiles. For instance, the concentrated dispersions (10 wt %) of Pd-PNS nanogels at 5% of CR released 27.5 ± 1.5% of total Pd(II) dose for 24 h and 62.4 ± 1.5% for 5 days E

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Figure 5. In vitro anticancer cell evaluation and mechanism study of Pt-PNS nanogels (CR = 5%) and Pd-PNS nanogels (CR = 5%) on sensitive MCF-7 and/or platinum-resistant MCF-7/Pt cells. The comparison of (A) cellular uptakes and (B) efflux of cisplatin, Pt-PNS nanogels, and Pd(II) and Pd-PNS nanogels between MCF-7 and MCF-7/Pt cells. n.s. represents no significance. Pt and/or Pd concentration was 0.03 mmol L−1 for (A) and (B). (C) Cell viability (CV) of MCF-7 and MCF-7/Pt treated with cisplatin, Pt-PNS nanogels (CR = 5%), and Pd(II) and Pd-PNS nanogels (CR = 5%). (D) Half-maximal inhibitory concentration (IC50) values and resistance factors derived from (C). (E) DNA damage, cell morphological variation, and nuclear shrinkage in MCF-7/Pt cell analysis by observing γH2A.X foci using confocal laser scanning microscopy (CLSM) (scale bar: 50 μm). (F) Morphology of 3D MCF-7/Pt CSC spheroids (scale bar: 20 μm) and (G) spheroid-forming number and area after treatment with different materials (untreated 3D MCF-7/Pt CSC spheroids were selected as control). Student’s t test, * P < 0.05, ** P < 0.01, *** P < 0.001.

1.2 and 48.7 ± 1.4% of total Pd(II) dose in the media of pH 6.5 and pH 7.4, respectively (Figure 4C). This was because hydrogen ions weakened the coordination bonds between Pd(II) ions and sulfonate groups, facilitating the release of Pd(II) in tumor acidic environments. 2.4. Cellular Uptake and Efflux of Pd-PNS Nanogels. It is well known that the hyposensitivity of resistant cancer cells to cisplatin was highly associated with downregulation of transport proteins (e.g., Ctr1) and upregulation of efflux proteins, such as P-glycoprotein (P-gp) and multidrug resistance-associated protein-1 (MRP-1), resulting in reduced

(Figure 4B). Accordingly, those of Pd-PNS nanogels at 100% of CR released only 19.3 ± 4.6% of total Pd(II) dose for 24 h and 42.4 ± 3.2% for 5 days, indicating reduced releasing rate of Pd-PNS nanogels with increasing CRs. This could be explained by the fact that the increasing CRs made the network structure of Pd-PNS nanogels more compact and effectively hindered the diffusion of Pd(II) ions from nanogel cores. In addition, Pd-PNS nanogels also showed the pH-dependent release of Pd(II) ions. The concentrated dispersions (10 wt %) of PdPNS nanogels at 5% of CR could release 71.5 ± 1.0% of total Pd(II) dose for 5 days in the media of pH 5.0 and only 62.4 ± F

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Figure 6. In vivo evaluation of overcoming of platinum resistance on MCF-7/Pt tumor-bearing BALB/c nude mice after regional chemotherapies. (A) Tumor growth profiles of MCF-7/Pt tumor-bearing BALB/c nude mice treated with different materials including saline, PNS, cisplatin, PtPNS nanogels, and Pd(II) and Pd-PNS nanogels (n = 5). (B) Photos and (C) weights of the tumors harvested from the mice after 14 days of different treatments (n = 5). (D) Histological sections stained using H&E and immunohistochemical sections labeled with TUNEL, Ki67, CD44, and CD133 after 14 days with different treatments (scale bar: 100 μm). (E) Comparison of average optical densities (ODs) of the slices as shown in (D). The administration dose of cisplatin or Pd(II) was 13.4 μmol kg−1. Student’s t test, * P < 0.05, ** P < 0.01, *** P < 0.001.

internalization of cisplatin.12,29,52 Therefore, the cellular uptake and efflux of K2PdCl4 and Pd-PNS nanogels were compared correspondingly to cisplatin and Pt-PNS nanogels (cisplatincoordination-cross-linking PNS nanogels, Figure S4) on MCF7-sensitive cells and MCF-7/Pt-resistant cells. Compared to the significant difference of cisplatin uptake, no difference was found in the uptake of K2PdCl4 between MCF-7-sensitive cells and MCF-7/Pt-resistant cells (Figure 5A). Interestingly, PtPNS nanogels seem to facilitate the cisplatin uptake of MCF7/Pt-resistant cells. However, there was still a difference in the platinum uptake for Pt-PNS nanogels between the sensitive and resistant cells. In all groups, Pd-PNS nanogels had the maximum internalization amount both by MCF-7-sensitive cells and MCF-7/Pt-resistant cells, and there was no difference between them. Furthermore, Pd-PNS nanogels also showed the minimum efflux amount by MCF-7/Pt-resistant cells among all treating groups (Figure 5B). These results suggested that Pd-PNS nanogels were unrecognized by platinumresistance-associated proteins, such as Ctr1, P-gp, and MRP1. To verify the reversal mechanism of platinum resistance, the platinum efflux amount was measured after MCF-7/Pt cells were co-cultured with the sample materials in the presence of

verapamil, an inhibitor for P-gp and MRP-1.53 The platinum efflux amount of cisplatin and Pt-PNS nanogels significantly decreased owing to the inhibitory effect of verapamil on P-gp and MRP-1 (Figure S5); however, the palladium efflux amount of K2PdCl4 and Pd-PNS nanogels in the presence of verapamil had no difference with those in the absence of verapamil, indicating an adverse platinum resistance of Pd(II) ions by enhanced intracellular accumulation. Furthermore, the endocytosis pathway of Pd-PNS nanogels was investigated by measuring intracellular Pd content in the presence of various inhibitors [5-(N-ethyl-N-isopropyl)amiloride (EIPA) (an inhibitor of micropinocytosis), chlorpromazine (CPZ) (an inhibitor of clathrin-mediated endocytosis), and incubation at 4 °C]. As shown in Figure S6, in contrast to highly adenosine 5′-triphosphate (ATP)-dependent endocytosis pathway of free cisplatin and K2PdCl4, Pt-PNS nanogels and Pd-PNS nanogels seem to show a complicated endocytosis pathway, which was mediated by micropinocytosis, clathrin, and ATP in an integrated manner. In addition, there were no differences of relative Pd(II) amount internalized by MCF-7 and MCF-7/Pt cells, suggesting distinct internalization pathways of Pd(II) from cisplatin. G

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Figure 7. In vivo prolonged retention and biocompatibility evaluations of Pt-PNS nanogels (CR = 5%) and Pd-PNS nanogels (CR = 5%) on MCF7/Pt tumor-bearing BALB/c nude mice. In vivo biodistribution of Pt and Pd in the major tissues, plasma, and tumors at (A) 1 day and (B) 14 days after different treatments (n = 3). The administration dose of cisplatin or Pd(II) was ca. 268 nmol per mouse. (C) Body weight curves of MCF-7/ Pt tumor-bearing BALB/c nude mice after different treatments including saline, PNS, cisplatin, Pt-PNS nanogels, and Pd(II) and Pd-PNS nanogels (n = 5). (D) Histological sections of the major tissues stained with H&E after 14 days of different treatments (scale bar: 200 μm, red arrows indicated the glomerular swelling). The evaluation of renal function 14 days postinjection of different materials from three serum biochemical levels, including (E) blood urea nitrogen (BUN), (F) creatinine (CREA), and (G) lactate dehydrogenase (LDH) (n = 5). Student’s t test, * P < 0.05, ** P < 0.01, *** P < 0.001.

2.5. In Vitro Cytotoxicity of Pd-PNS Nanogels on MCF-7/Pt-Resistant Cells and CSC Spheroids. In vitro cytotoxicities of Pd-PNS nanogels were compared to those of cisplatin, Pt-PNS nanogels, and K2PdCl4 on MCF-7-sensitive cells and MCF-7/Pt-resistant cells. The IC50 values of cisplatin, Pt-PNS nanogels, K2PdCl4, and Pd-PNS nanogels on MCF-7sensitive cells were 0.043, 0.065, 0.063, and 0.092 mmol L−1 respectively, whereas those on MCF-7/Pt-resistant cells were 0.249, 0.144, 0.075, and 0.095 mmol L−1 respectively (Figure 5C,D). The resistant factors (RFs), which were the ratio of IC50 between MCF-7-sensitive and MCF-7/Pt-resistant cells, were 5.781 for cisplatin, 2.259 for Pt-PNS nanogels, 1.192 for K2PdCl4, and 1.026 for of Pd-PNS nanogels. This indicated that Pd-PNS nanogels showed the strongest cytotoxicity on platinum-resistant cells, that is, an outstanding reversal effect on platinum resistance. DNA damage assays including immunofluorescence and western blotting also indicated that compared to cisplatin and Pt-PNS nanogels, Pd-PNS nanogels

and K2PdCl4 were able to induce more DNA double-stranded breaks (Figure 5E, γH2A.X channels, Figure S7),9,54 resulting in nuclear shrinkage and pyknosis of MCF-7/Pt-resistant cells [Figure 5E, 4′,6-diamidino-2-phenylindole (DAPI) channels]. Recently, CSCs or tumor-initiating/reinitiating cells were extensively reported to play a key role in cancer metastasis and recurrence, owing to their high resistance to chemotherapy.8−10,55 Therefore, the inhibition activities of Pd-PNS nanogels on tumor growth and proliferation were investigated with the CSC-enriched spheroids formed in 3D fibrin gels.56 The CSC-enriched spheroids treated by Pd-PNS nanogels were significantly smaller than those treated by cisplatin and Pt-PNS nanogels (Figures 5F and S8), indicating the good inhibitory effect of Pd-PNS nanogels on CSC growth. In addition, Pd-PNS nanogels also showed better inhibition activity on CSC proliferation than cisplatin and Pt-PNS nanogels, owing to fewer colony numbers of CSC-enriched H

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plasma after the treatment with cisplatin and K2PdCl4 were significantly higher than those treated with Pt-PNS and PdPNS nanogels. Moreover, higher tumor retention abilities of Pt-PNS nanogels and Pd-PNS nanogels were also observed after 14 days of treatment. The palladium contents were 11.1 ± 3.7% (K2PdCl4) and 49.7 ± 10.3% (Pd-PNS nanogels) (Figure 7B). It is well known that NIPAM-based polymers showed good biocompatibility.46,60 In the present work, the cytotoxicity of PNS polymers was first evaluated by the 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay with NIH 3T3 cells, MCF-7 cells, and MCF-7/Pt cells. The cell viability was above 80% after incubation with PNS polymers at the concentrations from 50 to 800 mg L−1 for 24 h (Figure S9), suggesting their good biosafety. Moreover, the cytotoxicity of Pd-PNS nanogels (IC50 was 0.096 mmol L−1) on NIH 3T3 cells was much lower than those of free K2PdCl4 (IC50 was 0.048 mmol L−1) and cisplatin (IC50 was 0.034 mmol L−1), indicating that the nanogels assembled with PNS efficiently decreased the toxicity of small molecular Pt and Pd compounds (Figure S10A). In addition, the hemolysis ratios (HRs) of Pd-PNS nanogels were lower than 0.7% (Figure S10B), which were far lower than those of Pd(II) and cisplatin (up to 2.5%). Based on the excellent prolonged tumor retention ability of Pd-PNS nanogels, the in vivo biosafety of Pd-PNS nanogels was evaluated using body weight, histological investigation, and serum biochemistry assay. Figure 7C shows that there was no obvious difference in the body weight of BALB/c nude mice after 14 days of treatment via intratumor injection. It was widely reported that platinum compounds often bring about severe side effects, such as nephrotoxicity, neurotoxicity and gastrointestinal toxicity.4,61,62 In Figure 7D, the glomerular swelling was found in the mice treated with cisplatin and K2PdCl4 and was not found in those treated with Pt-PNS and Pd-PNS nanogels, suggesting good nephrotoxicity suppression of PNS-assembled nanogels. In addition, renal function assays, including BUN, CREA, and LDH, also indicated that Pt-PNS and Pd-PNS nanogels could efficiently reduce the nephrotoxicity induced by small molecular platinum or palladium compounds (Figure 7E−G). This could be attributed to good local retention ability of PNS-assembled nanogels for metal ions.

spheroids after the treatment of Pd-PNS nanogels, as well as K2PdCl4 (Figures 5G and S8). 2.6. In Vivo Evaluation of Pd-PNS Nanogels on MCF7/Pt Tumor-Bearing BALB/c Nude Mice. The in vivo antitumor efficacy of Pd-PNS nanogels was evaluated on BALB/c nude mice bearing MCF-7/Pt-resistant tumors. A single dose of cisplatin, Pt-PNS nanogels (10% of PNS, 5% of CR), K2PdCl4, and Pd-PNS nanogels (10% of PNS, 5% of CR) were intratumorally injected at 13.4 μmol kg−1 when the tumor volume reached to ca. 100 mm3. As shown in Figure 6A, the tumor growth rate of the mice increased to only 1.5 ± 0.6% during 14 days after treatment with Pd-PNS nanogels, far less than that of those treated with K2PdCl4 (4.3 ± 1.1%), Pt-PNS nanogels (9.0 ± 2.3%), cisplatin (17.5 ± 2.8%), PNS (19.6 ± 4.1%), and saline (21.8 ± 4.7%), suggesting the long-term antitumor effect of Pd-PNS nanogels on MCF-7/Pt-resistant tumors due to the sustained release and intratumoral retention of palladium ions. Accordingly, the tumor weight of the mice was only 0.11 ± 0.03 g at 14 days after the treatment with PdPNS nanogels and reached up to 0.22 ± 0.05 g (K2PdCl4), 0.51 ± 0.09 g (Pt-PNS nanogels), 0.94 ± 0.14 g (cisplatin), 1.11 ± 0.22 g (PNS), and 1.11 ± 0.18 g (saline) at 14 days after the treatment (Figure 6B,C). The hematoxylin and eosin (H&E)-stained section of MCF-7/Pt tumors treated by PdPNS nanogels indicated that there were some wide necrotic areas, the disorganization of tumor tissues, and fewer surviving tumor cells, whereas many surviving tumor nidi were found in the tumors treated by the other materials, especially cisplatin, PNS, and saline. Moreover, the terminal deoxynucleotidyl transferase deoxyuridine-5′-triphosphate nick-end labeling (TUNEL)-staining slices of the mice treated by Pd-PNS nanogels showed the brightest green fluorescence, indicating their strong effect to induce apoptosis of tumor cells. Likewise, the lowest intensity of red fluorescence was found in the Ki67stained slices of the mice treated by Pd-PNS nanogels, showing their good ability in proliferation suppression of MCF-7/Pt tumor-resistant cells (Figure 6D,E). In addition, immunofluorescence analysis of CD44 and CD133, two CSC markers associated with proliferation, migration, and angiogenesis,10,57−59 suggested that the tumor slices of the mice treated by Pd-PNS nanogels had the lowest intensity of red fluorescence and highly significant differences compared with those treated by K2PdCl4, cisplatin, and Pt-PNS nanogels (Figure 6D,E), further confirming good CSC toxicity of PdPNS nanogels. 2.7. In Vivo Distribution and Biocompatibility of PdPNS Nanogels. The intratumoral retention of chemotherapeutics (especially water-soluble cisplatin and K2PdCl4) played a key role in the enhanced antitumor efficacy and reduced toxicity on normal tissues. The metal contents of platinum and palladium in major normal tissues, including heart, liver, spleen, lung, kidney, and plasma, and tumors were measured at 1 and 14 days after the mice were treated with cisplatin, Pt-PNS nanogels, K2PdCl4, and Pd-PNS nanogels using an inductively coupled plasma optical emission spectrometer (ICP-OES) (Figure 7A,B). As shown in Figure 7A, 81.7 ± 17.8% of Pt and 79.9 ± 12.2% of Pd were detained in tumors on the first day after treatment with Pt-PNS and PdPNS nanogels, respectively. In sharp contrast, only 36.6 ± 14.8% of Pt and 29.4 ± 15.8% of Pd were detained after the treatment with cisplatin and K2PdCl4, respectively. Besides, there were significant differences between both of them. Furthermore, the metal contents in major normal tissues and

3. CONCLUSIONS Bivalent palladium [Pd(II)] compounds were widely reported to have promising antitumor efficacy on platinum-resistant tumor cells owing to their coordination chemistry similar to platinum. To achieve a sustained release and long-term retention of highly water-soluble K2PdCl4 in regional chemotherapy, in this work, the temperature-sensitive triblock PNS polymer was first synthesized (MW was 64.1 kDa, and the SS/ NIPAM ratio was 1.0) and used as a temperature-sensitive ligand for the nanogel assembly coordinated with Pd(II) ions. By a simple method of coordination assembly, the resulting temperature-sensitive Pd-PNS nanogels, confirmed by FTIR and XPS, were constructed with a well-defined structure and composition in the CR range of 0−100% and achieved up to 80% of EEs and 40% of DLs. The hydrodynamic diameters of Pd-PNS nanogels decreased from 1097.1 ± 12.4 to 158.4 ± 6.5 nm and the ζ potentials increased from −41.9 ± 1.0 to −4.4 ± 1.2 mV as the CRs increased from 0 to 100%, owing to the formation of coordination bonds. Pd-PNS nanogels showed I

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were purchased from Sea Run Holdings, Inc., Freeport, Maine. All reagents were used as received, except for special instructions. All glassware used in the experiments was cleaned with a 1:1 deionized (DI) water/aqua regia solution and rinsed with ultrapure water twice. Milli-Q Ultrapure water (DI water) (18.2 MΩ) was used in all of the experiments. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of the Science and Technology Department (Hubei Province, China) and approved by the Animal Ethics Committee of Huazhong University of Science and Technology. 4.2. Synthesis of the PNS Triblock Polymer. The triblock polymer of PNS was synthesized by classical ATRP reaction. In brief, NIPAM (2.26 g, 20 mmol) was dissolved in 10 mL of the mixed solvent of DMSO/DI water (4/1 of v/v) contained in a Schlenk tube under stirring. Afterward, the reaction system was passed sequentially through liquid nitrogen refrigeration, vacuum, and argon-filling for two cycles. Then, BiBOEDS (30.5 μL, 0.1 mmol) and Me6TREN (54 μL, 0.2 mmol) were added into the reaction system quickly under an argon atmosphere. After another cycle of liquid nitrogen refrigeration, vacuum, and argon-filling, CuCl (20 mg, 0.2 mmol) was added to the frozen solvent to initiate ATRP. After 48 h of reaction at 60 °C, the thick and crude pNIPAM200 was obtained. Then, 20 mL of degassed DMSO solution of SS (4.582 g, 20 mmol) was added to the above reaction system using an argon-filled syringe and reacted for another 48 h at 110 °C. Afterward, the crude PNS triblock polymer was obtained and purified through dialyzing against DI water for 3 days. Finally, the refined PNS polymers were obtained through lyophilization and preserved in a vacuum desiccator. The molecular compositions of PNS were characterized using a nuclear magnetic resonance spectrometer (600 MHz, AV400, Bruker, Switzerland) with D2O as the solvent. The molecular weights were measured using Malvern Viscotek gel permeation chromatography (GPC) with 0.1 mol L−1 sodium nitrate aqueous solution containing a few drops of ammonia as the mobile phase at a flow rate of 0.9 mL min−1 at 30 °C. The concentrations of PNS dissolved in the mobile phase were all 2.0 mg mL−1, and PEG was used as the standard sample. 4.3. Preparation of Pd(II)-Cross-Linked PNS Nanogels (PdPNS Nanogels). First, K2PdCl4 was stoichiometrically mixed with AgNO3 ([AgNO3]/[K2PdCl4] = 4.0) for 12 h at 30 °C in dark for replacing chloride ions with nitrate ions to form K2Pd(NO3)4. Afterward, the resultant milky suspension was centrifuged (8000 rpm, 10 min) to remove AgCl, and the supernatant solution containing K2Pd(NO3)4 was further purified by passing through a 0.22 μm filter membrane. The concentration of K2Pd(NO3)4 ions was measured by ICP-OES (PerkinElmer Ltd.) and further reduced to 50 mmol L−1 for the later experiments. Pd(II)-coordination-cross-linking PNS nanogels (Pd-PNS nanogels) were prepared by the dropwise addition of the K2Pd(NO3)4 solution (50 mmol L−1) into PNS solution (10 wt %, pH 8.0), in terms of various coordination ratios (CRs). Taking 5% of CR as an example, 0.61 mL of K2Pd(NO3)4 solution was dropwise added into PNS polymer solution. After stirring and reacting at 25 °C for 24 h, the resultant Pd-PNS nanogels were purified and concentrated to 10 wt % by centrifugation (6000 rpm × 8 min) using an ultrafiltration tube (molecular weight cut-off: 10 000 Da) three times. As a control, cisplatin-coordination-cross-linking PNS nanogels (Pt-PNS nanogels) were prepared following the same protocol. 4.4. Structural Characterization of Pd-PNS Nanogels. The morphology of Pd-PNS nanogels was observed using a transmission electron microscope (TEM, Tecnai G2 20, FEI Corp., Netherlands) at 200 kV. The functional groups of K2Pd(NO3)4, PNS, and Pd-PNS nanogels were confirmed by Fourier transform infrared (FTIR) spectroscopy (VERTEX 70, Bruker Corp., Germany). The elemental compositions of K2Pd(NO3)4, PNS, and Pd-PNS nanogels and their bonding energy shifts were determined using X-ray photoelectron spectroscopy (XPS, AXIS-ULTRA DLD-600W, Shimadzu-Kratos Corp., Japan). 4.5. Temperature Sensitivity of Pd-PNS Nanogels. The transmittance of PNS and Pd-PNS nanogels (5 mg mL−1 of PNS concentration) was determined using a varying-temperature UV/vis

favorable thermosensitive sol−gel transition behavior and their LCSTs were below body temperature (37 °C), indicating that they had good syringeability. In vitro MTT assays, including the assay with CSC spheroids, indicated that Pd-PNS nanogels could inhibit the palladium efflux by P-gp and MRP-1 and thus enhance the DNA damage and toxicity to cisplatin-resistant MCF-7/Pt cells. Based on their temperature-sensitive sol−gel transition, furthermore, Pd-PNS nanogels showed sustained release profiles and long-term retention in tumors. Owing to these advantages of prolonged retention, escaping efflux, and efficient CSCs killing ability, Pd-PNS nanogels were promising to be developed as new temperature-sensitive delivery nanoplatforms for injectable regional chemotherapy on platinum-resistant cancers.

4. MATERIALS AND METHODS 4.1. Experimental Reagents and Animals. N-Isopropylacrylamide (NIPAM, purity: >98.0%, Tokyo Chemical Industry, Japan) was recrystallized from n-hexane. CuCl (Sinopharm Chemical Reagent Co., Ltd., China) was purified by dissolving in concentrated HCl solution and filtering after diluting with water before use. Tris(2dimethyl-aminoethyl)amine (Me6TREN, purity: >99.0%) was purchased from Alfa Aesar, Shanghai, China. Sodium chloride (NaCl, purity: ≥99.5%), Tween 20, concentrated hydrochloric acid (HCl, purity: 36−38%), concentrated nitric acid (HNO3, purity: 65.0− 68.0%), and dimethyl sulfoxide (DMSO, purity: >99.0%) were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. The standards of poly(ethylene glycol) (PEG) with weightaverage molecular weights (Mw) of 7290, 11 900, 33 600, and 72 000 Da; peak molecular weights (Mp) of 7750, 12 300, 37 100, and 69 800 Da; and number-average molecular weights (Mn) of 6770, 11 300, 33 000, and 70 000 Da were purchased from Shanghai Ziqibio Co., Ltd., Shanghai, China. Sodium p-styrenesulfonate (SS, purity: 90%), perchloric acid (HClO4, purity: 70.0−72.0%), potassium tetrachloropalladate (K2PdCl4, Pd ≥ 32.6%), and cis-diammineplatinum dichloride (cisplatin, Pt content >65.0%) were purchased from Shanghai Macklin Biochemical Co., Ltd., China. Platinum standard solution (1000 μg mL−1) and palladium standard solution (1000 μg mL−1) were purchased from Aladdin Reagent Co., Ltd., China. Silver nitrate (AgNO3, purity: >99.8%) was purchased from Aladdin Industrial Corporation. Bis[2-(2′-bromoisobutyryloxy)-ethyl]disulfide (BiBOEDS, purity: >97.0%), 5-(N-ethyl-N-isopropyl)amiloride (EIPA), and chlorpromazine (CPZ) were purchased from SigmaAldrich, St. Louis. Dulbecco’s modified Eagle’s medium (DMEM) medium, Roswell Park Memorial Institute (RPMI) 1640 medium, and fetal bovine serum (FBS) were purchased from Gibco BRL/Life Technologies, Grand Island, NY. Penicillin−streptomycin (100×) and SuperSignal West Femto Maximum Sensitivity Substrate (developing solution) were purchased from Thermo Fisher Scientific, Massachusetts. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) and enhanced bicinchoninic acid (BCA) protein assay kit (5000 T) were purchased from Yeasen Biotechnology Co., Ltd., Shanghai, China. 2-Amino-2-(hydroxymethyl)-propane-1,3-diol (Tris base, purity: 99.9%) and bovine serum albumin (BSA, purity: ≥98%) were purchased from BioFroxx, Germany. P-Histone H2A.X mAb (20E3) Rabbit mAb was purchased from Cell Signaling Technology (CST), Inc., Massachusetts. Fluorescein isothiocyanate (FITC)conjugated goat anti-rabbit IgG (H + L) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) were purchased from Servicebio Co., Ltd., Wuhan, China. 4′,6-Diamidino-2-phenylindole (DAPI), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Gel Quick Preparation Kit, and immunoprecipitation assay (RIPA) lysis buffer were purchased from Beyotime Biotechnology Co., Ltd., Shanghai, China. Nitrocellulose blotting membrane (Amersham Protran 0.45 μm NC, 0.45 μm, 300 mm × 4 m) was purchased from GE Healthcare Life Sciences. Verapamil hydrochloride (purity: 99.0%) was purchased from Finetech, Co., Ltd., Wuhan, China. Salmon fibrinogen (100 mg) and thrombin (1000 U) J

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viability (CV) of cells was measured by standard MTT assay and following eq 4

spectrophotometer (Lambda 35, PerkinElmer) at the wavelength of 500 nm. The hydrodynamic diameters and mean count rates of PdPNS nanogels (2 mg mL−1 of PNS concentration) with the increasing temperature from 25 to 55 °C or from 25 to 45 °C were determined using dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern Instruments Ltd., U.K.). The low critical solution temperatures (LCSTs) and volume phase transition temperatures (VPTTs) were defined, respectively, as the temperatures at the maximum slope of the curves of transmittance−temperature and size−temperature. The dynamic rheological properties of concentrated dispersions were evaluated by a stress-controlled rheometer (Kinexus ultra+, Malvern Instrument Ltd., U.K.) with a parallel plate (PP50, ⌀ = 50 mm, the gap was set at 0.5 mm) in the temperature range of 25−50 °C with the following parameters: The stress was 0.1 Pa, the frequency was 1.0 Hz, and the heating rate was 5 °C min−1. 4.6. Pd(II) Loading and in Vitro Release of Pd-PNS Nanogels. The Pd(II)-loading capacity of PNS was evaluated by two major parameters of drug-loading (DL) amount and encapsulation efficiency (EE). In brief, 50 μL of Pd-PNS nanogel dispersions with different K2Pd(NO3)4 feeding amounts was digested with the mixture solution of HNO3 (65−68%, 2.0 mL) and HClO4 (70−72%, 0.15 mL) at 350 °C on an electrical warming plate (TP-4, Longkou Zhongcun Chunhua Electric Appliance Factory, China). After 30 min, the digested solutions got clear and were diluted to 10 mL with DI water for measuring palladium concentration using ICPOES at the wavelength of 244.8 nm. The standard curve was measured with a series of palladium standard samples with concentrations of 0, 0.01, 0.1, 0.5, 1.0, 5.0, and 20.0 ppm. The DL and EE of PNS were calculated following eqs 1 and 2, respectively. DL% =

Cf · Vf C p·Vp

C ·V EE% = f f W0

CV =

HR =

(1)

ODs − ODn × 100% ODp − ODn

(5)

Here, ODs, ODp, and ODn are the OD values of samples, positive control, and negative control, respectively. 4.8. Cellular Uptake and Efflux of Platinum and Palladium by MCF-7 Cells and MCF-7/Pt Cells. To evaluate the cellular uptake, MCF-7 cells and MCF-7/Pt cells were seeded in six-well plates at a density of 3 × 105 cells per well in 2 mL of media. After incubation in a 5/95% CO2/O2 atmosphere at 37 °C for 12 h, the media were replaced with 2 mL of cisplatin, Pt-PNS, K2PdCl4, and Pd-PNS in serum-free DMEM media or RPMI 1640 media, at Pt or Pd concentration of 0.03 mmol L−1. After being cultured for another 24 h, the media were removed and the cells were washed with cold PBS three times. Then, the cells were digested with 0.5 mL of trypsin and collected for cell counting. Finally, the cells were digested with 2 mL of the mixture of HClO4 and HNO3 (v/v = 1/4) for measuring the cellular uptake using ICP-OES. Moreover, the endocytic pathways of different materials were investigated. In brief, MCF-7 cells and MCF-7/Pt cells were seeded in six-well plates at a density of 3 × 105 cells per well in 2 mL of media. After incubation in a 5/95% CO2/O2 atmosphere at 37 °C for 12 h, the cells were treated with PBS (Control), 50 μM EIPA (macropinocytosis inhibitor), and 10 μg mL−1 CPZ (clathrin-mediated uptake inhibitor) in serum-free RPMI 1640 media for 1 h at 37 or 4 °C. Afterward, the cells were incubated with different materials and the above inhibitors at 37 or 4 °C for another 2 h. Then, the cells were washed with cold PBS three times and collected for measuring intracellular drug concentration. To evaluate the cellular efflux, after incubation with different materials for 12 h, the cells were washed with cold PBS three times and divided into two parts. One part of cells was continuously cultured in fresh media for another 12 h, whereas the other part of cells was collected for measuring the initial platinum or palladium amount using ICP-OES. After 12 h, the cultured cells were washed with cold PBS three times and collected for measuring the final platinum or palladium amount using ICP-OES. The efflux amount of cisplatin or K2PdCl4 was calculated by subtracting the final amount from the initial amount of cisplatin or K2PdCl4. It is known that MCF-7/Pt cells highly express P-gp and MRP-1, which highly correlates with platinum resistance. To study whether these efflux

(2)

m

∑i = 1 CiVi C0V0

(4)

Here, ODs, ODb, and ODn are the OD values at the wavelength of 492 nm of samples, blank control, and negative control, respectively. The hemolysis test was performed as we reported previously.43 In brief, 1 mL of the whole blood of healthy BALB/c nude mice was collected from the orbit and added into 2 μL of 2% heparin sodium contained in a 10 mL EP tube with gentle blending. After adding 1 mL of saline, the mixture was centrifuged (1500 rpm × 10 min). Saline (5 mL) was added into the EP tube for re-suspension of erythrocytes after the supernatant was removed. The obtained suspension was further purified by centrifugation (1500 rpm × 20 min) three times. Finally, 200 μL of the purified erythrocytes was added into 9.8 mL of saline to prepare 2% erythrocyte saline suspension for measuring the hemolysis. After incubation at 37 °C for 10 min, 200 μL of 2% erythrocyte saline suspension was added into PNS saline dispersion (200 μL, 100 mg mL−1), cisplatin saline solution (4.66 mg mL−1, 200 μL), Pt-PNS saline solution [CR = 5%, 100 mg mL−1 (calculated by PNS), 200 μL], K2PdCl4 saline solution (5.07 mg mL−1, 200 μL), Pd-PNS saline solution [CR = 5%, 100 mg mL−1 (calculated by PNS), 200 μL], normal saline (200 μL, negative control), and ultrapure water (200 μL, positive control). The cocultured systems were centrifuged (1500 rpm × 10 min) after continuous incubation at 37 °C for 1 h. Besides, the supernatant was collected for measuring optical density (OD value) at the wavelength of 540 nm. The hemolysis ratio (HR) was calculated following eq 5

Here, W0 is the feeding amount of K2PdCl4 and Cf and Vf are the concentration and volume of K2PdCl4 contained in Pd-PNS nanogel dispersions, respectively. Cp and Vp are the concentration and volume of Pd-PNS nanogel dispersions. A self-made drug-eluting device was prepared for imitating the release behavior of Pd-PNS nanogels via intratumoral injection as we have reported.42,43 Briefly, 100 μL of Pd-PNS nanogel dispersions (10 wt %) with various CRs was injected into the release cell, and the sol− gel transition occurred at 37 °C for 5 min. Then, phosphate-buffered saline (PBS) with different pH values (5.0, 6.5, and 7.4) was used as an eluent to elute the released Pd(II) ions. The eluting solutions were collected every 12 h until 5 days, and they were sequentially numbered as 1, 2, 3,..., i. The Pd contents of these samples were measured using ICP-OES, and the accumulative releasing amount (AR) of Pd(II) ions was calculated following eq 3 AR =

ODs − ODb × 100% ODn − ODb

(3)

Here, Ci and Vi are the concentration and volume of Pd(II) ions from the sample i, respectively. C0 is the total Pd(II) concentration in PdPNS nanogels, and V0 is the volume of Pd-PNS nanogels. 4.7. In Vitro Biocompatibility Evaluation of PNS and Pd-PNS Nanogels. The cytotoxicity of PNS and Pd-PNS nanogels was evaluated on mouse embryonic fibroblast NIH 3T3, MCF-7, and MCF-7/Pt cells. Briefly, NIH 3T3, MCF-7, or MCF-7/Pt cells were incubated in 96-well plates at the density of 1 × 104 cells in 200 μL DMEM media (containing 10% FBS and 1% penicillin−streptomycin) per well. After incubation in a 5/95% CO2/O2 atmosphere at 37 °C for 12 h, the media were removed and cells were washed with cold PBS three times. Then, 200 μL of PNS solution dissolved in serumfree DMEM media with the concentrations of 50, 100, 200, 400, and 800 mg L−1 was added into corresponding wells. Each concentration was made in triplicate. After incubation for another 24 h, the cell K

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Chemistry of Materials proteins identified palladium, after incubation with different materials for 12 h, the cells were washed with cold PBS three times and then continuously cultured in fresh media containing verapamil (40 μg mL−1, an inhibitor of P-gp and MRP-1) for another 12 h.53 4.9. In Vitro Evaluation of the Overcoming Platinum Resistance of Pd-PNS Nanogels. The overcoming platinum resistance of Pd-PNS nanogels was evaluated on cisplatin-sensitive MCF-7 cells and platinum-resistant MCF-7/Pt cells. MCF-7 cells were cultured with DMEM media (containing 10% FBS and 1% penicillin−streptomycin), whereas MCF-7/Pt cells were cultured with RPMI 1640 media (containing 10% FBS and 1% penicillin− streptomycin). In brief, MCF-7 and MCF-7/Pt were incubated in 96-well plates at a density of 1 × 104 cells per well in 200 μL of media. After incubation in a 5/95% CO2/O2 atmosphere at 37 °C for 12 h, the media were replaced with 200 μL of cisplatin, Pt-PNS, K2PdCl4, and Pd-PNS dispersed in serum-free DMEM media or RPMI 1640 media in a series of concentrations. After being cultured for another 24 h, the media were removed and the cells were washed with cold PBS two times. Then, the cell viability was measured following a standard MTT assay and calculated following eq 4. Also, the resistant factors (RFs) of different materials were calculated following eq 6 RF =

MCF‐7/Pt (IC50) MCF‐7 (IC50)

with gentle shaking and then incubated with 5 mL of P-Histone H2A.X mAb (20E3) Rabbit (diluted 1000 times with 5% BSA TBS-T solution) at 4 °C overnight. After being washed with TBS-T three times (15 min at a time with gentle shaking), 5 mL of HRPconjugated goat anti-rabbit IgG (H + L) (diluted 5000 times with 5% BSA TBS-T solution) was added into every dish and incubated at room temperature for 40 min. After being washed with TBS-T three times (15 min at a time with gentle shaking), the positive protein bands were developed using developing solution and a Bio-Rad ChemiDoc XRS+ chemiluminescent system (Bio-Rad Laboratories, Hercules). 4.11. Cytotoxicity to 3D MCF-7/Pt CSC Spheroids. To investigate the cytotoxicity to CSCs, 3D MCF-7/Pt CSC spheroids were obtained following a previously reported method.56 Briefly, 1.0 mL of MCF-7/Pt cell suspensions (1.2 × 104 cells mL−1) in RPMI 1640 media was mixed in each well of 96-well plates with 1.0 mL of fibrinogen solutions in T7 buffer (2 mg mL−1, pH 7.4, 50 mM Tris, 150 mM NaCl) under ice-bath conditions. Thrombin (2 μL, 0.1 U μL−1) and 50 μL of the mixture of MCF-7/Pt cells and fibrinogen were added into each well of 96-well plates while stirring. Subsequently, 200 μL of RPMI 1640 (containing 10% FBS and 1% penicillin−streptomycin) was added into each well after incubation at 37 °C for 15 min. After being cultured for 4 days, the RPMI 1640 medium was replaced with sample solutions and continuously cultured for 24 h. Then, the morphology of 3D MCF-7/Pt CSC spheroids was observed and photographed using optical microscopy. 4.12. In Vivo Evaluation of the Overcoming Platinum Resistance of Pd-PNS Nanogels. In vivo evaluation of overcoming platinum resistance was studied on MCF-7/Pt tumor-bearing BALB/c nude mice. Briefly, an MCF-7/Pt subcutaneous tumor model was established in BALB/c nude mice (5 week old, female, SPF, 16 ± 2 g, Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) by inoculating MCF-7/Pt tumor cells in the right flank at a cell density of 1.0 × 106 cells per 100 μL. Until the tumors reached approximately 100 mm3, the MCF-7/Pt tumor-bearing BALB/c nude mice were divided into six groups randomly and five mice per group. Afterward, a single intratumoral injection of 50 μL of saline, PNS dispersion (100 mg mL−1 in saline), cisplatin solution (13.4 μmol kg−1 Pt content, in saline), Pt-PNS nanogel dispersion (13.4 μmol kg−1 Pt content, in saline), K2PdCl4 solution (13.4 μmol kg−1 Pd content, in saline), and Pd-PNS nanogel dispersion (13.4 μmol kg−1 Pd content, in saline) was injected into tumor tissues of the mice. The tumor volumes (V) were measured daily during 14 days of the experimental cycle using a Vernier caliper according to the equation of V = (L × W2)/2. At the end of the experiment, the mice were executed by cervical dislocation, and the major tissues and tumors were collected for histologic analysis and immunohistochemical analysis. 4.13. In Vivo Distribution and Serum Biochemistry Assay. An MCF-7/Pt tumor-bearing BALB/c nude mice model was established as mentioned above. PBS (50 μL), PNS nanogel dispersion (100 mg mL−1 in saline), cisplatin solution (13.4 μmol kg−1 Pt content, in saline), Pt-PNS nanogel dispersion (13.4 μmol kg−1 Pt content, in saline), K2PdCl4 solution (13.4 μmol kg−1 Pd content, in saline), and Pd-PNS nanogel dispersions (13.4 μmol kg−1 Pd content, in saline) were injected intratumorally as the tumor volume increased to ca. 100 mm3. Postinjection 1 and 14 days, three mice from each group were executed by cervical dislocation. The major tissues, blood, and tumors were harvested and digested for measuring the corresponding contents of Pt or Pd using ICP-OES. To perform the serum biochemistry assay, the plasma samples were collected from the mice 14 days postinjection and divided into two parts. One part of the plasma sample was used for measuring the contents of Pt or Pd, and the other part was used for the serum biochemistry assay. 4.14. Statistical Analysis. The data were expressed as mean ± standard deviation. The measurement data was analyzed statistically by the independent-sample t-test and double-factor variance analysis, and the enumeration data was treated with the χ2 test and Fisher’s exact probability test. * P < 0.05 was considered a statistically

(6)

Here, MCF-7/Pt(IC50) and MCF-7(IC50) indicate the IC50 values of different materials to MCF-7/Pt and MCF-7 cells, respectively. 4.10. DNA Damage Assay. The antitumor mechanism of K2PdCl4 was investigated by detecting DNA damage of MCF-7/Pt cells using cell immunofluorescence and western blotting. For cell immunofluorescence, MCF-7/Pt cells were seeded in confocal dishes at a density of 1 × 105 cells per dish in 2 mL of media. After being cultured at 37 °C for 12 h, the media were removed and replaced with 2 mL of serum-free RPMI 1640 media containing K2PdCl4 or Pd-PNS nanogels at the Pd concentration of 0.1 mmol L−1. After being cultured for another 24 h, the RPMI 1640 media were removed and the cells were washed with cold PBS three times. The treated MCF-7/ Pt cells were fixed with cold methanol at −20 °C for 20 min. After being washed with cold PBS three times, 0.5 mL of 3% BSA TBS-T [12.13 mg mL−1 Tris, 9 mg mL−1 NaCl, 6.67 mL L−1 concentrated HCl, and 2 mL L−1 Tween 20 (0.2%)] solution was added into each dish and incubated for 15 min for blocking. After removing 3% BSA TBS-T solution, 180 μL of P-Histone H2A.X mAb (20E3) Rabbit (diluted 150 times with 3% BSA TBS-T solution) was added into each dish and incubated at room temperature for 1.5 h, and then the cells were washed with TBS-T three times and 150 μL of FITCconjugated goat anti-rabbit IgG (H + L) (diluted 150 times with 3% BSA TBS-T solution) was added into every dish and incubated at room temperature for 30 min. After being washed with TBS-T three times, the MCF-7/Pt cells were stained with DAPI (5 μg mL−1, 150 μL) for 15 min at room temperature. After being washed with PBS six times, the cells were observed using confocal laser scanning microscopy (CLSM) to determine the degree of DNA damage. Western blotting was performed according to the previous report.63,64 MCF-7 and MCF-7/Pt were incubated in 12-well plates at a density of 5 × 105 cells per well in 2 mL of media. After being cultured at 37 °C for 12 h, the media were removed and replaced with 2 mL of serum-free RPMI 1640 media containing K2PdCl4 or Pd-PNS nanogels at the Pd concentration of 0.1 mmol L−1. After being cultured for another 24 h, the RPMI 1640 media were removed and the cells were washed with cold PBS three times. Then, the cells were incubated with 0.1 mL of RIPA lysis buffer at 4 °C for 30 min. Afterward, the lysed cells were collected into 1.5 mL EP tubes and centrifuged (12 000 rpm × 20 min) to obtain the total proteins at 4 °C. Protein quantification was performed using an enhanced BCA assay protein assay kit. The protein samples were mixed with loading buffer and boiled at 100 °C for 10 min to denature the proteins. Total proteins were separated using 10% SDS-PAGE followed by transferring to nitrocellulose blotting membranes. The membranes were blocked with 5% BSA TBS-T solution for 1 h at room temperature L

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Chemistry of Materials significant difference, and *** P < 0.001 was considered highly statistically significant.

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(5) Yang, X.; Du, X.; Liu, Y.; Zhu, Y.; Liu, Y.; Li, Y.; Wang, J. Rational Design of Polyion Complex Nanoparticles to Overcome Cisplatin Resistance in Cancer Therapy. Adv. Mater. 2014, 26, 931− 936. (6) Wang, W.; Liang, G.; Zhang, W.; Xing, D.; Hu, X. CascadePromoted Photo-Chemotherapy against Resistant Cancers by Enzyme-Responsive Polyprodrug Nanoplatforms. Chem. Mater. 2018, 30, 3486−3498. (7) Liang, X.; Meng, H.; Wang, Y.; He, H.; Meng, J.; Lu, J.; Wang, P. C.; Zhao, Y.; Gao, X.; Sun, B.; Chen, C.; Xing, G.; Shen, D.; Gottesman, M. M.; Wu, Y.; Yin, J.; Jia, L. Metallofullerene Nanoparticles Circumvent Tumor Resistance to Cisplatin by Reactivating Endocytosis. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 7449−7454. (8) Lytle, N. K.; Barber, A. G.; Reya, T. Stem Cell Fate in Cancer Growth, Progression and Therapy Resistance. Nat. Rev. Cancer 2018, 18, 669−680. (9) Xia, H.; Li, F.; Hu, X.; Park, W.; Wang, S.; Jang, Y.; Du, Y.; Baik, S.; Cho, S.; Kang, T.; Kim, D. H.; Ling, D.; Hui, K. M.; Hyeon, T. pH-Sensitive Pt Nanocluster Assembly Overcomes Cisplatin Resistance and Heterogeneous Stemness of Hepatocellular Carcinoma. ACS Cent. Sci. 2016, 2, 802−811. (10) Wang, H.; Agarwal, P.; Zhao, G.; Ji, G.; Jewell, C. M.; Fisher, J. P.; Lu, X.; He, X. Overcoming Ovarian Cancer Drug Resistance with a Cold Responsive Nanomaterial. ACS Cent. Sci. 2018, 4, 567−581. (11) Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J. The Next Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle Delivery, and Pt(IV) Prodrugs. Chem. Rev. 2016, 116, 3436−3486. (12) Li, Y.; Deng, Y.; Tian, X.; Ke, H.; Guo, M.; Zhu, A.; Yang, T.; Guo, Z.; Ge, Z.; Yang, X.; Chen, H. Multipronged Design of LightTriggered Nanoparticles To Overcome Cisplatin Resistance for Efficient Ablation of Resistant Tumor. ACS Nano 2015, 9, 9626− 9637. (13) Browning, R. J.; Reardon, P. J. T.; Parhizkar, M.; Pedley, R. B.; Edirisinghe, M.; Knowles, J. C.; Stride, E. Drug Delivery Strategies for Platinum-Based Chemotherapy. ACS Nano 2017, 11, 8560−8578. (14) Ma, P.; Xiao, H.; Li, C.; Dai, Y.; Cheng, Z.; Hou, Z.; Lin, J. Inorganic Nanocarriers for Platinum Drug Delivery. Mater. Today 2015, 18, 554−564. (15) Xiao, H.; Yan, L.; Dempsey, E. M.; Song, W.; Qi, R.; Li, W.; Huang, Y.; Jing, X.; Zhou, D.; Ding, J.; Chen, X. Recent Progress in Polymer-Based Platinum Drug Delivery Systems. Prog. Polym. Sci. 2018, 87, 70−106. (16) Wang, X.; Guo, Z. Targeting and Delivery of Platinum-Based Anticancer Drugs. Chem. Soc. Rev. 2013, 42, 202−224. (17) Arvizo, R. R.; Bhattacharyya, S.; Kudgus, R. A.; Giri, K.; Bhattacharya, R.; Mukherjee, P. Intrinsic Therapeutic Applications of Noble Metal Nanoparticles: Past, Present and Future. Chem. Soc. Rev. 2012, 41, 2943−2970. (18) Ndagi, U.; Mhlongo, N.; Soliman, M. E. Metal Complexes in Cancer Therapy - An Update from Drug Design Perspective. Drug Des., Dev. Ther. 2017, 11, 599−616. (19) Jia, P.; Ouyang, R.; Cao, P.; Tong, X.; Zhou, X.; Lei, T.; Zhao, Y.; Guo, N.; Chang, H.; Miao, Y.; Zhou, S. Review: Recent Advances and Future Development of Metal Complexes as Anticancer Agents. J. Coord. Chem. 2017, 70, 2175−2201. (20) Graf, N.; Lippard, S. J. Redox Activation of Metal-Based Prodrugs as a Strategy for Drug Delivery. Adv. Drug Delivery Rev. 2012, 64, 993−1004. (21) Fanelli, M.; Formica, M.; Fusi, V.; Giorgi, L.; Micheloni, M.; Paoli, P. New Trends in Platinum and Palladium Complexes as Antineoplastic Agents. Coord. Chem. Rev. 2016, 310, 41−79. (22) Alam, M. N.; Huq, F. Comprehensive Review on Tumour Active Palladium Compounds and Structure-Activity Relationships. Coord. Chem. Rev. 2016, 316, 36−67. (23) Amir, M. K.; Khan, S. Z.; Hayat, F.; Hassan, A.; Butler, I. S.; Rehman, Z. Anticancer Activity, DNA-Binding and DNA-Denaturing Aptitude of Palladium(II) Dithiocarbamates. Inorg. Chim. Acta 2016, 451, 31−40.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.9b00986.

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Transmittance changes with temperature of PNS, XPS of K2PdCl 4, and K2Pd(NO3)4; mean count rate vs temperature curves of Pd-PNS nanogels; characterization of Pt-PNS; efflux of Pd(II) by MCF-7/Pt after treatment with verapamil; cellular uptake pathways of Pd-PNS, 3D MCF-7/Pt CSC spheroids after treatment with Pd-PNS; cytotoxicity of PNS on mouse embryonic fibroblast NIH 3T3, MCF-7, and MCF-7/Pt cells; biocompatibility evaluations of Pt-PNS including cytotoxicity and hemolysis; and molecular weight and composition ratio of the PNS (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.Z.). *E-mail: [email protected] (X.Y.). ORCID

Hao Zhao: 0000-0002-5848-9457 Jiabao Xu: 0000-0002-3802-8311 Yanbing Zhao: 0000-0002-0675-6680 Author Contributions †

H.Z. and J.X. contributed equally to this work.

Author Contributions

H.Z. and J.X. conceived the project and designed the experiments. H.Z. and J.X. synthesized materials and performed in vitro experiments. H.Z., J.X., and W.H. carried out in vivo experiments. H.Z. and J.X. collected and analyzed the data. H.Z. and J.X. wrote the manuscript. H.Z., Y.Z., and X.Y. contributed to the revision. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant nos. 81673016 and 81773653) and the National Basic Research Program of China (grant no. 2018YFA0208903). We also thank the Analytical and Testing Center of HUST, the Research Core Facilities for Life Science (HUST) for the related analysis.

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