Nanoceria-Mediated Drug Delivery for Targeted Photodynamic

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Nanoceria-Mediated Drug Delivery for Targeted Photodynamic Therapy on Drug-Resistant Breast Cancer Hong Li, Cong Liu, Yiping Zeng, Yuhui Hao, Jiawei Huang, Zhangyou Yang, and Rong Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07338 • Publication Date (Web): 22 Sep 2016 Downloaded from http://pubs.acs.org on September 23, 2016

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ACS Applied Materials & Interfaces

Nanoceria-Mediated Drug Delivery for Targeted Photodynamic Therapy on Drug-Resistant Breast Cancer Hong Li, † Cong Liu, † Yi-Ping Zeng, † Yu-Hui Hao, † Jia-Wei Huang, † Zhang-You Yang, †,* and Rong Li †,* †Institute of Combined Injury, State Key Laboratory of Trauma Burns and Combined Injury,

Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Thi rd Military Medical University, Chongqing 400038, China. KEYWORDS: ceria nanoparticles, targeted photodynamic therapy, drug resistance, oncosis, breast cancers ABSTRACT Photodynamic therapy (PDT) has shown great potential for overcoming drug-resistant cancers. Here, we report a multifunctional drug delivery system based on chlorin e6 (Ce6)/folic acid (FA)-loaded branched polyethylenimine-PEGylation ceria nanoparticles (PPCNPs-Ce6/FA), which was developed for targeted PDT to overcome drug-resistant breast cancers. Nanocarrier delivery and FA targeting significantly promoted the cellular uptake of photosensitizers (PSs), followed by their accumulation in lysosomes. PPCNPs-Ce6/FA generated reactive oxygen species (ROS) after near infrared irradiation (NIR, 660 nm), leading to reduced P-glycoprotein (P-gp) expression, lysosomal membrane permeabilization (LMP) and excellent phototoxicity toward resistant MCF-7/ADR cells, even at ultralow doses. Moreover, we identified NIR-triggered lysosomal-PDT using the higher dose of PPCNPs-Ce6/FA, which stimulated cell death by plasma membrane blebbing, cell swelling and energy depletion, indicating an oncosis-like cell death pathway, despite the occurrence of apoptotic or autophagic mechanisms at lower drug doses. In

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vivo studies showed prolon ged blood circulation times, low toxicity in mice, and high tumor accumulation of PPCNPs-Ce6/FA. Additionally, using NIR-triggered PDT, PPCNPs-Ce6/FA displayed excellent potency for tumor regression in the MCF-7/ADR xenograft murine model. This study suggested that multifunctional PPCNPs-Ce6/FA nanocomposites are a versatile and effective drug delivery system that may potentially be exploited for phototherapy to overcome drug-resistant cancers, and the mechanisms of cell death induced by PDT should be considered in the design of clinical protocols. Introduction Drug resistance remains the major challenge in oncology with respect to treatment failure in metastatic cancer after repeated sessions of chemotherapy.1, 2 Several mechanisms have been confirmed to contribute to drug resistance such as alterations in the drug target, activation of prosurvival pathways, DNA damage repair, and ineffective induction of cell death.3, 4 Above all, alterations in drug efflux due to ATP-binding cassette (ABC) transporter proteins such as P-glycoprotein (P-gp), which is overexpressed in many tumors and can actively transport drugs out of cells, has most often been linked to drug resistance.5, 6 During the past decades, many efforts have been devoted to the inhibition of drug-efflux pumps.2 Resistance induced by one treatment might be overcome by another treatment.7 Therefore, several new treatment strategies, such as photodynamic therapy (PDT), have been developed to overcome drug-resistant cancers. PDT is a noninvasive medical technology based on photochemistry to treat numerous diseases such as solid tumors.8 During this process, light-activatable chemicals (photosensitizers, PSs), which accumulate in tumor cells, are energized by light of the appropriate wavelength and transfer photon energy to biological substrates to generate reactive oxygen species (ROS), leading to cytotoxicity.9-11 As a Pure Food and Drug Administration (FDA)-approved therapeutic modality, PDT exhibits highly localized tissue damage and relatively minimal side effects.12 Recently, PDT has been demonstrated to


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cause the photodestruction of ABC transporter protein through elevating the intracellular ROS and activation of Jun N-terminal kinase (JNK) or p38 mitogenactivated protein kinase (MAPK) pathway.7,

13-16

The reduction of ABC transporter protein expression facilitates the cytosolic

delivery of chemotherapeutic agents that do not otherwise readily enter cells, result in a synergistic effect with chemotherapy.17-19 This technique provides significant advantages for overcoming a variety of drug-resistant cancers, including head and neck cancers,20 breast cancers,21, 22 prostate cancers,23 and uterine sarcoma.18 However, the use of PDT is associated with different problems due to hydrophobic characteristics, a lack of selectivity and poor tumor targeting of the PSs, hindering the administration and efficiency of the treatment.12 To increase the overall efficacy of PDT, various nanosized carriers have been developed for the delivery of PSs by taking advantage of their high surface area per unit volume, selective targeting, and long circulation time.24-29 The use of nanocarriers, such as polymeric nanoparticles, magnetic nanoparticles, dendrimers, liposomes, and quantum dots, can deliver PSs to the tumor site in a more selective form with low toxicity to normal tissues.30-32 Rare-earth ceria nanoparticles (CNPs) have received considerable attention because of their pharmacological potential and excellent catalytic activities,33 and they have emerged as a fascinating and lucrative material in biological fields such as bioanalysis,34 biomedicine,35 and drug delivery.36 More importantly, CNPs behave optimal antioxidant properties at physiological pH but turn on their oxidases activity in the acidic microenvironment of tumors, which encourage the application of anti-tumor.37,

38

We have pioneered the development of PEGylated ceria

nanoparticles (PCNPs), which act as an ideal radiation-protective agent,39, 40 and recently reported branched polyethylenimine (BPEI)-coated PCNPs (PPCNPs) for the delivery of Ce6 to cervical cancer cells and imaging-guided synchronous photochemotherapy.41 We hypothesize that the excellent catalytic properties, the germination of electron transfer between nanoparticles and Ce6, and/or the high ability of oxygen store of nanoceria, since the lattice defects structure and the oxygen vacancies in nanoceria contribute to the active electronic configuration and facilitated loss/obtain of oxygen and/or its electrons,33, 42 may be responsible for the excellent phototoxicity



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of Ce6 conjugated CNPs. The subcellular targets of PSs impact the outcome of PDT,7 and lysosomes may be the optimal target to improve the efficacy of PDT, since the targeting of lysosomes with PDT may result in punching holes in the lysosomal membrane and eliminating the potentially protective cellular pathway.43 The direct lethal effects of PDT on cells were initially shown to results from the loss of a series of pro-survival proteins, leading directly to cell death.44 The autophagy stimulated by PDT may either function in a cytoprotective manner or induce autophagic cell death,45 depending on the type of ROS and the degree of oxidative injury.46 PDT with high doses may also lead to necrosis.7 In the present study, as indicated in Scheme 1, a multifunctional drug delivery system based on chlorin e6 (Ce6)/folic acid (FA)-loaded PPCNPs (PPCNPs-Ce6/FA) with satisfactory biocompatibility was synthesized. The nanocarrier significantly promoted tumor selectivity and enhanced the cellular uptake of PSs, followed by accumulation in the lysosomes of drug-resistant human breast cancers, leading to excellent PDT efficacy in vitro and vivo. In addition, we observed cell damage induced by PDT resulting from apoptotic or autophagic mechanisms at lower doses of PPCNPs-Ce6/FA, which evolved as an oncotic pattern of injury at higher doses accompanied by lysosomal membrane permeabilization (LMP), cellular membrane blebbing, cell and organelle swelling, cytoplasmic vacuole accumulation and intracellular adenosine triphosphate (ATP) depletion, indicating an oncosis-like manner of cell death.


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Scheme 1. Schematic illustration of the preparation of PPCNPs-Ce6/FA and the application of targeted PDT to overcome drug-resistant breast cancers. Results and Discussion Synthesis and Characterization. CNP synthesis and PEGylation were performed as described in our previous reports.39, 40 The BPEI, which can provide a positive charge, was chiefly conjugated onto PCNPs via the reaction between the carboxyl group of PEG and the amino group of BPEI.38, 47

Transmission electron microscope (TEM) imaging indicated that the PPCNPs were 3-5 nm in

size and exhibited excellent dispersion in water (Figure 1a). Ce6 and FA were then conjugated to PPCNPs via amidation between the carboxyl group of Ce6/FA and the amino group of BPEI. The ultraviolet (UV)-vis absorbance spectra analysis showed that PPCNPs did not exhibit



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characteristic absorption peaks of in the 400-800 nm range. The absorption spectrum of PPCNPs-Ce6 was similar to that of Ce6 (characteristic peaks: 404 nm and 550 nm), whereas that of PPCNPs-Ce6/FA had the characteristics of both Ce6 and FA (characteristic peak: 280 nm) (Figure 1b), indicated successful loading of Ce6 and FA in PPCNPs. The conjugations among Ce6, FA, and nanoparticles were further confirmed by fourier transform infrared (FT-IR) spectroscopy (Figure S1b). The concentrations of Ce6 and cerium (Ce) in PPCNPs-Ce6 were determined through a standard curve via UV-vis spectroscopy analysis and inductively coupled plasma-mass spectrometry (ICP-MS), respectively, and the loading efficiency of Ce6 vs Ce was approximately 37% (weight ratio). However, the fluorescence of Ce6 was dramatically quenched following the conjugation of Ce6 to PPCNPs (Figure 1c), which might be attributed to the electron transfer between Ce6 and PPCNPs. As expected, PPCNPs-Ce6 and PPCNPs-Ce6/FA exhibited excellent stabilities in various physiological solutions without showing any noticeable aggregation (Figure S1). Meanwhile, the hydrodynamic diameters of PPCNPs-Ce6/FA in phosphate-buffered saline (PBS), 1640 medium and 10% fetal bovine serum (FBS) were detected by a dynamic light scattering (DLS) test. The mean hydrodynamic sizes of PPCNPs-Ce6/FA in 1640 medium (52.9 nm) and 10% FBS (57.6 nm) were slightly increased compared with those in PBS (36.1 nm) (Figure S2a-c), indicated PPCNPs-Ce6/FA was stable in these solutions. The size change of nanoparticles might be attributed to the BPEI in the outer shell with positive charge which could interact with the small molecules or proteins in cell medium or FBS. In addition, the ζ potentials of the PPCNPs, PPCNPs-Ce6, and PPCNPs-Ce6/FA were 22.5±5.2 mV, 11.6±3.7 mV, and 8.8±2.4 mV respectively (Figure S2d), which were attributed to the -NH2+ of BPEI on the surface of nanoceria. The positive surface charges may provide more opportunities for cell uptake of nanoparticles.38 The generation of the ROS singlet oxygen (1O2) upon photoactivation of the PSs is critical for high levels cytotoxicity, and it directly impacts the outcome of PDT.48 The 1O2-producing


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capacities of free Ce6, PPCNPs-Ce6 and PPCNPs-Ce6/FA were detected by excitation of singlet-oxygen-sensor green (SOSG) fluorescence. The 1O2 production ability of PPCNPs-Ce6 and PPCNPs-Ce6/FA remained substantially high, at approximately 74% and 80% of the free Ce6, respectively (Figure 1d), even if the fluorescence of Ce6 was dramatically quenched by the conjugation of Ce6 to PPCNPs. These results suggested that PPCNPs could be used as a potential platform for loading Ce6 and FA to apply the available PDT.

Figure 1. Characterizations of various materials. (a) TEM imaging of PPCNPs. (b) UV-vis



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spectra of FA, Ce6, PPCNPs, PPCNPs-Ce6 and PPCNPs-Ce6/FA in PBS. (c) Fluorescence emission spectra of Ce6, PPCNPs, PPCNPs-Ce6 and PPCNPs-Ce6/FA. (d) SOSG fluorescence intensity at 530 nm of Ce6, PPCNPs, PPCNPs-Ce6 and PPCNPs-Ce6/FA at 3 min post-NIR irradiation (660 nm, 100 mW/cm2).

Figure 2. Cellular uptake and subcellular localization of PPCNPs-Ce6/FA. (a) DOX fluorescence in MCF-7 and MCF-7/ADR cells incubated with DOX (0.25 µg/mL) for different durations (measured by flow cytometry). (b-d) Flow cytometry measurements of Ce6 fluorescence in MCF-7/ADR cells incubated with free Ce6 (b), PPCNPs-Ce6 (c), and PPCNPs-Ce6/FA (d) at an equivalent Ce6 concentration (2 µM) for different durations. (e) Changes in the Ce6 fluorescence intensity in cells exposed to different treatments; *p

Nanoceria-Mediated Drug Delivery for Targeted Photodynamic

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