Nanolongan with Multiple On-Demand Conversions for Ferroptosis

Subscriber access provided by Iowa State University | Library

Article

Nanolongan with Multiple On-Demand Conversions for Ferroptosis-Apoptosis Combined Anticancer Therapy Weier Bao, Xianwu Liu, Yanlin Lv, Gui-Hong Lu, Feng Li, Fan Zhang, Bin Liu, Dan Li, Wei Wei, and Yuan Li ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b05602 • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Nanolongan with Multiple On-Demand Conversions for Ferroptosis-Apoptosis Combined Anticancer Therapy Weier Baoa,b,c,‡, Xianwu Liua,b,c, ‡, Yanlin Lv b, Gui-Hong Lu b, Feng Li b, Fan Zhang b, Bin Liua, Dan Lia, Wei Weib *, Yuan Lia * aBeijing

Advanced Innovation Center for Food Nutrition and Human Health, Key Laboratory of

Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, 100083, Beijing, China. bState

Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese

Academy of Sciences, 100190, Beijing, China. cCollege

of Life Science and Technology, Beijing University of Chemical Technology, 100029,

Beijing, China. ‡W. E. Bao and X. W. Liu contributed equally to this work.

Keywords: nanolongan, multiple on-demand responsive, NIR-responsive, ferroptosis, anticancer, combined therapy

ACS Paragon Plus Environment

1

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 43

ABSTRACT

As a type of programmed cell death, ferroptosis is distinct from apoptosis. Their combination thus provides a promising modality to significantly improve anticancer efficacy. To fully utilize this combination, we herein designed a nanolongan delivery system, which possessed a typical structure of one core (upconversion nanoparticles, UCNP) in one gel particle (Fe3+ cross-linked oxidized starch) with multiple on-demand conversions. The charge conversion of nanolongan surface in slightly acidic microenvironment enhanced circulation time for utilizing the EPR effect, enabled efficient uptake by tumor cells and induced subsequently lysosomal escape. As the core component, the UCNP with light conversion from near infrared light to ultraviolet light circumvented the impediment of limited penetration depth and enabled the reduction of Fe3+ to Fe2+. Accordingly, gel networks of nanolongan could be deconstructed due to this valence conversion, leading to the rapid release of Fe2+ and doxorubicin (Dox). In this case, Fenton reaction between Fe2+ and intracellular H2O2 generated potent reactive oxygen species for ferroptosis, while the co-released Dox penetrated into nucleus and induced apoptosis in a synergistic way. As a result, superior anticancer therapeutic effects were achieved with little systemic toxicity, indicating our nanolongan could serve as a safe and high-performance platform for ferroptosis-apoptosis combined anticancer therapy.

As most cancer patients are diagnosed at an advanced stage, chemotherapy remains the standard care in the clinic. After this treatment, tumor cells, in most cases, are expected to be killed via apoptosis.1 However, the therapeutic effect of utilizing chemo-drug alone has been disappointing due to low accumulation in tumor regions, side effects to normal tissues, and increasingly serious multidrug resistances.2 To improve the therapeutic effect, researchers have


2

Page 3 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

gradually explored the possibility to combine chemotherapy with other therapeutic modalities, which can address different targets.3, 4 For example, radiotherapy has been widely used in association with docetaxel, cisplatin, or 5-fluorouracil, which can prolong survival time and reduce recurrence rate.5, 6 In yet another aspect, mono-antibody, such as bevacizumab,7 trastuzumab,8 and cetuximab,9 have also been synergistically applied with series of chemo-drugs, and the drug specificity and resistance can be ameliorated.10 These clinical practices demonstrate the importance of finding therapeutic modalities for the combination with chemotherapy. Recently, ferroptosis has been discovered as a type of programmed cell death.11 In this process, Fenton reaction between Fe3+ and intratumoral H2O2 efficiently generates potent reactive oxygen species (ROS),12-14 leading to the accumulation of lipid peroxides.15, 16 As ferroptosis is distinct from apoptosis, their combination sheds a light on the way to fight against cancer. Unfortunately, such a promising strategy has rarely been studied. In order to achieve this combination therapy, the prerequisite is to co-deliver the drug pair (i.e., chemo-drug and iron element) into tumor.17 One of the most popular approaches for delivery is the construction of nanocarriers, which can passively enrich at tumor site through the enhanced permeability and retention (EPR) effect.18 With further functionalization, nanocarriers can also be endowed with the capacity of active targeting, which will further increase the delivery performance.19, 20 In spite of these achievements, a series of special considerations should be taken for the development of nanocarriers used for combination therapy. In terms of delivery performance, the drug pair should be precisely transported to the cytoplasm, where the iron element can encounter H2O2 to promote the Fenton reaction21 and the chemo-drug can target subcellular sites. In this aspect, the primary strategy of prolonging circulation and improving cancer cells uptake have not yet fully succeeded.22, 23 The nanocarriers should be further


3

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 43

endowed capacity to escape from the lysosome, where they are always entrapped after internalization.24 As the final mission for the nanocarriers, the release profiles should also be deliberated, especially for the iron element. To induce maximum phospholipid bilayer peroxidation, a high local ROS concentration should be achieved. This requires a rapid release of iron from the nanocarriers, and the form of Fe2+ is more favorable due to its superior performance in the Fenton reaction.25-27 Through these means, a large amount of singlet oxygen (O2•) and hydroxyl radicals (•OH) can be efficiently generated before these short-lived species (in microseconds levels) lose their activity.28, 29 In this work, we designed a nanolongan delivery system, which possessed a typical structure of one core in one gel particle (denoted as G) (Scheme 1). Typically, an upconversion nanoparticle (UCNP, denoted as U) and Doxorubicin (Dox) were encapsulated in an oxidized starch-based gel nanoparticle, which was sequentially crosslinked by Fe3+ ion and further decorated with Polyethyleneimine (PEI, denoted as P) and 2,3-dimethylmaleic anhydride (DMMA, denoted as D). Such a structure possessed charge, light and valence conversions, which could address the aforementioned concerns for ferroptosis-apoptosis combined anticancer therapy. In details, DMMA offered a negative charged surface after intravenous injection, which could prolong the circulation time and provide more opportunities to reach tumor site via EPR effect. Upon exposure to slightly acidic microenvironment at tumor site, the nanolongan was reversed to positive charge due to exfoliation of DMMA.30, 31 This conversion not only facilitated tumor internalization, but also induced a subsequent proton-sponge effect for lysosomal escape. With further near infrared light (NIR, denoted as L) irradiation, the UCNP featured with NIR to ultraviolet light (UV) conversion enabled the reduction of Fe3+ to Fe2+. This valence conversion deconstructed the gel network and achieved the rapid release of the drug pair. As a result, Fe2+


4

Page 5 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

reacted with H2O2 in cytoplasm to generate ROS for ferroptosis, whereas Dox diffused to the nucleus to induce apoptosis. These salient features unequivocally proclaim such a nanolongan with multiple on-demand conversions could serve in efficient ferroptosis-apoptosis combined anticancer therapy.

Scheme 1. Schematic illustration of nanolongan with multiply conversions and the corresponding anticancer mechanism. (A) The Fe3+ cross-linked structure of nanolongan carrier, containing UCNP as the core and Dox absorbed in the polymer shell. The nanolongan was formed by coordination of carboxyl groups on oxidized starch polymers with Fe3+ and further decorated with PEI and DMMA. (B) Corresponding anticancer mechanism for nanolongan. ① Passive accumulation of DGU: Fe/Dox nanolongan with a long circulation and improved EPR effect. ② pH-activated surface shift of negative DGU: Fe/Dox to positive GU: Fe/Dox nanolongan at tumor site (pH=6.8) for enhancing cellular uptake. ③ Lysosome escape of


5

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 43

GU: Fe/Dox nanolongan by proton sponge effect. ④ NIR-responsive deconstruction of nanolongan under the action of UCNP. ⑤ Apoptosis of released Dox in the nucleus. ⑥ Ferroptosis of ROS by the Fenton reaction of reduced Fe2+ with tumor cellular H2O2 at cytoplasm.

RESULTS AND DISCUSSION Characterizations of nanolongan delivery system. In our previous study, we developed a polysaccharide microgel based on the coordination between COO- groups in oxidized polysaccharides (Figure S1) and Fe3+ ions.32, 33 On the absorption of a UV photon, the Fe (Ⅲ) complexes undergo a charge transfer from the ligand to the metal center. During this process, one electron of the metal center is reduced, and an electron of the donor (coordination ligand) is oxidized,34, 35 leading to the conversion of Fe3+ to Fe2+.36, 37 In this case, DG: Fe microgel without UCNP could be deconstructed after UV irradiation due to decreased binding strength between Fe2+ and COO- groups, while the microgel remained complete under NIR irradiation (Figure S2). This response not only could tackle the problem of delivery instable Fe2+, but also showed the potential for light-triggered release. However, such an excitation with UV light suffered from the limited penetration depth in vivo. To circumvent this impediment, UCNPs capable of converting NIR photons into UV photons were encapsulated in the gel as light transducers (Figure S3).38, 39 Under NIR irradiation, reduced Fe2+ and loaded drug thus could be rapidly released due to the gel (contained UCNP) deconstruction (Figure S4), serving for the on-demand ferroptosisapoptosis combination therapy (Scheme 1). Considering nanosize requirements for utilizing the EPR effect and more space for payloads, we herein proposed to construct a typical structure of one UCNP core in one nanogel. By optimizing Dox loading efficiency and UCNP quantity in G


6

Page 7 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(Figure S5 and Figure S6), we successfully obtained the desired nanolongan structure. After Dox loading, the nanolongan was decorated with DMMA with the assistance of PEI, which could be verified by slightly increased particle size and significantly changed zeta potentials (Table S1). The resultant DGU: Fe/Dox nanocarriers were systematically characterized. As shown in Figure 1A and Figure S7, each dark UCNP was individually surrounded with a light gel shell of Fe3+ cross-linked oxidized starch, exhibiting a typical “nanolongan” structure. This typical architecture was also confirmed by elemental mapping (Figure 1B), corresponding line scanning (Figure 1C) and energy dispersive X-ray spectroscopy (EDX) (Figure 1D). Further quantitative analysis revealed the weight percentages of each component (Figure 1E), wherein the ratios of Fe and Dox were 11.84% and 10.07%, respectively. The DGU: Fe/Dox nanolongan had an approximately 125.8 nm diameter (Figure 1F) with a narrow size distribution (PDI=0.127), which favored the EPR effect. During one week storage, little change was observed to the particle size and zeta potential (Figure 1G), indicating a good colloidal stability for intravenous injection. Owing to UCNP encapsulation, two light windows with 350 nm and 475 nm peaks could be observed upon NIR irradiation (Figure 1H). Notably, UV emission with NIR irradiation enabled the reduction of Fe3+ to Fe2+ in vivo, which endowed the capacity for light-triggered release.


7

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 43

Figure 1. Characterizations of nanolongan carriers. (A) TEM image of nanolongans. (B) Element mapping images of nanolongan (C, Fe, O, Na, Yb, Y and Tm). (C) The distribution of


8

Page 9 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Fe and UCNP in nanolongan. (D) EDX element analysis of nanolongan. (E) The weight percentage of constituent components in nanolongan. (F) Size distribution of nanolongan in pH=7.4 Tris-HCl buffer (PDI=0.127). (G) Variations of zeta potential and average size of nanolongan in cell culture media over 7 days. (H) Fluorescence emission spectrum under NIR excitation and image of nanolongan solution under NIR laser irradiation. Data represent mean ± SD (n=3). Effects of charge conversion on cellular fate. As extravasation from the blood stream is a stochastic behavior, it has become a consensus that a longer blood circulation time can offer nanoparticles more opportunities to reach tumor site via the EPR effect. To evaluate nanoparticle stealth performance, equivalent numbers of nanolongans with different surface charges were individually incubated with J774A.1 macrophage cells for 24 h. As shown in Figure 2A, GU (without DMMA decoration) nanoparticles (indicated by red fluorescent dots) were largely internalized by the macrophage, which is a common feature for positive charged nanoparticles. Such a plentiful uptake by the macrophage would limit the time window for passive accumulation. On the contrary, the intracellular dots became much fewer in the presence of negative charged DGU formulation. Correspondingly, the internalization amount was less than 10% compared with GU, indicating a longer circulation time after intravenous injection. For verification, an additional in vivo verification experiment was performed by intravenously injecting mice with both types of formulation and then monitoring the time-elapsed change of blood concentration. As expected, the half-life of DGU was 3.6 folds longer than GU in vivo (Figure 2B), which could provide a sufficient period to utilize the EPR effect. Once the DGU arrived at the tumor site, they should be, in turn, efficiently internalized by tumor cells, which highly correlated with their subsequent killing effect. Owing to the slightly


9

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 43

acidic microenvironment at the tumor site, the negative charged DMMA could be exfoliated, exposing positive charged surface sourced from doped PEI (Figure S8). In this case, contrary requirement on the cellular uptake could be fulfilled. To demonstrate our proposal, we continued to evaluate the cellular uptake of DGU by breast carcinoma cells (4T1) at different pH environment (Figure 2C). At general physiological environment (pH=7.4), only a small amount of DGU were internalized into the 4T1 cells due to the repulsive force between the DGU and cell membrane. Once incubated at mimic tumor microenvironment (pH=6.8), the nanolongan rapidly switched to positive charged GU within 30 min (Figure 2D), and cellular uptake was improved up to 14.8 folds. In this case, much more Fe and Dox could be delivered into cancer cells to improve ferroptosis-apoptosis combined therapy. After internalization, foreign nanoparticles are always sequestered in lysosomal compartments. However, the cytoplasm and nucleus are desired locations where Fe ions and Dox could induce ferroptosis and apoptosis, respectively. In this case, only when GU escaped from lysosomes, could the payloads arrive at their subcellular targets. To clarify this, we next sought to reveal the cellular fate of the GU (Figure 2E). Taking 12 h as an example, a number of GU (indicated by red dots) were found in the cytoplasm, and the co-localization rate dropped to less than 40%. Such an efficient lysosomal escape could be attributed to the proton-sponge effect. Once in the acidic lysosome (pH=4.5-5.5), the positive charged PEI on GU became protonated. This would trigger a concurrent influx of chloride ions, leading to osmotic swelling and the physical rupture of the lysosomal membrane. For further verification, the effect of GU on the hemolysis of rabbit red blood cells (RBCs) was investigated. Similarly, we did observe a pHdependent hemolysis (Figure 2F), again demonstrating the superior capability of GU for membrane deconstruction in acidic microenvironment.


10

Page 11 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 2. Effects of charge conversion on cellular fate. (A) CLSM images and flow cytometry analysis of GU and DGU in J774A.1 cells after 24 h. (B) Pharmacokinetic profiles of GU and DGU in blood. (C) CLSM images and flow cytometry analysis of DGU in 4T1 cells after 24 h incubation at different pH. (D) The zeta potential of DGU in pH=6.8 and 7.4 Tris-HCl buffer


11

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 43

over 120 minutes. (E) CLSM image of GU escaping from lysosomes in 4T1 cells (Lyso-Tracker, green). Overlay analysis showed the co-localization information of lysosomes and GU. (F) RBC hemolysis for GU in Tris-HCl buffer of different pH after 30 min incubation. GU and DGU were labeled by Cy5 and presented as red dots in (A), (C) and (E). Green represents the cell membrane and blue indicates the nucleus in (A) and (C). Data represent mean ± SD (n=3). *P < 0.05, **P < 0.01. Evaluations of NIR responsive ability of nanolongan. Having achieved cytoplasm delivery, the rapid release of Fe2+ and Dox is the final requirement for our nanolongan to achieve best therapeutic effect. As shown in Figure 3A, a complete deconstruction of nanolongan was observed upon NIR irradiation, whereupon the particle size expanded from 125.8 nm to approximately 1225.9 nm (Figure 3B and 3C). Such a sensitivity could be attributed to the aforementioned Fe3+ reduction under UV light (Figure S9) transduced by UCNP. Correspondingly, we observed the rapid release of Fe2+ and Dox (Figure 3D and 3E), even in a controllable manner (Figure S10). Based on bleaching experiment of methylene blue (MB), Fe2+ exhibited higher Fenton reactivity for catalyzing H2O2 to •OH than Fe3+ (Figure 3F). For in vivo demonstration, we further investigated the production of ROS in 4T1 cellular environment by using DCFH probe (Figure 3G). Once upon NIR irradiation, the cells became much brighter, indicating the released Fe2+ had reacted with H2O2 in the cytoplasm to generate large amounts of ROS. Meanwhile, we also monitored Dox distribution and quantitatively analysis with high content equipment (Figure 3H and 3I). Without NIR irradiation, most Dox signals remained clear and presented as sharp dots because the GU: Fe/Dox remained stable in the cytoplasm. Once irradiated, the signal became dispersed and blurred, and the intensity at nuclear areas gradually increased, suggesting that Dox had been released and penetrated into the cell nucleus.


12

Page 13 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 3. Evaluations of NIR responsive ability of nanolongan. (A) Schematic illustration of nanolongan in response to NIR. The release of Dox and the generation of ROS were due to the reduction of Fe3+ to Fe2+, resulting from catalytic UV emitted by NIR under the action of UCNP. (B) TEM images of nanolongans before and after NIR irradiation. (C) Size distribution of nanolongan before and after NIR irradiation. (D) The transformation efficiency of Fe3+ to Fe2+ in nanolongan under NIR irradiation. (E) In vitro Dox release from nanolongan triggered by NIR irradiation. (F) Absorption intensity of methylene blue (MB) monitored at 664 nm with equal concentration of Fe3+ or Fe2+ over 6 h. Due to the presence of •OH, MB was bleached, which


13

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 43

was indicated by a discoloration from dark blue to almost white. The lower the MB intensity was, the higher the Fenton activity was. (G) Generation of ROS in the 4T1 cytoplasm by using DCFH after NIR irradiation. (H) Fluorescence intensity of released Dox in nucleus with or without NIR irradiation. (I) CLSM images showing Dox (red) entrance into nucleus (blue) before and after NIR irradiation. The nanolongans used in the above experiments were pretreated in pH=6.8 Tris-HCl buffer for the charge conversion. Data represent mean ± SD (n=3). *P < 0.05, **P < 0.01. In vitro ferroptosis-apoptosis and combined cytotoxicity. Encouraged by the plentiful ROS and Dox nucleus entry, we next investigated the ferroptosis and apoptosis of 4T1 cells in vitro. To mimic slightly acidic tumor microenvironment, we incubated the formulation with cells in the pH=6.8 cell culture medium. It is reported that ferroptosis can inhibit glutathione peroxidase 4 (GPX4), 40-42 a phospholipid peroxidase, which results in a lethal accumulation of lipid peroxides. Meanwhile, lipoxygenase enzyme (FACL4), which plays important role on the synthesis of long chain fatty acids for cell membrane, can also be suppressed.43, 44 In this case, GPX4 and FACL4 were utilized as two typical indicators for ferroptosis (Figure 4A and Figure S11). As a typical apoptosis inducer, Dox alone or encapsulated showed little effect on the expressions of GPX4 and FACL4. Owing to the low Fenton activity of crosslinked Fe3+, the expressions of these two indicators became slightly decreased in DGU: Fe and DGU: Fe/Dox groups. Once upon NIR irradiation, we could observe a significant suppression in DGU: Fe+L and DGU: Fe/Dox+L groups. Such a potent ferroptosis sourced from this released Fe2+ was further verified by the mitochondrial membrane shrinkage and normal nucleus (Figure S12).For the evaluation of apoptosis, we also carried out an Annexin V-FITC/PI assay via flow cytometry (Figure 4B). Free Dox could initiate certain apoptosis, while DGU: Fe alone had little effect.


14

Page 15 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Once DGU: Fe was utilized as carrier for Dox (DGU: Fe/Dox), the ratio of apoptotic cells got further increased due to the efficient internalization. After exposure at NIR irradiation, more cells became necrotic in the DGU: Fe/Dox+L group, which could be attributed to the rapid release of Dox. Next, we evaluated the combined cytotoxicity by CCK8 assay. As shown in Figure 4C and Figure S13, all formulations exhibited a dose-dependent cytotoxicity to 4T1 and MCF-7 cells. The cytotoxicity increased in the sequence of DGU: Fe, DGU: Fe+L, Dox, DGU: Fe/Dox, and DGU: Fe/Dox+L. Taking DGU: Fe/Dox+L group for the example, only less than 10% cells survived due to cooperation of ferroptosis and apoptosis. These results were further verified by using Calcein-AM and ethidium homodimer-1 as probes to indicate the live and dead 4T1 cells, respectively (Figure 4D). The red fluorescence from dead cells became intense in various degrees after being treated with different formulations, and almost all cells were dead in the DGU: Fe/Dox+L group. Notably, aforementioned cytotoxicity could be significantly weakened at neutral cell culture medium (Figure S14). Such a pH induced compromise, in turn, demonstrated the importance of utilizing DMMA to on-demand reverse charge for cancer cell uptake and subsequent therapeutic effect.


15

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 43


16

Page 17 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 4. Ferroptosis-apoptosis effects and corresponding cytotoxicity of various formulations. (A) The expression levels of ferroptosis representative proteins (GPX4 and FACL4) determined by simple western immunoblots. (B) The Annexin V-FITC/PI apoptosis detection analysis of 4T1 cells after treatment with different formulations after 24 h incubation by flow cytometry. (C) CCK-8 cytotoxicity analysis of 4T1 and MCF-7 cells treated with different formulations after 24 h incubation. (D) Live/dead cytotoxicity analysis of 4T1 cells after treatment with different formulations after 24 h incubation. Green represents the live cells and red indicates the dead cells. The nanolongans used in the above experiments were pretreated in pH=6.8 Tris-HCl buffer for the charge conversion. Data represent mean ± SD (n=3). *P < 0.05, **P < 0.01. In vivo biodistribution of nanolongan. Only when the payloads were precisely and plentifully delivered to tumor tissues, can they exert a therapeutic effect. So we continued to monitor the biodistribution of different nanolongan formulations in a 4T1-xenografted mouse model. To ascertain this, we replaced Dox with a fluorescent Cy7 dye and then traced the nanolongan in vivo after intravenous injection (Figure 5A). Without DMMA decoration, GU with positive charge was rapidly and largely enriched at the liver, and the signal at tumor site was not satisfactory. On the contrary, the fluorescence signal obtained from the liver clearly decreased, while the signal at the tumor site markedly and continuously increased with elapsing time, indicating the importance of DMMA decoration in prolonging circulation time and increasing tumor cell uptake. According to the in vivo imaging, the signal-to-noise (S/N) ratio in the tumor was also calculated (Figure 5B). After injection of GU, the ratio value was quite small (~0.5) and slightly increased at later time points. In contrast, the ratio in the DGU group constantly increased and reached 8.0 at 24 h. As a result, the fluorescence intensity of the excised


17

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 43

tumor in DGU group was 8.1 times higher compared with GU group (Figure 5C and Figure 5D). Similar results could also be found by the quantitative analysis of UCNP in tumor, again confirming a significantly improved tumor targeting delivery (Figure 5E).

Figure 5. Improved accumulation of nanolongan at tumor sites in 4T1 xenograft tumor mice model. (A) In vivo imaging of biodistribution of GU and DGU in tumor bearing mice model. (B) Signal-to-noise ratio in the tumors of various groups at 24 h postinjection. (C) Ex vivo images of tumors and other tissues at 24 h postinjection. (D) Fluorescence intensity of Cy7 replaced drug in visceral organs and tumor collected at 24 h postinjection. (E) Normalized


18

Page 19 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

UCNP in tumors of various group at different time intervals after postinjection. The amounts of UCNP in the tumors of DGU and GU at different times were normalized with the amount of UCNP in the GU group tumor at 24 h. Data represent the mean ± SD (n=6). *P < 0.05, **P < 0.01. In vivo therapeutic effect and safety. The abovementioned results prompted us to evaluate the therapeutic effects on 4T1 xenograft mice. Compared with PBS group, the treatment with DGU: Fe or Dox alone showed an unsatisfactory effect on tumor inhibition (Figure 6A). Once upon NIR irradiation (DGU: Fe+L), the released Fe2+ reacted with intratumoral H2O2 to induce ferroptosis, which could be verified by significantly decreased GPX4 and FACL4 expression level (Figure 6B). Correspondingly, a significantly inhibited tumor growth was observed. Owing to the cooperation of Dox induced apoptosis, which could be validated by the TUNEL assay (Figure 6C). DGU: Fe/Dox+L treatment further enhanced the therapeutic effect, and even achieved completely tumor elimination at higher dose (Figure S15), resulting in 100% survival rate at 55 days (Figure S16 and Figure S17). Consistent with aforementioned data, Ki 67 expression, a cell proliferation indicator, in tumor tissue decreased in the order of PBS, DGU: Fe, Dox, DGU: Fe+L, and DGU: Fe/Dox+L (Figure 6D). As metastasis to distant lung and bone are always observed for breast cancer in clinic, we also evaluated the antimetastases performance. Metastatic cells in lung and metastasis-induced erosion in bone occurred to different extents for other groups, while no sign of metastasis was observed in the mice treated with DGU: Fe/Dox+L (Figure 6E, 6F and Table S2).


19

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 43

Figure 6. In vivo combination of ferroptosis and apoptosis for anticancer therapy. (A) Growth inhibition of 4T1 tumor after treatment with the different formulations during 48 days.


20

Page 21 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(B) Immunohistochemical analysis of FACL4 and GPX4 in tumor tissue for ferroptosis after 48 days. (C) TUNEL analysis of tumor tissue indicating the cell apoptosis. (D) Ki 67 analysis of tumor tissue indicating cell proliferation. (E) Hematoxylin and eosin (HE) staining showing lung metastasis indicated by the yellow arrows. (F) CT scanning images showing bone metastasis at homolateral tibia (indicated by the red circles). In addition to the therapeutic effects, we also evaluated the safety of our nanolongan-based delivery system. As shown in Figure 7A, treatment with Dox exhibited typical toxicity in the heart, indicated by the cardiomyocyte vacuolation. In contrast, few histological abnormalities in main organs were found in DGU: Fe/Dox+L group due to the good delivery capacity to tumor. Meanwhile, blood samples were collected for the determination of serum biochemistry of AST, ALT, BUN, LDH, and ALP (Figure 7B). Among these parameters, AST, ALT and LDH were found with abnormally enhanced. On the contrary, the values returned to normal ranges in the DGU: Fe/Dox+L group, which suggested this treatment had effectively protected the mice from hepatic or other organ damage and further confirmed the safe use of our nanolongan for ferroptosis-apoptosis combined anticancer therapy.


21

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 43

Figure 7. In vivo toxicity evaluations of nanolongan. (A) H&E-stained slice images of major organs from different groups after 48 days. (B) Hematological analysis of treated mice after 48 days. Gray areas represent the normal range of different biosafety indicators.


22

Page 23 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

CONCLUSION In summary, we successfully constructed nanolongans with triple on-demand conversions for ferroptosis-apoptosis combined anticancer therapy. The charge conversion of nanolongan surface sourced from DMMA decoration not only provided long circulation for utilizing EPR effect, but also enabled the efficient uptake by tumour cells and subsequent lysosome escape. As the core, the UCNP with light conversion from NIR to UV circumvented the impediment of limited penetration depth and enabled the reduction of Fe3+ to Fe2+. Accordingly, the gel network of nanolongan could be deconstructed due to this valence conversion, leading to the rapid release of Fe2+ and Dox. As a result, we achieved superior anticancer therapeutic effect with the combination of ferroptosis and apoptosis, while few side effects were observed. These results together voted our nanolongan could serve as a safe and high-performance modality for ferroptosis-apoptosis combined anticancer therapy. MATERIALS AND METHODS Materials. The oxidized starch of degree of oxidation 90% (DO90 starch) was synthesized according to the method reported previously.32, 33 Doxorubicin (Dox, 99.5%), Ferrous sulfate (FeSO4·7H2O, 99.5%), 2, 3-dimethylmaleic anhydride (DMMA, 99.5%) were purchased from Sigma-Aldrich, USA. Polyethyleneimine (PEI, 50%), 2′, 7′-Dichlorofluorescin diacetate (DCFHDA) were purchased from Solarbio Biochemical Co. (Beijing, China). The liproxstatin-1 was purchased from Selleck Chemicals, USA. The upconversion nanoparticle (NaYF4: Yb/Tm, ultraviolet light emission, UCNP) was purchased from Suzhou yansheng bio-tech co. ltd. Trypsin-EDTA, Alexa Flour 488-phalloidin and Hoechst 33342 were purchased from Life technologies, USA. The fluorescent hydrophilic dyes Cy5 and Cy7 were purchased from Fanbo Biochemical Co. (Beijing, China). Two breast cancer lines (4T1 and MCF-7) and mouse


23

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 43

Macrophages (J774A.1) were purchased from Beijing Xiehe Hospital. Balb/c mice were obtained from Vital River Laboratories (Beijing, China). 980 nm laser meter (LWIRL980-10WF, Beijing) was obtained from Beijing Laserwave Optoelectronics Technology Co., Ltd. (Beijing, China). The intensity of NIR laser used in the all experiments were 1.1 W/cm2 rather than the nominal intensity of 2.0 W/cm2. Deconstruction of the nanolongan was initiated using an UV light irradiation instrument (ZF 1A, Shanghai) with a middle wavelength at 368 nm. The intensity of the light irradiating the sample was 60 mW/cm2. Preparation of oxidized starch nanogel (G). Firstly, DO 90 starch and Ferrous sulfate FeSO4 (DO 90 starch to Ferrous sulfate weight ratio, 100:18, DO 90 starch concentration 25 mg/mL) were dissolved in 2 mL pH=3 ultrapure water. Then the mixed solution was dropped into oil phase, which contained 22 mL paraffin, 44 mL petroleum ether and 2.15 g emulsifier PO-500. Then the water in oil emulsion was homogeneous mixed under a high-speed dispersion machine (IKA T18, Germany) at a speed of 16000 rpm for 5 min. Next, the COO- groups of DO 90 was crosslinked with Fe3+ obtained from oxidation of Fe2+ by pumping oxygen slowly into the water-in-oil emulsion at 40 °C for 30 min . Then G: Fe nanoparticles were obtained by washing out the oil phase with petroleum ether and ethyl alcohol respectively for three times. Finally, the G: Fe particles were obtained after air drying overnight. Preparation of Fe3+ cross-linked oxidized starch microgels. Firstly, DO 90 starch and Ferrous sulfate FeSO4 (DO 90 starch to Ferrous sulfate weight ratio, 100:7, DO 90 starch concentration 100 mg/mL) were dissolved in 1 mL pH=3 ultrapure water. Then the mixed solution was dropped into oil phase, which contained 20 mL paraffin and 0.75 g emulsifier Span 80. Then the water in oil emulsion was homogeneously mixed under magnetic stirring apparatus (IKA RTC, Germany) at a speed of 250 rpm for 30 min. Next, the COO- groups of DO 90 was


24

Page 25 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

crosslinked with Fe3+ obtained from oxidation of Fe2+ by pumping oxygen slowly into the waterin-oil emulsion at 35 °C for 30 min. Then microgels were obtained by washing out the oil phase with Hexane and methanol respectively at a speed of 3000 rpm for 2 min for three times. Finally, the microgels were obtained after air drying overnight. Preparation and characterization of microlongan. 5 mg UCNP (positive charge) were mixed with 10 mg negatively charged G: Fe microgels in 5 mL pH=3 solution by electrostatic interaction for 30 min. Then these UG: Fe were coated by 30% PEI solution (DO 90 starch to PEI weight ratio, 10:16, DO 90 starch concentration 1.49 mg/mL). Next, DMMA was added into mixed solution which was stable at pH=8.0 for 3 hours (DO 90 starch to DMMA weight ratio, 1:10, DO 90 starch concentration 0.65 mg/mL). Then the unabsorbed UCNP and DMMA in the supernatant were removed by centrifugation at 3000 rpm for 2 min (DHS MC22R, China). Finally, the pH-sensitive DGU: Fe microgels in the sediment were obtained. The images of microgels were observed by CLSM (Leica TCS SP5, Germany). Before imaging, microgels decorated with PEI-FITC and DMMA were incubated in pH=6.8 Tris-HCl buffer for 60 minutes. The solution was centrifuged at 3000 rpm for 5 min and resuspended by water. The NIR light induced morphological changes of microgels before and after NIR irradiation was monitored by live cell imaging system (PerkinElmer Ultra VIEW VoX, USA). Preparation of nanolongan. 5 mg UCNP (positive charge) were mixed with 10 mg negatively charged G: Fe in 5 mL pH=3 solution by electrostatic interaction for 30 min. Then 3.5 mg hydrophilic Dox (positive charge) were homogeneously mixed with the UG: Fe solution by electrostatic absorption for another 30 min. Then these UG: Fe/Dox were coated by 30% PEI solution (DO 90 starch to PEI weight ratio, 10:16, DO 90 starch concentration 1.49 mg/mL). Next, DMMA was added into mixed solution which was stable at pH=8 for 3 hours (DO 90


25

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 43

starch to DMMA weight ratio, 1:10, DO 90 starch concentration 0.65 mg/mL). Then the unabsorbed Dox, UCNP and DMMA in the supernatant were removed by centrifugation at 12000 rpm for 5 min at 4 °C (DHS MC22R, China). Finally, the pH-sensitive DGU: Fe/Dox nanoparticles in the sediment were obtained after washing with pH=7.4 Tris-HCl buffer and centrifuging for three times. To facilitate the subsequent fluorescent imaging and measurement, hydrophilic positively charged dyes Cy5 and Cy7 were loaded in DGU: Fe/Dox instead of Dox for cellular and in vivo imaging experiments respectively using the same procedure. Drug loading (DL) efficiency: The concentration of Dox was determined by automatic microplate reader (Tecan Infinite M200, Switzerland). The nanolongan was broken by ultrasonication (BRANSON Digital Sonifier 250, USA) to obtain the embedded Dox. The detection wavelength for Dox was 480 nm (excitation)/580 nm (emission). The DL of DGU: Fe/Dox was determined as follows. Briefly, the amount of Dox in solution was determined by automatic microplate reader. The DL% of GU: Fe/Dox was calculated according to Equation (1) DL% 

Doxweight  100% GUweight+Doxweight

(1)

The DL% of GU: Fe built with different quantity of UCNP were calculated. TEM characterization of nanolongan. The unstained DGU: Fe/Dox sample was dropped on the copper grid for three replicates. The morphology of the DGU: Fe/Dox was observed by TEM at 120 kV (JEOL JEM-1400, Japan). SEM characterization of nanolongan. The sample was the same as TEM used for the elements analysis by scanning electron microscope (SEM) (JEOL JSM 6700F, Japan). Colloidal stability of nanolongan. Particle size distribution and zeta-potential for different formulations were analyzed by dynamic light scattering (Malvern Nano-ZS 90, UK) at 25 °C. Each sample was repeated for three times. To study the colloidal stability at physiological mimic


26

Page 27 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

condition, DGU: Fe/Dox were suspended in fetal bovine serum (Hyclone) at a final DO 90 concentration of 100 μg/mL, and the size and zeta potentials were measured every day for 7 days. Fluorescence emission spectrum of nanolongan. The Fluorescence emission spectrum of U and DGU: Fe/Dox nanoparticles were measured by Steady state fluorescence spectrometer (Edinburgh instruments FSL980-STM, UK). Full-wavelength emission scanning of different nanoparticles were excited by 980 nm laser. The nanoparticles concentration was guaranteed to be less than 10-5 mol/L to prevent fluorescence quenching. Each sample was repeated for three times. Charge conversion of pH responsive nanolongan under different pH. Before measuring zeta potential, DGU: Fe/Dox was incubated in pH=6.8 and 7.4 Tris-HCl buffer respectively over 120 minutes. DGU: Fe/Dox was collected at time point (0 min, 15 min, 30 min, 60 min, 90 min, 120 min). The solution was centrifuged at 12000 rpm for 5 min and resuspended by water. The zeta potential of DGU: Fe/Dox over 120 minutes were analyzed by dynamic light scattering at 25 °C. Each sample was repeated three times for obtaining parallel measurement results. The qualitative evaluation of cellular uptake by CLSM. For the cellular uptake study, J774A.1 and mouse breast cancer cell lines 4T1 cells were incubated with different formulations (DGU and GU). 4T1 cells or J774A.1 cells were seeded onto 35 mm glass-bottom dishes at a density of 1×105 cells in 1 mL of culture media. The cellular uptake of 0.1 mg/mL Cy5 labeled various formulations (Dox was replaced by Cy5 in DGU: Fe/Dox and GU: Fe/Dox) were observed by CLSM (Leica TCS SP5, Germany). The cells were fixed by 4% paraformaldehyde after washing away the adhered nanolongan. Then, the cell membrane and nuclei were stained


27

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 43

with Alexa Fluor 488-phalloidin (Green) and Hoechst 33342 (Blue), respectively. The corresponding fluorescence images were taken by CLSM (Leica TCS SP5, Germany). The quantitative evaluation of cellular uptake by Flow cytometry. Flow cytometry (Beckman Coulter CyAn ADP, USA) was applied to further quantify the cellular uptake of the GU and DGU. 4T1 cells or J774A.1 cells were seeded in 24-well plate for 12 h allowing cell attachment. The cells were blended with 0.1 mg/mL Cy5 labeled nanolongans for another 12 h. Subsequently, cells were washed by cold PBS, and the uptake amount was determined on flow cytometer (FCM). Data were obtained from 15000 cells for each sample. The co-localization of nanolongan and lysosomes by CLSM: The cultural conditions for 4T1 cells were described above and GU: Fe/Cy5 were added at a DO 90 concentration of 0.1 mg/mL after 6 h incubation. Lyso-Tracker Green at a concentration of 50 nM was added to label lysosomes in the cells, and they were co-incubated for 20 min at 37 °C before observation. LysoTracker Green was excited at 488 nm and Cy5 in GU: Fe/Cy5 was excited at 664 nm, respectively. The corresponding fluorescent images at 500-545 nm and 660-710 nm were taken by CLSM. The lysosome escape of nanolongan by hemolysis assay. Such an efficient lysosome escape could be attributed to the proton-sponge effect, confirmed by hemolysis assay. Rabbit red blood cells (RBCs) were isolated from whole blood collected from rabbit at 3000 rpm for 10 min and washed 3 times with sterile PBS. RBCs were suspended at 5 % (v/v) in pH=7.5, 6.5, 5.5 and 4.5 Tris-HCl buffers to a final volume of 1 mL. Then 0.1 mg/mL GU: Fe were mixed with the RBCs solution. Subsequently, after 1 h incubation at 37 °C, the mixed solutions were centrifuged for 10 min at 2000 rpm at 4 °C. Dissolved hemoglobin in supernatants was measured by multimode reader at 540 nm. The percentage of hemolysis was calculated as:


28

Page 29 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Hemolysis% 

Asample  Ablank Atriton  Ablank

100% (2)

Where Ablank is RBCs in Tris-HCl as the negative group; Atriton is RBCs in ultrapure water as the positive group. After calculating the hemolysis of GU: Fe, the sediment was re-suspended for imaging observation of the morphology of RBCs by live cell imaging system (PerkinElmer Ultra VIEW VoX, USA). The morphology changes of nanolongans under NIR irradiation. The NIR light induced morphological changes of nanolongan before and after NIR irradiation (2.0 W/cm2, 60 min) were measured by TEM (JEOL JEM-1400, Japan). NIR-triggered Dox release. DGU: Fe/Dox was dissolved in pH=3 water to 2 mg/mL. Then the solution was irradiated with continuous NIR. After that, the released Dox in irradiated solution was filtrated by centrifugation at 12000 rpm for 5 min. The released Dox in supernatant from nanolongan triggered with continuous NIR irradiation and pulsed NIR irradiation (2.0 W/cm2) was measured by automatic microplate reader (Tecan Infinite M200, Switzerland) at different time intervals for 24 hours. NIR-triggered transformation of Fe3+ to Fe2+. The concentration of Fe2+ and Fe3+ ions in the nanolongan during NIR irradiation was determined by the Fe-phenanthroline method. Fe2+ can form a stable complex with 1,10-phenanthroline which has a maximum absorbance at 510 nm. The absorbance of the complex was monitored using automatic microplate reader (Tecan Infinite M200, Switzerland) at 25 °C. NIR-triggered Dox diffusion into cancer cell nucleus. The Dox diffusion into 4T1 cell nucleus was observed by CLSM. 4T1 cells were cultured as described above, and GU: Fe/Dox were added at a DO 90 concentration of 0.1 mg/mL allowing the endocytosis after 6 h


29

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 43

incubation. The Dox release from GU: Fe/Dox (red) in cytoplasm was triggered by continuous NIR irradiation (2.0 W/cm2, 5 min). Then, the cell nucleus were labeled by DAPI (blue). GU: Fe/Dox adhering to the cell surface and redundant dye were removed by washing with cold PBS buffer for three times. The corresponding fluorescent images were taken by CLSM. The ratio of fluorescence intensity in cytoplasm and cell nucleus was calculated to indicate NIR-triggered Dox release in cytoplasm, respectively. Similarly, the same experimental result was obtained in MCF-7 cells. NIR-triggered Fe3+ reduction into Fe2+. In order to prove which kind of light could reduce Fe3+, nanolongan was illuminated by different lights (NIR, UV and VIS) respectively. The ion valence transformation of Fe3+ to Fe2+ was measured with phenanthroline method by automatic microplate reader at different time intervals for 20 hours. Phenanthroline could react with Fe2+ form a stable orange-red complex in solution compared with Fe3+. The concentration of Fe2+ was calculated in the solution by the absorbance at 510 nm. The ability of Fe3+/Fe2+ to generate ROS in vitro. Before measuring generation of ROS in 4T1 cells, the ability for generating hydroxyl radicals by Fe3+ or Fe2+ ions was evaluated and compared by MB bleaching assay. 200 μL solution of MB (10 mg/L) and Fe3+(FeCl3) or Fe2+ (FeSO4 •7H2O) with equal Fe concentration 0.178 mol/L reacted with H2O2 (200 μM, pH=6.5) in 96-well plate for 0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 3 h and 6 h, respectively. After selective •OH trapping by MB, the absorption intensity at 664 nm at each time point was measured. Bleaching of MB, due to the presence of •OH in a sample, was indicated by a discoloration from a dark blue color to an almost white color. Fe2+ reacted with H2O2 in vitro to generate ROS. NIR-triggered ROS generation by CLSM. The cultural conditions for 4T1 cells were described above, and GU were added at a DO 90 concentration of 0.1 mg/mL after 6 h


30

Page 31 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

incubation. The ROS were reacted with DCFH-DA for 20 min in 37 °C before NIR irradiation. Not fluorescent DCFH can be converted to fluorescent DCF by ROS oxidation. Based on the Fenton reaction, Fe2+ could react with H2O2 to produce ROS more strongly and actively than Fe3+.The UV emitted by NIR under the action of UCNP, catalyzing the reduction of Fe3+ into Fe2+. Therefore, The ROS in cytoplasm from Fenton reaction raised from reduced Fe2+ in nanolongan were triggered by continuous NIR irradiation (2.0 W/cm2, 5 min). Then the cellular ROS oxidized DCF can be used as indicator for ROS production. DCF was excited at 488 nm. The corresponding fluorescent images of cellular DCF at excitation wavelength of 510-555 nm were taken by CLSM. In vitro cellular ferroptosis of nanolongan by simple western immunoblots. In brief, 4T1 cells were seeded in 6-well plates at a density of 1 × 106 cells per well for 24 h. Then the cells were treated with different nanolongan formulations (equivalent Dox concentration 20 μg/mL and corresponding additives). The expressions of FACL4 and GPX4 proteins were evaluated by western blot for ferroptosis. Cytoplasm protein was extracted by Minute TM Cytoplasmic and Nuclear Extraction Kit (Invent Biotechnologies, USA). Protein concentration is measured by the BCA method. The protein was incubated with rabbit anti-mouse polyclonal antibody (1:1000, Santa Cruz, USA). The protein antibody complexes were detected using the HRP (Horseradish peroxidase) conjugated secondary antibody (1:5000) (Earthox, USA). Protein bands were visualized using automatic protein expression analysis system (Wes System, USA). In vitro cellular ferroptosis of nanolongan by TEM. Different treated cells were fixed with 1 mL general fixative (containing 2.5% glutaraldehyde in 0.1 M Phosphate buffer) at 4 °C for 2 hours. After dehydration, cells were embedded in epoxy resin and the resin was stored at 55 °C for 48 h to allow resin polymerization. The embedded samples were then sliced with a


31

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 43

thickness of 50-70 nm. Finally, the cell sections were stained with 5% uranyl acetate for 15 min and 2% lead citrate for 15 min before TEM imaging. In vitro cellular apoptosis of nanolongan. 4T1 cells were seeded in 24-well plates at a density of 1 × 105 cells per well. After 24 h incubation, the cells were incubated with different nanolongan formulations for 24 h (equivalent Dox concentration 20 μg/mL and corresponding additives). Then the culture medium was refreshed and the cells were incubated with fresh medium at 37 °C without NIR irradiation or with NIR irradiation (2.0 W/cm2, 5 min). The cells without any treatment were tested as the negative control. Then all of the cells were washed with cold PBS for three times, digested by trypsin (EDTA depleted), and collected by centrifugation. After being washed with PBS for three times, the cells were resuspended in 0.5 mL of Annexin binding buffer. After that, all cells were stained in PI (Propidium iodide is a nuclear staining reagent that can stain DNA) and Annexin-V-FITC (Annexin V is a member of the intracellular protein annexin family that selectively binds to the phosphatidylserine of the everted cell membrane in a calcium-dependent manner) containing binding buffer for 15 min and finally detected by flow cytometry (FCM). Data were obtained from 15000 cells for each sample. CCK-8 cytotoxicity assay for nanolongan. The cytotoxicity was determined using the CCK-8 (Beyotime, China) assay. Briefly, 4T1 cells were seeded in 96-well plate at a density of 1.5×104 cells in 100 μL of culture media for 24 h allowing cell attachment. As for DGU: Fe/Dox+L+inhibitor group, the cells were pretreated with liproxstatin-1 (22nM). The cells were blended with different Dox formulations (with the equivalent Dox concentration ranging from 0 μg/mL to 20 μg/mL) for 24 h. The cells were irradiated by the NIR irradiation at 2.0 W/cm2 for 5 min. CCK-8 test solution was added to each well of the plate (The volume of the test solution in each well is one tenth of the total volume) and incubated for another 1-4 h. After selective CCK-


32

Page 33 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

8 reduction by viable cells, the absorption intensity at 450 nm was measured. Percentage of the viability was normalized according to the untreated cells. Live-dead cytotoxicity assay for nanolongan. The cytotoxicity was measured by Live/dead cell viability assay (Invitrogen). 4T1 cells were cultured as described above, then different Dox formulations (equivalent Dox concentration 20 μg/mL and corresponding additives) were added for 24 h. The cells were stained by Calcein-AM/EthD-1 staining working solution for 20 min at 37 °C. Calcein AM/EthD-1 staining working solution was prepared by mixing 0.5 μL Calcein AM solution (4 mM) and 2 μL EthD-1 solution (2 mM) into 1 mL DPBS. Green (494/517 nm) viable cells can be stained by Calcein AM, while red (528/617 nm) dead cells can be stained by EthD-1. In vivo tumor targeting evaluation of nanolongan. Balb/c mice (4-6 weeks old) were bought from Vital River Laboratories (Beijing, China) and used for animal experiments directly. The tumors were obtained by injecting female mice with 4T1 cells (1.0 × 106 cells in 100 μL of PBS) into the ventral mammary fat pad of the female Balb/c mice. In order to observe the biodistribution of the nanolongan in vivo, Dox was replaced by hydrophilic dye Cy7 in DGU: Fe/Dox and GU: Fe/Dox. 100 μL of DGU or GU of DO 90 concentration of 100 μg/mL was intravenously injected to 4T1 tumor-bearing female Balb/c and their biodistribution was observed using an in vivo imaging system (Kodak FX Pro, Japan) at 1 h, 4 h, 12 h and 24 h. Then the tumors and organs were excised and imaged. At the same time, after dissolving tumor and liver of different time intervals by concentrated hydrochloric acid, the amount of UCNP was measured by ICP-MS (Perkinelmer NexION 300X, USA). The amount of UCNP in the tumor for GU group at 24 h was set as “1” for comparison. Then the amounts of UCNP in the tumors of


33

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 43

DGU and GU at different times were normalized with the amount of UCNP in the GU group tumor at 24 h. In vivo pharmacokinetic evaluation of nanolongan. To study the pharmacokinetic behavior of GU and DGU, blood samples were collected at predestined time intervals (0.5, 1, 2, 4, 6. 8, 12, and 24 h) using a heparinized tube after injection of GU: Fe/Cy5 and DGU: Fe/Cy5 (DO 90 starch concentration of 100 μg/mL). All the blood samples were centrifuged at 4000 g for 10 min at 4 °C. Then the fluorescence intensity of equal serum volume were measured by automatic microplate reader at 664 nm. Relative fluorescence intensity was normalized according to the untreated mice. In vivo anticancer efficacy evaluation. For investigating their antitumor effect of different Dox formulations, tumor-bearing mice were treated when the average tumor volume reached approximately 100 mm3 (at day 10). The mice were randomly divided into six groups (each group n = 6). Relative tumor volumes (V) were measured for six groups of mice that were intravenously administered with PBS, Dox, DGU: Fe, DGU: Fe+L, DGU: Fe/Dox+L (1 mg/kg and 2.0 W/cm2, 5 min) and DGU: Fe/Dox+L (High) (2 mg/kg and 2.0 W/cm2, 5 min) once every other day until day 26. Tumor volumes were calculated by: V

L W 2 2

(3)

Where L is the longest and W is the shortest tumor diameter (mm). Immunohistochemical section evaluations of nanolongan. TUNEL apoptosis detection kit was used for nucleus apoptosis of tumor tissues. Immunohistochemical analysis was used to evaluate Ferroptosis in tumor. The secondary antibody to GPX4 (orange) and FACL4 (red) were labeled. In the end, Ki 67 detection was used for measuring the proliferation of tumor by automatic multispectral imaging System (PerkinElmer Vectra II, USA).


34

Page 35 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Anti-metastasis effect evaluations of nanolongan. Because 4T1 mammary adenocarcinoma possesses spontaneously metastasis, the metastasis of lungs and bone tissues need to be evaluated. The five treated groups were analyzed for evaluating the antimetastasis effect in vivo. The bone erosion at homolateral tibia caused by tumor metastasis was imaged by using a micro CT scanner (Caliper Life Sciences Quantum FX, USA). Lung metastasis in different treatment groups was evaluated by hematoxylin-eosin staining. Safety evaluation of triple conversions nanolongan anticancer therapy. To further evaluate the safety of different formulations in vivo, the body weight changes were recorded. Additionally, the serum levels of urea nitrogen (BUN), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) were analyzed by using an automated analyzer (Hitachi Ltd Hitachi-917, Japan). The organs of different formulation were sliced and stained by hematoxylin-eosin staining. Animal care. All animal experiments were performed in compliance with the guide of care and use of laboratory animals. The animal protocol was approved by the Institutional Animal Care and Use Committees at Institute of Process Engineering, Chinese Academy of Sciences. Statistical analysis. Statistical evaluations of data were performed using the Student’s t test. All results were expressed as mean ± standard error unless otherwise noted. *P < 0.05, **P < 0.01. AUTHOR INFORMATION Corresponding Authors *E-mail (Yuan Li): [email protected] *E-mail (Wei Wei): [email protected] ORCID


35

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 43

Yuan Li: 0000-0002-6285-3522 Wei Wei: 0000-0002-6244-3187 Author Contributions ‡W. E. Bao and X. W. Liu contributed equally to this work. ACKNOWLEDGEMENT The financial supports from the National Natural Science Foundation of China (No. 31471577, 31772014, 21622608) and the National Key R&D Program of China (2017YFA0207900) are gratefully acknowledged. Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org. REFERENCES 1.

Jia, J.; Zhu, F.; Ma, X.; Cao, Z. W.; Li, Y. X.; Chen, Y. Z. Mechanisms of Drug

Combinations: Interaction and Network Perspectives. Nat. Rev. Drug Discovery 2009, 8, 111– 128. 2.

Greco; Francesca; Vicent; María, J. Combination Therapy: Opportunities and Challenges

for Polymer-Drug Conjugates as Anticancer Nanomedicines. Adv. Drug Delivery Rev. 2009, 61, 1203–1213. 3.

Looney, W. B.; Trefil, J. S.; Hopkins, H. A.; Kovacs, C. J.; Ritenour, R.; Schaffner, J. G.

Solid Tumor Models for Assessment of Different Treatment Modalities: Therapeutic Strategy for Sequential Chemotherapy with Radiotherapy. Proc. Natl. Acad. Sci. U. S. A. 1977, 74, 1983– 1987. 4.

Long, D.; Liu, T.; Tan, L.; Shi, H.; Liang , P.; Tang, S.; Wu, Q.; Yu, J.; Dou, J.; Meng, X.


36

Page 37 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

a Multi-Synergistic Platform for Tumor Therapy by Mild Microwave Irradiation-Activated Chemotherapy and Enhanced Ablation. ACS Nano 2016, 10, 9516–9528. 5.

Lorch, J. H.; Goloubeva, O.; Haddad, R. I.; Cullen, K.; Sarlis, N.; Tishler, R.; Tan, M.;

Fasciano, J.; Sammartino, D. E.; Posner, M. R. Induction Chemotherapy with Cisplatin and Fluorouracil Alone or in Combination with Docetaxel in Locally Advanced Squamous-Cell Cancer of The Head and Neck: Long-Term Results of The TAX 324 Randomised Phase 3 Trial. Lancet Oncol. 2011, 12, 153–159. 6.

Schein, P. S.; Schein, P. S. a Comparison of Combination Chemotherapy and Combined

Modality Therapy for Locally Advanced Gastric Carcinoma. Cancer 1982, 49, 1771–1777. 7.

Friedman, H. S.; Prados, M. D.; Wen, P. Y.; Mikkelsen, T.; Schiff, D.; Abrey, L. E.;

Yung, W. K. A.; Paleologos, N.; Nicholas, M. K.; Jensen, R. Bevacizumab Alone and in Combination with Irinotecan in Recurrent Glioblastoma. J. Clin. Oncol. 2009, 27, 4733–4740. 8.

Papaldo, P.; Fabi, A.; Ferretti, G.; Mottolese, M.; Cianciulli, A. M.; Di, C. B.; Pino, M.

S.; Carlini, P.; Di, C. S.; Sacchi, I. A Phase II Study on Metastatic Breast Cancer Patients Treated with Weekly Vinorelbine with or without Trastuzumab According to HER2 Expression: Changing The Natural History of HER2-Positive Disease. Ann. Oncol. 2006, 17, 630–636. 9.

Van, C. E.; Köhne, C. H.; Hitre, E.; Zaluski, J.; Chang, C. C.; Makhson, A.; D'Haens, G.;

Pintér, T.; Lim, R.; Bodoky, G. Cetuximab and Chemotherapy as Initial Treatment for Metastatic Colorectal Cancer. N. Engl. J. Med. 2009, 360, 1408–1417. 10.

Gianni, L.; Romieu, G. H.; Lichinitser, M.; Serrano, S. V.; Mansutti, M.; Pivot, X.;

Mariani, P.; Andre, F.; Chan, A.; Lipatov, O. AVEREL: a Randomized Phase III Trial Evaluating Bevacizumab in Combination with Docetaxel and Trastuzumab as First-Line Therapy for HER2-Positive Locally Recurrent/Metastatic Breast Cancer. J. Clin. Oncol. 2013, 31, 1719–


37

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 43

1724. 11.

Dixon, S. J.; Lemberg, K. M.; Lamprecht, M. R.; Skouta, R.; Zaitsev, E. M.; Gleason, C.

E.; Patel, D. N.; Bauer, A. J.; Cantley, A. M.; Yang, W. S. Ferroptosis: an Iron-Dependent Form of Nonapoptotic Cell Death. Cell 2012, 149, 1060–1065. 12.

He, C.; Jiang, S.; Jin, H.; Chen, S.; Gan, L.; Yao, H.; Wang, X.; Peng, M.; Ji, Z.; Lin, Y.

Mitochondrial Electron Transport Vhain Identified as a Novel Molecular Target of SPIO Nanoparticles Mediated Cancer-Specific Cytotoxicity. Biomaterials 2016, 83, 102–114. 13.

Tang, Z.; Zhao, P.; Ni, D.; Liu, Y.; Zhang, M.; Wang, H.; Zhang, H.; Gao, H.; Yao, Z.;

Bu, W. Pyroelectric Nanoplatform for NIR-ΙΙ Triggered Photothermal Therapy with Simultaneous Pyroelectric Dynamic Therapy. Mater. Horiz. 2018, 5, 946–952. 14.

Tang, Z.; Liu, Y.; He, M.; Bu, W. Chemodynamic Therapy: Tumour Microenvironment-

Mediated Fenton and Fenton-Like Reactions. Angew. Chem., Int. Ed. 2018, 5,664–677. 15.

D'Herde, K.; Krysko, D. V. Ferroptosis: Oxidized PEs Trigger Death. Nat. Chem. Biol.

2016, 13, 4–10. 16.

Gorrini, C.; Harris, I. S.; Mak, T. W. Modulation of Oxidative Stress as an Anticancer

Strategy. Nat. Rev. Drug Discovery 2013, 12, 931–947. 17.

Meng, H.; Xue, M.; Xia, T.; Ji, Z.; Tarn, D. Y.; Zink, J. I.; Nel, A. E. Use of Size and A

Co-Polymer Design Feature to Improve The Biodistribution and The Enhanced Permeability and Retention Effect of Doxorubicin-Loaded Mesoporous Silica Nanoparticles in a Murine Xenograft Tumor Model. ACS Nano 2011, 5, 4131–4144. 18.

Nel, A.; Ruoslahti, E.; Meng, H. New Insights into "Permeability" as in The Enhanced

Permeability and Retention Effect of Cancer Nanotherapeutics. ACS Nano 2017, 11, 9567–9569. 19.

Byrne, J. D.; Betancourt, T.; Brannon-Peppas, L. Active Targeting Schemes for


38

Page 39 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Nanoparticle Systems in Cancer Therapeutics. Adv. Drug Delivery Rev. 2008, 60, 1615–1626. 20.

Yang, G.; Wang, J.; Wang, Y.; Li, L.; Guo, X.; Zhou, S. An Implantable Active-

Targeting Micelle-in-Nanofiber Device for Efficient and Safe Cancer Therapy. ACS Nano 2015, 9, 1161–1174. 21.

Dunford, H. B. Oxidations of Iron(II)/(III) by Hydrogen Peroxide: from Aquo to Enzyme.

Coord. Chem. Rev. 2002, 233, 311–318. 22.

Chambers, E.; Mitragotri, S. Prolonged Circulation of Large Polymeric Nanoparticles by

Non-Covalent Adsorption on Erythrocytes. J. Controlled Release 2004, 99, 111–119. 23.

Kwon, G.; Suwa, S.; Yokoyama, M.; Sakurai, Y.; Okano, T.; Kataoka, K. Enhanced

Tumor Accumulation and Prolonged Circulation Times of Micelle-Forming Poly (Ethylene Oxide-aspartate) Block Copolymer-Adriamycin Conjugates. J. Controlled Release 1994, 29, 17– 23. 24.

Paillard, A.; Hindre, F. C. C.; Benoit, J. P.; Garcion, E. The Importance of Endo-

Lysosomal Escape with Lipid Nanocapsules for Drug Subcellular Bioavailability. Biomaterials 2010, 31, 7542–7554. 25.

Pratt, D. A.; Tallman, K. A.; Porter, N. A. Free Radical Oxidation of Polyunsaturated

Lipids: New Mechanistic Insights and The Development of Peroxyl Radical Clocks. Acc. Chem. Res. 2011, 44, 458–467. 26.

Dixon, S.; Lemberg, K.; Lamprecht, M.; Skouta, R.; Zaitsev, E.; Gleason, C.; Patel, D.;

Bauer, A.; Cantley, A.; Yang, W. S. Ferroptosis: an Iron-Dependent Form of Nonapoptotic Cell Death. Cell 2012, 149, 1060–1072. 27.

Zhang, C.; Bu, W.; Ni, D.; Zhang, S.; Li, Q.; Yao, Z.; Zhang, J.; Yao, H.; Wang, Z.; Shi,

J. Synthesis of Iron Nanometallic Glasses and Their Application in Cancer Therapy by a


39

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 43

Localized Fenton Reaction. Angew. Chem., Int. Ed. Engl. 2016, 128, 2141–2146. 28.

Lim, S. H.; Thivierge, C.; Nowaksliwinska, P.; Han, J.; Bergh, H. V. D.; Wagnières, G.;

Burgess, K.; Hong, B. L. In Vitro and in Vivo Photocytotoxicity of Boron Dipyrromethene Derivatives for Photodynamic Therapy. J. Med. Chem. 2010, 53, 2865–2871. 29.

Hatz, S.; Lambert, J. D.; Ogilby, P. R. Measuring The Lifetime of Singlet Oxygen in a

Single Cell: Addressing The Issue of Cell Viability. Photochem. Photobiol. Sci. 2007, 6, 1106– 1116. 30.

Kamada, H.; Tsutsumi, Y.; Yoshioka, Y.; Yamamoto, Y.; Kodaira, H.; Tsunoda, S.;

Okamoto, T.; Mukai, Y.; Shibata, H.; Nakagawa, S. Design of a pH-Sensitive Polymeric Carrier for Drug Release and Its Application in Cancer Therapy. Clin. Cancer Res. 2004, 10, 2545– 2550. 31.

Habeeb, A. F. S. A.; Atassi, M. Z. Enzymic and Immunochemical Properties of

Lysozyme Evaluation of Several Amino Group Reversible Blocking Reagents. Biochemistry 1970, 9, 4939–4944. 32.

Chen, X.; Wang, S.; Lu, M.; Chen, Y.; Zhao, L.; Li, W.; Yuan, Q.; Norde, W.; Li, Y.

Formation and Characterization of Light-Responsive TEMPO-Oxidized Konjac Glucomannan Microspheres. Biomacromolecules 2014, 15, 2166–2171. 33.

Shi, M.; Bai, J.; Zhao, L.; Yu, X.; Liang, J.; Liu, Y.; Nord, W.; Li, Y. Co-Loading and

Intestine-Specific Delivery of Multiple Antioxidants in pH-Responsive Microspheres Based on TEMPO-Oxidized Polysaccharides. Carbohydr. Polym. 2017, 157, 858–865. 34.

Graedel, T. E.; Weschler, C. J.; Mandich, M. L. Influence of Transition Metal Complexes

on Atmospheric Droplet Acidity. Nature 1985, 317, 240–242. 35.

Zhu, X.; Prospero, J. M.; Savoie, D. L.; Millero, F. J.; Zika, R. G.; Saltzman, E. S.


40

Page 41 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Photoreduction of Iron(III) in Marine Mineral Aerosol Solutions. J. Geophys. Res.: Atmos. 1993, 98, 9039–9046. 36.

Peng, F.; Li, G.; Liu, X.; Wu, S.; Tong, Z. Redox-responsive Gel-Sol/Sol-Gel Transition

in Poly(acrylic acid) Aqueous Solution Containing Fe(III) Ions Switched by Light. J. Am. Chem. Soc. 2008, 130, 16166–16167. 37.

Tao, Z.; Peng, K.; Fan, Y.; Liu, Y.; Yang, H. Multi-stimuli Responsive Supramolecular

Hydrogels Based on Fe and Diblock Copolymer Micelle Complexation. Polym. Chem. 2016, 7, 1405–1412. 38.

Xu, J.; Xu, L.; Wang, C.; Rong, Y.; Qi, Z.; Xiao, H.; Dong, Z.; Zhu, W.; Rui, P.; Zhuang,

L. Near-Infrared-Triggered Photodynamic Therapy with Multi-Tasking Upconversion Nanoparticles in Combination with Checkpoint Blockade for Immunotherapy of Colorectal Cancer. ACS Nano 2017, 11, 4463–4474. 39.

Xiang, J.; Xu, L.; Gong, H.; Zhu, W.; Wang, C.; Xu, J.; Feng, L.; Cheng, L.; Peng, R.;

Liu, Z. Antigen-loaded Upconversion Nanoparticles for Dendritic Cell Stimulation, Tracking, and Vaccination in Dendritic Cell-Based Immunotherapy. ACS Nano 2015, 9, 6401–6411. 40.

Angeli, J. P. F.; Schneider, M.; Proneth, B.; Tyurina, Y. Y.; Tyurin, V. A.; Hammond, V.

J.; Herbach, N.; Aichler, M.; Walch, A.; Eggenhofer, E. Inactivation of The Ferroptosis Regulator Gpx4 Triggers Acute Renal Failure in Mice. Nat. Cell Biol. 2014, 76, 1180–1191. 41.

Xie, Y.; Hou, W.; Song, X.; Yu, Y.; Huang, J.; Sun, X.; Kang, R.; Tang, D. Ferroptosis:

Process and Function. Cell Death Differ. 2016, 23, 369–379. 42.

Mai, M.; Freigang, S.; Schneider, C.; Conrad, M.; Bornkamm, G. W.; Kopf, M. T Cell

Lipid Peroxidation Induces Ferroptosis and Prevents Immunity to Infection. J. Exp. Med. 2015, 212, 555–568.


41

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

43.

Page 42 of 43

Doll, S.; Proneth, B.; Tyurina, Y. Y.; Panzilius, E.; Kobayashi, S.; Ingold, I.; Irmler, M.;

Beckers, J.; Aichler, M.; Walch, A. ACSL4 Dictates Ferroptosis Sensitivity by Shaping Cellular Lipid Composition. Nat. Cell Biol. 2017, 13, 91–98. 44.

Yuan, H.; Li, X.; Zhang, X.; Kang, R.; Tang, D. Identification of ACSL4 as a Biomarker

and Contributor of Ferroptosis. Biochem. Biophys. Res. Commun. 2016, 478, 1338–1343


42

Page 43 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Nanolongan with Multiple On-Demand Conversions for Ferroptosis-Apoptosis Combined Anticancer Therapy Weier Baoa,b,c,‡, Xianwu Liua,b,c, ‡, Yanlin Lv b, Gui-Hong Lu b, Feng Li b, Fan Zhang b, Bin Liua, Dan Lia, Wei Weib *, Yuan Lia *

Nanolongan with typical one-core-in-one-gel structure and multiple on-demand conversions was synthesized for ferroptosis-apoptosis combined anticancer therapy.


43

Nanolongan with Multiple On-Demand Conversions for Ferroptosis

Recommend Documents