A Simple, Yet Multifunctional, Nanoformulation for Eradicating Tumors

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A simple yet multifunctional nanoformulation for eradicating tumors and preventing recurrence with safely low administration dose Yan Liu, Yawen Xu, Zezhong Zhang, Yingying Huo, Dexin Chen, Wei Ma, Kang Sun, Gulen Yesilbag Tonga, Guangdong Zhou, Daniel S. Kohane, and Ke Tao Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b02053 • Publication Date (Web): 31 Jul 2019 Downloaded from pubs.acs.org on July 31, 2019

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A simple yet multifunctional nanoformulation for eradicating tumors and preventing recurrence with safely low administration dose Yan Liu1‡, Yawen Xu2‡, Zezhong Zhang3, Yingying Huo2, Dexin Chen1, Wei Ma3, Kang Sun1, Gulen Yesilbag Tonga4, Guangdong Zhou2*, Daniel S. Kohane4, Ke Tao1* 1 State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, P. R. China; 2 Department of Plastic and Reconstructive Surgery, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200235, P. R. China; 3 School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, P. R. China; 4 Laboratory for Biomaterials and Drug Delivery, Division of Critical Care Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA Corresponding Author *G. Z.: E-mail: [email protected]. *K. T.: Phone: 86-21-3420-2956, Fax: 86-21-3420-2745, E-mail: [email protected].

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ABSTRACT

Designing simple-structured nanomedicine without lacking key functionalities and therefore avoiding incomplete damage or relapse of tumor with safe dose of administration is pivotal for successful cancer nanotherapy. We herein presented a nanomedicine of photodynamic therapy (PDT) that simply assembled amphiphilic macromolecules of poly-L-lysine conjugating with photosensitizers onto hydrophobic upconverting nanoparticles. We demonstrated that the nanoformulation, in spite of its simple structure and synthesis, simultaneously possesses multiple features, including substantial payload of photosensitizers, avid cellular internalization both in vitro and in vivo, efficient diffusion and broad distribution in tumor lesion, and potent fatality for cancer stem cells that are refractory to other therapy modalities. Owing to the combination of these functionalities, the tumors in mice were eradicated and no relapse was observed at least in 40 days, just with an extremely low intraperitoneal injection dose of 5.6 mg/kg. Our results suggested a strategy for designing multifunctional nanomedicines with simple construct and efficacious therapeutic response and presented the promising potential of PDT in radical cure of cancer.

KEYWORDS: upconversion nanoparticles, photodynamic therapy, poly-L-lysine, cancer stem cells, tumor penetration


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Many functionalities of nanomedicines have been recognized as crucial for achieving positive outcomes for cancer nanotherapy, such as high drug loading capability,1-2 targeting to tumor sites,3-7 internalization into cancer cells,8-10 broad penetration in tumors,11-12 precise and sensitive triggered release of therapeutics,13-16 overcoming drug resistance,17-23 etc. Owing to that lacking any individual functionalities may result in sub-optimal therapy or the recurrence of tumor,24-25 multifunctional nanomedicine that covers diverse characteristics attracted intensive attentions.2627

However, numerous challenges remained in the design of multifunctional nanomedicines.28-29

For example, properties that enhance cellular uptake may improve potency,30-31 they also hinder tumor penetration as they will enhance uptake by cells relatively close to microvessels.32 Meanwhile, the modification of nanomedicines with drug resistance modulators such as Pglycoprotein inhibitor,33 that generally designed for overcoming the refractory of cancer cells, would also result in the difficulty of arriving at tumor sites and the risk of side-effect because the proteins were also expressed in most normal organs tasked with protecting them from toxins.34 More importantly, multifunctionality, if it is not unachievable, generally increases the complexity of nanomedicines, resulting in the issues of cost, operation, repeatability, and therefore the impediment to the translation.35-38 As consequences of these remained challenges, incomplete regression and recurrence of tumor were always observed in pre-clinical experiments of nanotherapeutics. Photodynamic therapy (PDT), that employs photosensitizers triggered by light to generate toxic reactive oxygen species (ROS) specifically at tumor sites,39-40 has proven to be an effective treatment of various cancers over 30 years of clinical experience.41-43 In spite of this, taking upconverting nanoparticles (UCNs)-based PDT nanoformulations as a typical example, the injection doses (ID) for animal studies varied in the range as high as 20~225 mg/kg, however, still resulting in tumor recurrence


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in many cases.44-45 In contrast, the survey on the biocompatibility of UCNs demonstrated the safety with the ID only below 20 mg/kg.46 Thus, improving the potency to radically eliminate tumor, and minimizing the dose to a safe range are still highly desired,47 and nanomedicines possessing effective diverse functionalities could be a possible solution. To this end, we herein conjugated poly-L-lysine (PLL) with a hydrophobic photosensitizer, protoporphyrin IX (PpIX), forming an amphiphilic macromolecule, and then self-assembled the conjugates on the surface of hydrophobic UCNs (in our case, NaYF4:Yb,Er@NaGdF4) in the same pot, thus obtaining a simple-structured UCNs-based PDT nanoformulation (Figure 1a). Such a nanoconstruct was proposed based on the following hypotheses: 1) Significant dose of photosensitizers could be loaded, otherwise the hydrophobic UCNs could not transfer to hydrophilic since the conjugate would be too hydrophilic to coat on the nanoparticles. 2) The nanomedicine could be efficiently internalized into cancer cells with the help of PLL, as PLL has been proved to be an efficient agent for introducing drugs, siRNA and DNA into cells, with excellent biocompatibility.48-50 3) ROS generated inside cells was recently reported to inhibit the production of adenosine triphosphate and compromise the function of transmembrane efflux pumps.51 Owing to that the drug efflux by membrane transporters was considered as the main origin of drug resistance,18 PDT may hold intrinsic capability to overcome the refractory of cancer cells as long as the nanomedicines are internalized. 4) Before exposing to light irradiation, avid cellular uptake and active efflux of nanomedicines by transporters would result in an efficient transcytosis process. We hypotheses that this process could continuously happen cell by cell so that improving cellular uptake would benefit for the tumor penetration. With these characteristics combined, improved therapeutic outcome could be expected.


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The conjugation between PLL and PpIX was conducted with a facile EDC-NHS activation process and was evidenced by the newly appeared peaks in the spectra of high-performance liquid chromatography-mass spectrometry (HPLC-MS), as shown in Figure S1. Then a chloroform solution of as-prepared UCNs, protected with oleic acid or sodium oleate,52 was directly added in the reacting solution of the PLL-PpIX. The feeding weight ratio between PpIX and UCNs were controlled as 30%, 50%, and 70%, respectively. After the evaporation of chloroform, the nanoparticles were transferred to hydrophilic, and the successful coating of the conjugates was evidenced by elemental mapping, in which both carbon and nitrogen were found on the nanoparticles (Figure 1b). TEM images of the conjugations were shown in Figure S2, in which an organic layer around UCNs was observed despite the low contrast. Additionally, as shown in Figure 1c, the conjugate of PLL-PpIX in a free form cannot be concentrated by centrifugation, whereas PLL-PpIX that mixed with UCNs was almost completely centrifuged down with dense color of PpIX (the centrifuged nanoformulation was denoted as sample UCNs@PLL-PpIX), by which the loading of PpIX on UCNs was clearly demonstrated. The average hydrodynamic size of resultant UCNs@PLL-PpIX was about 190 nm, and the zeta potential was about +27 mV (Figure S3). The payload of PpIX on UCNs were determined via two different approaches. The first one was to compare UV-Vis absorption of samples with a standard concentration-absorption curve of pure PpIX solution (Figure S4), and the dose of UCNs were calibrated by means of determining the content of gadolinium by inductively coupled plasma atomic emission spectrometer (ICPAES). By this method, the ratio between PpIX and UCNs was identified as 28.61%, 34.25%, and 43.61% for 30%, 50%, and 70% PpIX feeding, respectively, as shown in Figure 1d. An alternative approach was also conducted, in which thermogravimetric analysis (TGA) was used


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to determine the total loading percentage of PLL-PpIX conjugates and nuclear magnetic resonance (NMR) was utilized to identify the ratio between PLL and PpIX (Figure S5 and Table S1). The ratio was accordingly calculated as 25.21%, 37.41%, 44.32%, respectively. The differences between these two methods are less than 3%, indicating their reliability. It should be mentioned that the weight ratio between photosensitizers and UCNs in the reported cases was generally lower than 14%.44, 53-56 With the significant increase of loading capacity, we found that the fluorescent peak of UCNs at ~540 nm was almost completely quenched, as shown in Figure 1e, indicating an efficient energy transfer from UCNs to the loaded photosensitizers (PpIX). Additionally, other functional moieties bearing carboxyl groups can be modified onto PLL together with photosensitizes. As an instance, folic acid (FA), as a typical tumor active targeting ligand, was conjugated on PLL together with PpIX (denoted as PLL-PpIX-FA), as verified by HPLC (Figure S6). The existence of FA after coating on UCNs (UCNs@PLL-PpIX-FA) was also proved by the UV-Vis absorbance with the emergence of the characteristic peak of FA at ~280 nm, as shown in Figure S7. ROS generation of the nanoparticles was evaluated by using 1,3-diphenyl-isobenzofuran (DPBF) as an indicator, as the absorbance peak of DPBF at ~430 nm would be gradually quenched with the continuous generation of ROS.57-58 The result showed that the higher payload of PpIX resulted in a faster decay of DPBF absorbance (Figure 1f). It should be noted that for 70% or 50% PpIX feeding, the absorbance peak of DPBF decreases to less than 40% of the original intensity in 10 min. Taking our previous work as a reference,53 30 min of irradiation is required for the same decrease while keeping all conditions the same. Despite the fact that the ROS generation efficiency depends on various parameters such as the power density of laser, the concentration of both DPBF and nanoparticles, the fluorescence intensity of UCNs, etc., a rough


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assessment can still be made based on the reported cases showing that the decrease of absorbance for 15~30% in 5 minutes.58-60 While in the current work, DPBF absorbance decreased for ~56% with 5 min irradiation, indicating the significant ROS generation. We also found that further conjugation with FA has almost no influence on ROS generation (Figure S8). Meanwhile, the generation of singlet oxygen (1O2), measuring with 9,10-anthracenediylbis(methylene) dimalonic acid (ABDA) as an indicator, is similar to that of ROS, as shown in Figure S9. We next evaluated the cellular uptake of the nanoformulation. Figure 2a presented that the fluorescence of the nanoparticles in terms of both PpIX and UCNs were localized around the nucleus of Hela cells, which indicated the successful internalization after incubation. Then the cellular uptake of UCNs@PLL-PpIX and UCNs@PLL-PpIX-FA under different concentration was quantified (Figure 2b), showing that the internalized UCNs@PLL-PpIX was about 2.4 µg UCNs per 104 cells and that of UCNs@PLL-PpIX-FA increased to ~2.9 µg UCNs/104 cell, when the incubation concentration of UCNs was 280 µg/ml. For studying the interaction between nanoparticles and those cells with drug refractory, cancer stem cells (CSCs) were chosen as a model, because CSCs are able to survive from chemo- or radiotherapy and are responsible for tumor relapses observed after treatment.25, 61 For identifying the CSCs in the cell population and evaluating the cellular uptake, we used anti-CD133 as a marker for CSCs62-64 and compared the cell counting results before and after being marked. As shown in Figure 2c, stem-like cells were identified in a percentage of ~3.55% (3.34%+0.21%) in blank cells, which is comparable to the reported cases.65-67 For those cells incubated with nanoparticles, the percentage of cells being labeled with anti-CD133 is around 3.7%~3.8%, indicating the nanoparticles has no influence on the overexpression of CD133 of CSCs. More


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importantly, the percentage of CSCs detected with the fluorescence of PpIX (3.51% and 3.70%, respectively for different incubating concentrations) were almost as same as those of blank cells, indicating almost all the CSCs were loaded with nanoparticles. Further quantitively comparison demonstrated that CSCs uptake the formulation in a similar manner to non-stem cancer cells, as shown in Figure 2d. We then assessed the accumulation of nanoparticles in the tumor lesion at 1.5 h after injection. In intratumoral injection mode, UCNs@PLL-PpIX accumulated in tumor in a percentage of above 30% ID•g-1 (Figure 3a, red bar). For intraperitoneal injection, UCNs@PLL-PpIX accumulated at tumor site is about 0.14% ID•g-1 (Figure S10), whilst because of the positive targeting effect of folic acid, UCNs@PLL-PpIX-FA was targeted to lesion with the ratio of ~0.19% ID•g-1 (red bar of Figure 3b, and Figure S10). Despite the fact that the targeting effect is not as high as the reported average level,68 the fluorescence of FA conjugated nanoparticles still was observed in the histological slices not only for intratumor injection, but also for intraperitoneal injection. More importantly, the fluorescence is almost overlay with the cell (Figure 3c). For further identifying this, we digested the extra cellular matrix of tumor section, isolated the cells and measured the dose of remained nanoparticles. The ratio between nanoparticles inside cells and all those in the tumor (shown in Figure 3a and 3b) is ~64% and ~80% for intratumoral injection and intraperitoneal injection, respectively, which implies the significant internalization in vivo. The distribution of nanoparticles in tumor tissue was evaluated by means of microCT, with thin silica layer coated UCNs (UCNs@SiO2) as the control. As shown in Figure 3d, both UCNs@PLL-PpIX and UCNs@PLL-PpIX-FA were located in most area of the tumor in 1 hour and further expanded in 3 hours. In contrast, UCNs@SiO2 were positioned much less than the


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experimental nanoparticles at both 1 hour and 3 hours, which means that the nanoformulation distributed much broader than UCNs@SiO2. Additionally, we randomly cut each tumor into 8 nubbles, and the dose of UCNs in each of the nubs were recorded. The results were schematically illustrated as Figure 3e, showing that the UCNs@SiO2 were dispersed in the tumor with a huge deviation from 10% to 100% ID/g, whereas the amount of UCNs@PLL-PpIX-FA was concentrated in the range of 30%-40% ID/g, which demonstrated the homogeneous distribution and the improved penetration of the nanoformulation in tumor. The therapeutic response was demonstrated in vitro at first. First of all, both kinds of nanoparticles did not show obvious cytotoxicity in dark when the concentration of UCNs were increased to 280 µg/ml. After exposing to a 980 nm laser for 10 minutes, the cell viability decreases in the extent relating to the concentration, as shown in Figure 4a. Taking 140 µg/ml UCNs as an example, the cell viability decreased to 30 min irradiation as being summarized,44 only 5.6 mg/kg intraperitoneal ID and 15 min irradiation with 980nm laser (1 W/cm2) was applied, which significantly lowered the intensity of therapy and reduced the risk of side-effects. Thus, this work presented that multifunctionalities can be obtained in a simple-structured nanomedicine leading to an efficacious therapeutic response, which indicates the potential of nanomedicine-based PDT for fulfilling unmet medical demands. The power density (1 W/cm2) of the laser in our work somewhat results in the side-effect of the skin. Further improving the therapeutic index so that lowering the intensity of irradiation may still remain one of the purposes for nanoformulations in future work.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available from the ACS Publications website or from the authors. Experimental section; TEM and Zeta potential of UCNs@PLL-PpIX; HPLC-MS spectra and 1H NMR spectra of of PLL-PpIX and PLL-PpIX-FA conjugations; the concentration-absorption calibration curve of PpIX; TGA curves, drug loading assessment, UV-Vis absorption spectra, and ROS generation of UCNs@PLL-PpIX and UCNs@PLL-PpIX-FA; biodistribution studies; the intracellular ROS generation of UCNs@PLL-PpIX-FA; temperature change of tumors after


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laser irradiation; TUNEL analysis of tumor tissues; body weight assessments; H&E analysis of major organs. AUTHOR INFORMATION Corresponding Author *G. Z.: E-mail: [email protected] *K. T.: Phone: 86-21-3420-2956, Fax: 86-21-3420-2745, E-mail: [email protected] ORCID Ke Tao: 0000-0002-8014-8587 Author Contributions ‡Y. L. and Y. X. contributed equally to this work. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing ﬁnancial interest. Acknowledgements This work was financially supported by National Science Foundation of China (Project No. 31671027 and No. 31671004). We thank Instrumental Analysis Center of SJTU for assistance with the instrumentation.


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(49) Pongrac, I. M.; Dobrivojevic, M.; Ahmed, L. B.; Babic, M.; Slouf, M.; Horak, D.; Gajovic, S. Improved biocompatibility and efficient labeling of neural stem cells with poly(L-lysine)coated maghemite nanoparticles. Beilstein J. Nanotechnol. 2016, 7, 926-936. (50) Zhang, X.; Oulad-Abdelghani, M.; Zelkin, A. N.; Wang, Y.; Haikel, Y.; Mainard, D.; Voegel, J. C.; Caruso, F.; Benkirane-Jessel, N. Poly(L-lysine) nanostructured particles for gene delivery and hormone stimulation. Biomaterials 2010, 31, 1699-1706. (51) Wang, H.; Gao, Z.; Liu, X.; Agarwal, P.; Zhao, S.; Conroy, D. W.; Ji, G.; Yu, J.; Jaroniec, C. P.; Liu, Z.; Lu, X.; Li, X.; He, X. Targeted production of reactive oxygen species in mitochondria to overcome cancer drug resistance. Nat. Commun. 2018, 9, 562-577. (52) Chen, Z.; Chen, H.; Hu, H.; Yu, M.; Li, F.; Zhang, Q.; Zhou, Z.; Yi, T.; Huang, C. Versatile synthesis strategy for carboxylic acid−functionalized upconverting nanophosphors as biological labels. J. Am. Chem. Soc. 2008, 130, 3023-3029. (53) Chen, D.; Tao, R.; Tao, K.; Chen, B.; Choi, S. K.; Tian, Q.; Xu, Y.; Zhou, G.; Sun, K. Efficacy dependence of photodynamic therapy mediated by upconversion nanoparticles: subcellular positioning and irradiation productivity. Small 2017, 13, 1602053. (54) Prieto, M.; Rwei, A. Y.; Alejo, T.; Wei, T.; Lopez-Franco, M. T.; Mendoza, G.; Sebastian, V.; Kohane, D. S.; Arruebo, M. Light-emitting photon-upconversion nanoparticles in the generation of transdermal reactive-oxygen species. ACS Appl. Mater. Interfaces 2017, 9, 4173741747. (55) Wang, D.; Zhu, L.; Pu, Y.; Wang, J.-X.; Chen, J.-F.; Dai, L. Transferrin-coated magnetic upconversion nanoparticles for efficient photodynamic therapy with near-infrared irradiation and luminescence bioimaging. Nanoscale 2017, 9, 11214-11221. (56) Wong, P. T.; Tang, S.; Cannon, J.; Chen, D.; Sun, R.; Lee, J.; Phan, J.; Tao, K.; Sun, K.; Chen, B.; Baker, J. R., Jr.; Choi, S. K. Photocontrolled release of doxorubicin conjugated through a thioacetal photocage in folate-targeted nanodelivery systems. Bioconjugate Chem. 2017, 28, 3016-3028. (57) Li, B.; Lin, L.; Lin, H.; Wilson, B. C. Photosensitized singlet oxygen generation and detection: Recent advances and future perspectives in cancer photodynamic therapy. J. Biophotonics 2016, 9, 1314-1325. (58) Hou, Z.; Zhang, Y.; Deng, K.; Chen, Y.; Li, X.; Deng, X.; Cheng, Z.; Lian, H.; Li, C.; Lin, J. UV-emitting upconversion-based TiO2 photosensitizing nanoplatform: near-infrared light mediated in vivo photodynamic therapy via mitochondria-involved apoptosis pathway. ACS Nano 2015, 9, 2584-2599. (59) Cui, S.; Chen, H.; Zhu, H.; Tian, J.; Chi, X.; Qian, Z.; Achilefu, S.; Gu, Y. Amphiphilic chitosan modified upconversion nanoparticles for in vivo photodynamic therapy induced by near-infrared light. J. Mater. Chem. 2012, 22, 4861–4873. (60) Tian, G.; Ren, W.; Yan, L.; Jian, S.; Gu, Z.; Zhou, L.; Jin, S.; Yin, W.; Li, S.; Zhao, Y. Red-emitting upconverting nanoparticles for photodynamic therapy in cancer cells under nearinfrared excitation. Small 2013, 9, 1929-1938. (61) Clevers, H. The cancer stem cell: premises, promises and challenges. Nat. Med. 2011, 17, 313-319. (62) Qi, W.; Zhao, C.; Zhao, L.; Liu, N.; Li, X.; Yu, W.; Wei, L. Sorting and identification of side population cells in the human cervical cancer cell line Hela. Cancer Cell Int. 2014, 14, 3-13.


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(63) Hou, H. W.; Warkiani, M. E.; Khoo, B. L.; Li, Z. R.; Soo, R. A.; Tan, D. S.; Lim, W. T.; Han, J.; Bhagat, A. A.; Lim, C. T. Isolation and retrieval of circulating tumor cells using centrifugal forces. Sci. Rep. 2013, 3, 1259. (64) Singh, S. K.; Hawkins, C.; Clarke, I. D.; Squire, J. A.; Bayani, J.; Hide, T.; Henkelman, R. M.; Cusimano, M. D.; Dirks, P. B. Identification of human brain tumour initiating cells. Nature 2004, 432, 396-401. (65) Rao, Q.-X.; Yao, T.-T.; Zhang, B.-Z.; Lin, R.-C.; Chen, Z.-L.; Zhou, H.; Wang, L.-J.; Lu, H.-W.; Chen, Q.; Di, N.; Lin, Z.-Q. Expression and functional role of ALDH1 in cervical carcinoma cells. Asian Pac. J. Cancer Prev. 2012, 13, 1325-1331. (66) Wang, K.; Zeng, J.; Luo, L.; Yang, J.; Chen, J.; Li, B.; Shen, K. Identification of a cancer stem cell-like side population in the Hela human cervical carcinoma cell line. Oncol. Lett. 2013, 6, 1673-1680. (67) Yang, B.; Lu, Y.; Zhang, A.; Zhou, A.; Zhang, L.; Zhang, L.; Gao, L.; Zang, Y.; Tang, X.; Sun, L. Doxycycline induces apoptosis and inhibits proliferation and invasion of human cervical carcinoma stem cells. PLoS One 2015, 10, e0129138. (68) Wilhelm, S.; Tavares, A. J.; Dai, Q.; Ohta, S.; Audet, J.; Dvorak, H. F.; Chan, W. C. W. Analysis of nanoparticle delivery to tumours. Nat. Rev. Mater. 2016, 1, 16014. (69) Liu, L.-H.; Qiu, W.-X.; Li, B.; Zhang, C.; Sun, L.-F.; Wan, S.-S.; Rong, L.; Zhang, X.-Z. A Red Light Activatable Multifunctional Prodrug for Image-Guided Photodynamic Therapy and Cascaded Chemotherapy. Adv. Funct. Mater. 2016, 26, 6257-6269. (70) Ding, B.; Shao, S.; Yu, C.; Teng, B.; Wang, M.; Cheng, Z.; Wong, K. L.; Ma, P.; Lin, J. Large-Pore Mesoporous-Silica-Coated Upconversion Nanoparticles as Multifunctional Immunoadjuvants with Ultrahigh Photosensitizer and Antigen Loading Efficiency for Improved Cancer Photodynamic Immunotherapy. Adv. Mater. 2018, 30, e1802479. (71) Kue, C. S.; Kamkaew, A.; Lee, H. B.; Chung, L. Y.; Kiew, L. V.; Burgess, K. Targeted PDT agent eradicates TrkC expressing tumors via photodynamic therapy (PDT). Mol. Pharmaceutics 2015, 12, 212-222. (72) Lucky, S. S.; Muhammad Idris, N.; Li, Z.; Huang, K.; Soo, K. C.; Zhang, Y. Titania coated upconversion nanoparticles for near-infrared light triggered photodynamic therapy. ACS Nano 2015, 9, 191-205.


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Figure 1. The synthetic routes and characterization of nanoformulations. a) Schematic illustration of the preparation of nanoformulations; b) TEM imaging of the nanoformulation with elemental mapping of fluorine (blue), yttrium (red), gadolinium (Green) and carbon (purple), respectively; c) Photographs of UCNs@PLL-PpIX and PLL-PpIX in water before and after centrifugation; d) Loading percentage of PpIX measured by two different methods; e) UV-Vis absorption of PpIX, and the fluorescence spectra of UCNs and the nanoformulations; f) ROS generation indicating by the decay of DPBF absorption at ~430 nm. The result from our previous work53 tested under the same conditions was used as the reference.


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Figure 2. The interaction between cells and nanoformulations. a) The internalization of nanoformulations identified by multichannel confocal fluorescence micrographs for DAPI, PpIX, UCNs and their overlay, scale bar: 100 µm; b) The cellular uptake of UCNs@PLL-PpIX and UCNs@PLL-PpIX-FA with incubation at different concentration; c) Cell sorting with labeling of anti-CD133 and nanoparticles (NP) in terms of PpIX by cytometry. From left to right: blank control, only with anti-CD133, with nanoparticles incubation at concentration of 3.5 μg/ml and 35 μg/ml, respectively; d) The cellular uptake of nanoparticles by the cells that labelled with anti-CD133 (cancer stem cells) and that cannot be marked with anti-CD133 (non-stem cancer cells).


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Figure 3. The intratumoral distribution of nanoaprticles. The accumulation dose in tumor tissue and the remained dose of nanoparticles in tumor cells after collagenase digestion at 1.5 h after different treatments: a) UCNs@PLL-PpIX with intratumoral injection and b) UCNs@PLLPpIX-FA with intraperitoneal injection; Data are presented as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001. c) Multichannel confocal fluorescence micrographs of the histological slices of tumor in control group, intratumor injection group of UCNs@PLL-PpIX, and intraperitoneal injection group of UCNs@PLL-PpIX-FA with eosin staining, respectively, scale bar: 80 µm; d) The distribution of nanoparticles in tumor scanning by microCT at 1 hour and 3 hours after local administration, respectively, scale bar: 4 mm; e) The local dose of nanoparticles per gram tissue in eight nubbles randomly cut from each tumor.


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Figure 4. The photodynamic toxicity of nanoformulations for Hela cells. a) The viability of HeLa cells incubated with different samples without NIR irradiation and with the irradiation for 10 min, respectively; b) The live-staining of the incubated cells by calcein AM without irradiation and with irradiation, respectively, scale bar: 100 µm; c) The labeling of anti-CD133 for the blank cells, cells incubated with UCNs@PLL-PpIX-FA, cisplatin, and treated by the nanoparticles (70 μg/ml) with laser irradiation, respectively.


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Figure 5. The in-vivo therapeutic effects of nanoformulations. a) The evolution of the normalized tumor volume without injection (upper panel), with intratumor injection (middle panel) and intraperitoneal injection (lower panel), respectively. Data are presented as mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. b) The photographs of the mice after therapy for different days. A 15-min irradiation was performed for (+) Laser at 1.5 h after injection in the 1st day.


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ToC figure


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A Simple, Yet Multifunctional, Nanoformulation for Eradicating Tumors

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