Mucosal Penetrating Bioconjugate Coated Upconverting

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A mucosal penetrating bio-conjugate coated upconverting nanoparticles that integrate biological tracking and photodynamic therapy for gastrointestinal cancer treatment Zhaoxu Meng, Liping Zhang, Zhonggui He, and He Lian ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00359 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 6, 2018

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ACS Biomaterials Science & Engineering

A mucosal penetrating bio-conjugate coated upconverting nanoparticles that integrate biological tracking and photodynamic therapy for gastrointestinal cancer treatment Zhaoxu Meng1, Liping Zhang1, Zhonggui He2, He Lian1*

1

Department of Biomedical Engineering, Faculty of Medical Instrumentation, Shenyang Pharmaceutical University, Shenyang, 110016, China

2

Wuya college of Innovation, Shenyang Pharmaceutical University, Shenyang, 110016, China

Corresponding author: Dr. He Lian* Email: [email protected]

ACS Paragon Plus Environment

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ACS Biomaterials Science & Engineering 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

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Abstract

The integration of non-invasive photodynamic therapy (PDT) and imaging in the gastrointestinal tract is extremely challenging because of the limited light penetration in traditional PDT, and physiological

barriers

such

as

inorganic

salts

and

mucous

layers.

Herein,

D-α-

tocopherol polyethylene glycol 1000 succinate-succinic acid-mercapto-ethylamine (TPGS-SH) was used to functionalize upconverting nanoparticles (UCNPs) and endow the modified UCNPs@TPGS-SH nanoparticles with excellent hydrophilic properties, biocompatibility, gastrointestinal stability and mucus penetration. After loading the photosensitizer zinc phthalocyanine

(ZnPC)

onto

UCNPs@TPGS-SH,

the

resulting

nanosystem

ZnPC-

UCNPs@TPGS-SH converted near infrared light (NIR) to visible light, which activated ZnPC to generate singlet oxygen that showed a strong killing effect in Caco-2 cells. In-situ mucosal penetration assay showed that ZnPC-UCNPs@TPGS-SH accumulated more in the enterocytes of duodenum, jejunum and ileum, and less in the mucosal layer compared to ZnPCUCNPs@TPGS. Moreover, cellular uptake and in-situ tracing experiments confirmed that the florescent light emitted by UCNPs upon upconversion of deep penetrating NIR light could also serve as an imaging probe. These findings demonstrate the promising potential of this theranostic nanosystem for simultaneous deep penetrating biological tracing and PDT in gastrointestinal cancer.

Keywords: mucosal penetration; upconverting nanoparticles; biological imaging; photodynamic therapy; gastrointestinal cancer


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1. Introduction Gastrointestinal cancer results in high morbidity and mortality, with an annual death count of 8.2 million1. It is the second leading cause of cancer related deaths worldwide. Every year2, more than 4 million new cases emerge, which is greater than the sum of lung and breast cancer cases3. Although advanced detection and screening techniques have been developed, the prevention and treatment of gastrointestinal cancer is still a major challenge. Photodynamic therapy (PDT) is a treatment strategy based on photochemical and photobiological reactions which are triggered by photosensitizers, the appropriate wavelength of light, and molecular oxygen. PDT can oxidize tissues and cells, causing irreversible DNA damage, abnormal proliferation, and eventually leading to apoptosis4-7. PDT has been developed for gastrointestinal cancer in the past decades. Much of the research has focused on developing delivery systems such as polysaccharide and chlorin e6 complex8, aloe emodin-encapsulated nano-liposomes9, a muco-adhesive thermo-responsive system containing methylene blue10, and oxygen self-enriching perfluorocarbon nano-droplets11. Although this mild and non-invasive therapeutic modality has attracted a lot of attention and has been approved by many countries for cancer treatment, it is still beset with significant challenges and problems, such as the limited penetration depth of the irradiation light12,

13

.

Fortunately, the upconverting nanoparticles (UCNPs) that absorb photons of lower energy and emit a photon of higher energy have been developed in recent years, and can solve this problem. The light wavelength absorbed by the UCNPs lies in the near-infrared (NIR) region which allows deeper tissue penetration, while the light emitted by UCNPs can overlap with the excitation range of many photosensitizers14, 15 such as the phthalocyanine zinc (ZnPC) used in this study.


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However, the UCNPs capped with oleic acid as well as most photosensitizers are generally hydrophobic16-18. Furthermore, the mucosal surface of the gastrointestinal tract greatly hinders the cellular internalization of foreign nanoscale particles

19-21

. This phenomenon is a result of a

natural defense mechanism wherein the viscoelastic gel formed by entangled mucin fibers can identify and adhere to bacteria and other microorganisms with the help of highly glycosylated, negatively charged oligosaccharide domains, and finally clear them through mucus layer renewal and intestinal peristalsis22,

23

. Therefore, a functional modification is needed to stabilize the

UCNPs, solubilize the photosensitizers, and enable mucosal penetration. Recently,

we

developed

a

dual

functional

material

–D-α-

tocopherol polyethylene glycol 1000 succinate-succinic acid-mercaptoethylamine (TPGS-SH) – that exhibits mucosal permeability and P-gp inhibition function24. The reproducible synthesis process, ability to self-assemble, enhanced drug solubilization, and excellent mucosal penetration effects of TPGS-SH have been well documented. As a kind of amphiphilic conjugate, TPGS-SH has been successfully used for the oral delivery of paclitaxel in our previous study. In the present study, we introduced TPGS-SH as a surface modification material for the first time in functionalizing UCNPs to construct a nanotheranostic system of ZnPC-UCNPs@TPGSSH for simultaneous biological tracing and PDT for gastrointestinal cancer. The system was fabricated by a controllable and maneuverable ultrasound-based method. The inner hydrophobic core of the system consisted of UCNPs and the photosensitizer ZnPC that was inserted through an ultra-sonication induced assembly process, and both were stabilized by the hydrophobic segment of TPGS-SH. The outer PEG-SH shell, which is the hydrophilic end of TPGS-SH, could endow the system with water solubility and biocompatibility, mucus penetration, and gastrointestinal stability. When irradiated by a 980nm laser light, UCNPs converted NIR light to


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visible light, which induced a PDT effect by activating ZnPC to generate ROS (reactive oxygen singlet), and simultaneously helped with real-time biological imaging of deeper tissues (figure 1).

Figure 1 Schematic diagram showing the preparation of TPGS-SH functionalized UCNPs for ZnPC loading and upconversion effect (a), and the description intestinal distribution of UCNPs@TPGS-SH, mucosal penetration, and subsequent biological tracer and photodynamic therapy (b).

2. Materials and methods


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2.1 Materials YCl3·xH2O (x ≈ 6, 99.99%), YbCl3·xH2O (x ≈ 6, 99.99%), ErCl3 (99.99%), NaF (99.99%), 1-octadecene (ODE, 90%), and oleic acid (OA, 90%) were purchased from Macklin Biochemical Co., Ltd (Shanghai, China). D-α-tocopherol poly-ethyleneglycol 1000 succinate (TPGS), succinic anhydride (SA), β-mercaptoethylamine hydrochloride (MEA·HCl), and zinc phthalocyanine (ZnPC) were purchased from Aladdin Biochemical Technology Co., Ltd (Shanghai, China). Propidium iodide (PI), 4, 6-diamidino-2-phenylindole (DAPI), and Rhodamine-labeled phalloidin were purchased from Sigma (St. Louis, MO, USA). All other chemicals and reagents were of analytic grade and used without further purification. 2.2 Synthesis of thiolated TPGS (TPGS-SH) The mucosal penetrating bio-conjugate TPGS-SH was synthesized by coupling βmercaptoethylamine with carboxylated TPGS activated by succinic anhydride as we previously reported24. Briefly, triethylamine and 4-dimethylaminopyridine (DMAP) acted as catalytic agents to open the succinic anhydride ring and formed an ester bond with the terminal hydroxyl group of TPGS. This carboxylation reaction was performed at 30 ℃ for 24 h. Afterwards, carboxylated TPGS and

the high efficiency catalyst 1-[Bis(dimethylamino)methylene]-1H-

1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) was added to MEA·HCl in DMF solution, the mixture was heated to 40 ℃ with stirring for 10 h to form amide bond . The whole operation process was carried out under nitrogen protection and the final product TPGSSH conjugate was obtained by freeze-drying. The chemical structure of TPGS-SH conjugate is provided in the supplementary information (figure 1s). 2.3 Synthesis of OA-capped NaYF4:Yb/Er (20/2 mol%) UNCPs


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The OA-capped NaYF4:Yb/Er UCNPs (OA-UCNPs) were synthesized by the solvo-thermal synthesis method25, 26 using oleic acid as the stabilizing agent as described below. 2.3.1 The synthesis of reaction precursors YCl3·6H2O (7.8 mM), YbCl3·6H2O (2 mM) and ErCl3 (0.2 mM) were dissolved in absolute ethanol in a 250 mL flask, and stearic acid (30 mM, 8.5344 g) was added. The mixture was heated to 82 ℃ till the reflux state was achieved, and 20 mL NaOH was added dropwise with vigorous stirring for 30 min. The reaction mixture was continuously stirred at 82 ℃ for another 40 min. The resulting product was filtered, washed with water and absolute ethanol, and dried at 60 ℃ for 12 h to obtain the rare earth stearate precursor. 2.3.2 The synthesis of UCNPs Rare earth stearate precursor (1mM) and NaF (10mM) were added to a solvent containing 10 mL water, 15 mL ethanol and 5 mL oleic acid. After sonication for 15 min, the mixture was placed in a hydrothermal reactor at 200 ℃ for 24 h. After the reaction system was cooled to 60 ℃, the mixture was centrifuged (1000 rpm) for 3 min. A white precipitate was obtained which was washed thrice with chloroform/ethanol solvent mix (1:6, v/v), thrice with water/ethanol solvent mix (1:2, v/v), and once with absolute ethanol. The UCNPs were finally obtained after drying the mixture at 60 ℃ for 12 h. 2.4 TPGS-SH surface modification and characterization Briefly, 15 mg OA-UCNPs was added into 15 mL TPGS-SH aqueous solution (20 mg/mL) with vigorous stirring and the mixture was sonicated using a probe-type sonicator (200 w, 3 sec pulses every 3 s for a duration of 20 min). The mixture was then centrifuged for 10 min, the


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upper aqueous layer was taken out and washed twice with deionized water. Finally, the UCNPs@TPGS-SH product was re-suspended in water and stored at 4 ℃. The size and morphology of the UCNPs was observed with a high-resolution transmission electron microscope (TEM, Tecnai G20, USA). The crystal structure was characterized by a D/Max 2500 PC X-ray diffractometer (Rigaku, Tokyo, Japan) and size distribution was analyzed using a Zetasizer (Nano ZS, Malvern Co., UK). Thermogravimetric analysis (TGA) was conducted with a thermos-gravimetry analyzer (TG-DTA, Japan) from 45 to 500℃ at a heating rate of 10℃/min under a nitrogen flow. Infrared spectra was collected by infrared spectrometry (Bruker IFS55, Germany) and other optical measurements were performed at room temperature by a fluorescence spectrophotometer (Edinburgh FLSP920, UK) externally equipped with a 980 nm laser and a UV-Vis spectrophotometer (Agilent CARY 300, USA). 2.5 ZnPC loading efficiency and Upconversion fluorescence Different amounts of ZnPC (80-320 µM) were dispersed in a small volume of DMSO, and the solution was added into a 5 mL UCNPs@TPGS-SH aqueous solution with constant stirring. After probe sonication (Scientz JY92-IID, Ningbo, China) for 20 min, the mixture was centrifuged at 3500 rpm for 5 min to remove free ZnPC. The ZnPC-UCNPs@TPGS-SH nanoparticles were then collected, washed, and re-suspended in deionized water by sonication to obtain a homogeneous colloidal solution. To determine the drug loading efficiency of ZnPC, a standard curve of absorbance versus concentration was made by measuring the absorbance value of ZnPC at 654 nm on a UV-Vis spectrophotometer (Agilent CARY 300, USA). The ZnPC loaded onto UCNPs@TPGS-SH was dissolved in DMSO and diluted in the standard curve range for absorbance measurement. Drug loading (DL) ratio was calculated as follows: Amount of


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ZnPC (mg)/Amount of ZnPC-UCNPs@TPGS-SH ×100%. The upconversion fluorescence spectra of ZnPC-UCNPs@TPGS-SH with different drug loading ratios were also measured by the fluorescence spectrophotometer. Samples with the highest drug loading ratio were used for cell and animal experiments. 2.6 Determination of singlet oxygen production The production of singlet oxygen was determined using 1, 3-Diphenylisobenzofuran (DPBF) as the probe. The generation of 1O2 converts DPBF to DBB, which can be detected by decreased absorbance at 420 nm27. Briefly, 5 mg/mL ZnPC-UCNPs@TPGS-SH was mixed with 50 mM DPBF and irradiated by a 980 nm laser for different periods of time. The consumption of DPBF was calculated by measuring the absorbance change of the samples at 420 nm. 2.7 Size and fluorescence stability of UCNPs@TPGS-SH Classical simulated gastrointestinal media, simulated gastric fluid (SGFsp, pH 1.2), simulated intestinal fluid (SIFsp, pH 6.8), simulated gastric fluid containing pepsin (SGF, pH 1.2), and simulated intestinal fluid containing trypsin (SIF, pH6.8) were prepared according to Chinese Pharmacopoeia to determine the size and fluorescence stability of UCNPs@TPGS-SH. For the tests, 5 mL of UCNPs@TPGS-SH was added to an equal volume of SGF or SIF media, and incubated at 37℃. After 0, 3, and 6 h, 3 mL of the samples were taken out to measure the upconversion fluorescence spectra and particle size by DLS. Day-light and UCL images of UCNPs@TPGS-SH (0.5 mg/mL) in SGF and SIF media were also collected after the 6 h incubation. 2.8 In situ tracing of ZnPC-UCNPs@TPGS-SH in the intestine


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All animal experiments in this study were performed according to the Guidelines for the Care and Use of Laboratory Animals approved by the Ethics Committee of Animal Experimentation of Shenyang Pharmaceutical University. To track the in-situ behavior of UCNPs@TPGS-SH in the intestine, 2 mL ZnPC-UCNPs@TPGS-SH was orally administrated to Sprague-Dawley (SD) rats. After 1 or 2 h, the rats were sacrificed, their duodenum, jejunum and ileum segments were isolated, everted and frozen at -80℃ in the embedding agent. The frozen intestines were sectioned into slices of 10 µm thickness, fixed on cationic resinous slides with 4% para-formaldehyde and permeabilized with 1% Triton X-100. The cell nuclei were stained with DAPI for 5 min and the tissue sections were observed under a confocal laser scanning microscope externally equipped with a 980 nm laser. 2.9 In-vitro studies 2.9.1 Cell culture Human colon carcinoma cell line Caco-2 purchased from the American Tissue Culture Collection (Rockville, MD) were cultured in Delbecco's modified eagle medium (DMEM) containing 4,500 mg/L glucose, 20 mM HEPES, 10% fetal bovine serum (FBS), 100 µg/mL streptomycin and 100 U/mL penicillin, and then incubated in an atmosphere of relative humidity of 90% at 37℃ with 5% CO2 (v/v). 2.9.2 Cytotoxicity Cell viability was determined by the MTT assay. Caco-2 cells were seeded into 96-well plates at a density of 3×104 cells per well and cultured for 24 h in a moist atmosphere with 5% CO2 (v/v). The culture medium was replaced by different concentrations of UCNPs@TPGS-SH


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dispersed in fresh serum-free media, and the cells were incubated for an additional 12, 24 or 48 h. At the designated intervals, 50 µL MTT (2 mg/mL) was added to each well and after a 4 h incubation at 37 ℃, the medium was discarded and 200 µL DMSO was added. The plates were shaken for 30 min at room temperature, and the absorbance of each well was recorded at 570 nm with a microplate reader. 2.9.3 Photodynamic therapy of cancer cells Caco-2 cells were seeded into 96-well plates at a density of 3×104 cells per well and cultured for 24 h. After cell attachment, PBS, void UCNPs@TPGS-SH or ZnPCUCNPs@TPGS-SH were added at different concentrations and incubated for 12 h. The cells were then exposed to a 980 nm laser (800 mW) for different durations (0, 5, 10 and 15 min). Following a 24 h incubation, cell viability was determined by the MTT assay as described. 2.9.4 Cellular uptake Caco-2 cells were seeded on pre-sterilized coverslips in 6-well plates at a density of 1×105 cells per well and cultured for 7 days. After washing with HBSS solution, the cells were incubated with ZnPC-UCNPs@TPGS-SH dispersed in fresh serum-free culture medium (500 µg/mL) for 1, 2 or 4 h. The cells were then washed thrice with cold PBS solution and fixed with 70% ethanol for 15 min. The coverslips were fixed on a slide with a sealing liquid for observation under a fluorescence microscopy (BX53, Olympus, Japan). The mean fluorescence intensity was calculated with the ImagePro-Plus software as (IOD SUM) / (area sum). 2.10 In situ mucosal penetration study


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SD rats weighing 230-250g were fasted overnight and injected intraperitoneally with a urethane solution (1 g/kg). The abdominal cavity was exposed by making a ventrimeson incision and ZnPC-UCNPs@TPGS-SH or ZnPC-UCNPs@TPGS dispersed in Krebs Ringer modified buffer solution (1 mg/mL) were perfused into a 10 cm long intestinal segment that included the duodenum, jejunum and ileum. After 2 h, 2-cm segments were sheared and washed gently with deionized water to remove the unabsorbed UCNPs@TPGS-SH. The mucosal layer and enterocytes were then separated into two EP tubes for grinding in a homogenization machine with 1mL deionized water. Afterwards, yttrium ions (Y3+) were dissolved by adding appropriate amount of digestive juice (composed by nitric acid, hydrochloric acid and perchloric acid) at 150 ℃ for 2 h, and detected by an inductively coupled plasma-atomic emission spectrometry.

3. Results 3.1 Preparation and characterization of UCNPs@TPGS-SH The procedure for the preparation of UCNPs@TPGS-SH is described in figure 1. OAcapped UCNPs were firstly synthesized and then coated with TPGS-SH through ultra-sonication method. The solvo-thermal synthesis method was used to obtain NaYF4:Yb/Er (20/2 mol%) nanoparticles and OA was chosen as the stabilizing agent to form coordination bonds with rare earth ions. The OA-capped UCNPs could be easily coated with TPGS-SH through the hydrophobic forces between OA and vitamin E succinate (VES), the hydrophobic segment of TPGS-SH. The coating process made the OA-capped UCNPs soluble in water. The size and morphology of OA-capped UCNPs and UCNPs@TPGS-SH were observed by transmission electron microscopy as shown in figures 2a and 2b. The image of OA-UCNPs


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showed a uniform particle size distribution of around 25 nm, but a clear aggregation state was present due to the hydrophobic interaction of oleic acid on the particle surface. In contrast, no obvious particle aggregation was seen in the image of UCNPs@TPGS-SH. Dynamic light scattering (DLS) measurements determined the particle size of UCNPs@TPGS-SH to be 26.4± 1.2 nm (figure 2c), which was consistent with the homogeneous size distribution observed in the TEM images. XRD analysis was carried out to determine the crystal structure of UCNPs@TPGS-SH (figure 2d). Almost all of the diffraction peaks corresponded with the standard spectra, indicating a uniform hexagonal crystal structure of UCNPs@TPGS-SH which can ensure an excellent upconversion fluorescence. TG and FTIR spectra analysis were also performed to verify the successful coating of TPGS-SH on the UCNPs surface. In the TG curve for OA-UCNPs, only an 11.82% loss of mass was observed from 260 ℃ to 400 ℃. However, the UCNPs@TPGS-SH curve showed a more significant loss of 21.59% from 250 ℃ to 500 ℃ (figure 2e), which was attributed to the thermal decomposition of TPGS-SH. The FTIR spectra (figure 2f) of OA-UCNPs showed peaks at 2914 and 2851 cm-1 representing the stretching vibration of C-H bond, and at 1563 and 1463 cm-1 corresponding to the carboxyl stretching vibration of OA. After coating with TPGS-SH, an obvious stretching vibration was observed at 2920 cm-1, and new peaks at 1732 and 1637 cm-1 were attributed to the vibration of C=O bond and amideⅡband. In addition, a strong new peak at 1101 cm-1 corresponding to C-O vibration was also found.


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Figure 2 TEM images of (a) OA-UCNPs and (b) UCNPs@TPGS-SH. (c) Size distribution of UCNPs@TPGS-SH from DLS analysis. (d) XRD spectrum of UCNPs@TPGS-SH, with vertical lines representing the standard pattern of NaYF4:Yb,Er (JCPDS No. 28-1192). (e) Thermogravimetric (TG) curves and (f) FT-IR spectra of OA-UCNPs and UCNPs@TPGS-SH.


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3.2 ZnPC loading and optical properties The UCNPs@TPGS-SH had hydrophilic PEG-SH segments extending outward, and the water-insoluble VES segments and the OA capped UCNP forming the hydrophobic core, which enabled the loading of the photosensitizer ZnPC through hydrophobic interactions. In addition, several other hydrophobic anti-cancer drugs could also be loaded into the inner layer of UCNPs@TPGS-SH. The upconversion luminescence spectrum of UCNPs@TPGS-SH under 980 nm laser irradiation and the UV-Vis absorption spectrum of ZnPC in ethanol are shown in Figure 3a. The strongest emission peaks of UCNPs@TPGS-SH were located at 540 nm and 654 nm, which was consistent with the maximum absorption wavelength of ZnPC (664 nm), indicating the potential activation of ZnPC by the upconverted red light. As shown in figure 3b and 3c, with increasing volume of ZnPC, drug loading capacity also increased (the highest drug loading ratio was 3.05%) while the fluorescence emission intensity of ZnPC-UCNPs@TPGS-SH between 650660nm (the emission peak of UCNPs) decreased gradually, indicating resonance transference between UCNPs and the loaded ZnPC. In contrast, no significant decrease in fluorescence intensity was observed in OA-UCNPs and blank UCNPs@TPGS-SH (figure 3d). The day-light photos of UCNPs@TPGS-SH and ZnPC-UCNPs@TPGS-SH, and the UCL images of UCNPs@TPGS-SH are shown in figure 3e, 3f, and 3g.


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Figure 3 (a) UV-Vis absorption spectrum of ZnPC in ethanol, UCL spectrum of UCNPs@TPGSSH in water. (b) Drug loading efficiency of ZnPC-UCNPs@TPGS-SH with different concentrations of ZnPC. (c) UCL spectrum of ZnPC-UCNPs@TPGS-SH with different loading efficiency of ZnPC (λex = 980 nm). (d) Upconversion luminescence (UCL) spectrum of OAUCNPs, UCNPs@TPGS-SH (λex = 980 nm). (e, f) Day-light and UCL images of


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UCNPs@TPGS-SH, and (g) Day-light image of ZnPC-UCNPs@TPGS-SH. (h) Production of single oxygen (1O2) by ZnPC-UCNPs@TPGS-SH upon irradiation by a 980 nm laser using DPBF as the probe.

3.3 Determination of singlet oxygen production DPBF, a commonly used singlet oxygen quencher, was used to monitor the generation of singlet oxygen by ZnPC-UCNPs@TPGS-SH in real time. Singlet oxygen generated during the continuous irradiation of UCNPs by 980 nm laser was measured as the decreased absorbance of DPBF at 420 nm. As expected (figure 3h), singlet oxygen production was proportional to the irradiation time; a rapid decline (about 43%) in OD420

nm

was observed within 30 min of

irradiation, decreasing further by 56.2% at 60 min. In the control groups, i.e. non-irradiated void UCNPs@TPGS-SH and irradiated void UCNPs@TPGS-SH, only a very slight decrease in DPBF absorption intensity was observed, indicating a lack of singlet oxygen generation. These results indicate that ZnPC-UCNPs@TPGS-SH could easily generate singlet oxygen through resonance energy transference, making it an excellent carrier for NIR-induced PDT. 3.4 Size and upconversion fluorescence stability The UCNPs@TPGS-SH displayed good size stability in the four different simulated gastrointestinal media within 6 h, as no obvious sediment was seen (figure 4a), and the mean particle size remained steady at around 25 nm (figure 4b). This could be attributed to the presence of hydrophilic PEG segments in the outer layer of the particles that hinder any contact and aggregation between the UCNPs. The fluorescence stability of UCNPs@TPGS-SH was first recorded by day-light photography, and significant green fluorescence was still observed after a 6 h incubation with SIF or SGF (figure 4a). To determine the fluorescence stability of


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UCNPs@TPGS-SH in SGF and SIF more accurately, a fluorescence spectrometer externally equipped with a 980 nm laser was used. As shown in figure 4c, after 6 h incubation with SGF, the fluorescence intensity of UCNPs@TPGS-SH at 540 nm decreased by 27%, possibly due to the erosion of the UCNPs by hydrogen ions in the medium which lead to fluorescence quenching. In contrast, when UCNPs@TPGS-SH was incubated in SIF medium, about 90% of the fluorescence could be retained within 3 h, and more than 85% of the fluorescence still existed after 6 h incubation (figure 4d). Thus, the PEG layer provided by TPGS-SH could effectively prevent the erosive impact of inorganic salts and digestive enzymes on the UCNPs and ensure fluorescence stability in the gastrointestinal environment.

Figure 4 (a) Day-light and UCL images of UCNPs@TPGS-SH (0.5 mg/mL) in SGF and SIF media after incubation for 6 h. (b) Particle size stability of UCNPs@TPGS-SH in different media


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after different incubation times (mean ± SD, n = 3). Fluorescence stability of UCNPs@TPGS-SH in SGF (c) and SIF (d) solution within 6 h. SGFsp: simulated gastric fluid; SGF: simulated gastric fluid containing pepsin; SIFsp: simulated intestinal fluid; SIF: simulated intestinal fluid containing trypsin) 3.5 Intestinal tracing and PDT effect on tumor cells Confocal laser scanning microscopy was used to track the intestinal bio-distribution of ZnPC-UCNPs@TPGS-SH in real time after oral administration. DAPI was used to stain the nuclei to further improve localization. The fluorescent images of different intestinal segments were collected at the designated times as shown in figure 5a. After 1 h, fluorescence signals of ZnPC-UCNPs@TPGS-SH only accumulated at the termini of the intestinal villi and microvilli while no detectable fluorescence was observed in the basal side of the intestine. After 2 h however, brighter fluorescence signals were observed in the epithelial cells of the duodenum, jejunum and ileum, along-with deeper penetration and accumulation at the basal side. This increase in fluorescence intensity corresponded to the increased absorption of UCNPs, making the ZnPC-UCNPs@TPGS-SH an excellent marker for in vivo tracking and imaging. The potential PDT effect of ZnPC-UCNPs@TPGS-SH was assessed by the MTT assay. When Caco-2 cells were incubated with different concentrations of the void UCNPs@TPGS-SH without NIR light for 12, 24 or 48 h, no obvious cytotoxicity was observed and the cell viability was above 90%, indicating the bio-safety of UCNPs@TPGS-SH (figure 5b). To confirm the PDT effect of ZnPC induced by energy resonance transfer, the cells were incubated with PBS, void UCNPs@TPGS-SH or ZnPC-UCNPs@TPGS-SH under NIR light. While the control groups retained 90% cell viability even after a 15 min irradiation, the cell viability of the ZnPC-


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UCNPs@TPGS-SH group rapidly decreased to 45.42% after only 10 min irradiation (figure 5c), indicating that photosensitizers and lasers with appropriate wavelengths are the two prerequisites for triggering PDT. It was also noteworthy that increasing the irradiation time improved the photodynamic effect since the tumor cell killing after 15 min irradiation was about 2.5 times higher than after 10 min irradiation (figure 5c). However, this increasing tumor cell killing effect was not consistent as the cell death rate increased by only 5.21% when the irradiation time was 15 minutes. The effect of particle concentration on cell viability was also investigated. As shown in figure 5d, UCNPs@TPGS-SH by itself could not induce obvious cell death even after 10 min irradiation whereas ZnPC-UCNPs@TPGS-SH could effectively inhibit cell growth after NIR light exposure, and the inhibitory effect increased in a dose dependent manner.


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Figure 5 (a) Fluorescence and upconversion luminescence images (λex = 980 nm) of rat intestine after oral administration of ZnPC-UCNPs@TPGS-SH for 1 and 2 h. The histological sections were stained with DAPI (blue). Photo-thermal therapy of the prepared UCNPs on Caco-2 cells: (b) Cell viability after incubation with void UCNPs@TPGS-SH at different concentrations for 12, 24 and 48 h. (c) Cell viability upon incubation with PBS solution, void UCNPs@TPGS-SH and ZnPC-UCNPs@TPGS-SH at the concentration of 100 µg/mL after irradiation (800 mW) for 5, 10 and 15 min. (d) Cell viability after incubation with void UCNPs@TPGS-SH and ZnPC-


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UCNPs@TPGS-SH at different concentrations after irradiation (800 mW) for 10 min (mean ± SD, n=3). 3.6 Cellular uptake of ZnPC-UCNPs@TPGS-SH To determine the effect of incubation time on cellular uptake, Caco-2 cells were treated with ZnPC- UCNPs@TPGS-SH for 1, 2 or 4 h and then observed under fluorescence microscopy. As shown in figure 6 (a-c), the fluorescence intensities increased gradually with longer incubation times, which was also confirmed by ImagePro-Plus software (figure 6d). These results indicate that ZnPC-UCNPs@TPGS-SH could effectively accumulate inside the tumor cells, which is essential for PDT. In addition, the fluorescent UCNPs@TPGS-SH could also be considered as a signal probe for real-time imaging in tumor therapy.

Figure 6 Fluorescence microscopy images showing the cellular uptake in Caco-2 cells incubated with ZnPC-UCNPs@TPGS-SH for 1 h (a), 2 h (b) and 4 h (c). Scale bars represent 50 µm. Meanfluorescence intensity (d) of ZnPC-UCNPs@TPGS-SH after incubation for different times.


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3.7 In situ mucosal penetration study The mucosal penetration of ZnPC-UCNPs@TPGS-SH was probed in SD rats by measuring the yttrium ion (Y3+) concentrations in the mucus layer and enterocytes 2 h after intra-enteral administration. As shown in figure 7, the ZnPC-UCNPs@TPGS-SH treated intestines accumulated more yttrium ion in the enterocytes of duodenum, jejunum and ileum, but less in the mucosal layer. In contrast, ZnPC-UCNPs@TPGS had a weaker mucus penetrating ability, as shown by the lower concentration of yttrium ions in enterocytes and a higher concentration in the mucus layer (P < 0.05). These results demonstrate that the PEG layer alone could only exert a limited mucus penetrating ability, and most of the UCNPs were still blocked by mucin fibers. On the other hand, when the PEG layer was terminally modified with sulfhydryl group, it could effectively pass through the mucus layer, and enter the epithelial cells through disulfide bond interaction between the sulfhydryl groups and the cysteine-rich areas in mucus layer.

Figure 7 Bio-distribution of ZnPC-UCNPs@TPGS-SH and ZnPC-UCNPs@TPGS in mucus and enterocytes in duodenum, jejunum and ileum segments in rats as determined by ICP-AES (mean ± SD, n=3).


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4. Discussion Currently, a large number of nanoscaled platforms with diverse drug loading and targeting capacities have been developed for cancer therapy, and they are designed such that they can be tracked using sophisticated imaging techniques like magnetic resonance imaging photoacoustic tomography30,

31

28, 29

,

, and near infrared imaging32. The amalgamation of nanoscale

therapy and diagnosis is a new field known as ‘nano-theranostics’33. However, the application of nanotheranostics in gastrointestinal cancer treatment is still beset with many challenges which limit the function of the nanoparticles, mainly the complex physiological environment in the gastrointestinal tract contributed by inorganic salts and digestive enzymes, and the physical hindrance presented by the mucus layer20, 21. Keeping these limitations in mind, we constructed a water-soluble and mucus penetrating ZnPC-UCNPs@TPGS-SH for gastrointestinal cancer therapy. In addition, we also integrated imaging and PDT properties into this construct. A TPGS-SH conjugate was made by attaching β-mercaptoethylamine to the terminus of TPGS which endowed it with properties essential for oral nanomedicine delivery, such as enhanced mucosal permeability, strong emulsifying and solubilizing ability, favorable P-gp suppression and biocompatibility, which had been confirmed by our previous work. Herein, the TPGS-SH conjugate was coated on the surface of UCNPs and enabled the latter to effectively pass through the intestinal mucus via the disulfide bond exchange reaction, and accumulate in the enterocytes. Upon irradiation by a 980 nm laser, ZnPC-UCNPs@TPGS-SH converted NIR light into visible light and activated the photosensitizer to generate ROS, thus integrating intestinal tracing, enterocytes imaging, and photodynamic therapy (Figure 1).


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It has been widely reported in the literature that UCNPs can be synthesized through the high temperature thermal decomposition method using rare earth oleates or rare earth trifluoroacetates as reaction materials14, 34, 35. However, the complicated synthesis process of rare earth oleate precursors and their highly viscous states at normal temperature makes the preparation process difficult. In addition, the high boiling point organic solvents used in the thermal decomposition method, such as trioctylphosphine and octadecylene, are also toxic. To avoid these problems, we adopted a two-step thermal-solvent synthesis method as reported before25, 26. The rare earth stearate precursor was first synthesized using rare earth chloride as raw material and absolute ethanol as the solvent. The relatively simple operation process described in the method section and the powdered solid state overcomes the shortcomings of the rare earth oleate precursors. The rare earth stearate precursor was then used to synthesis UCNPs that exhibited high crystallinity, uniform particle size, good dispersibility and controllable topography, all of which are depicted in figures 2, 3 and 4. UCNPs@TPGS-SH exhibited good size stability in the SGF and SIF media, but a relatively obvious fluorescence quench was detected after a 6 h incubation in the SGF media. Nevertheless, 73% of the fluorescence intensity was still retained. In addition, since the gastric emptying of rat occurs within 30 min, the fluorescence quenching in SGF media will not have a significant impact on subsequent intestinal imaging and PDT in-vivo. TPGS-SH modified UCNPs exhibiting good hydrophilicity, biocompatibility and mucosal penetration could effectively pass through the mucus layer, be absorbed by epithelial cells and produce strong upconversion fluorescence signal under NIR light irradiation for in-situ imaging (figure 5a). The fluorescence intensity of ZnPC-UCNPs@TPGS-SH expectedly changed with time. Therefore, tracking the distribution of nanotheranostic particles in the intestine in real time


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can provide an important time basis for PDT, thus allowing clinicians to develop precise treatment plans. Cell viability analysis indicated that cell death was mainly caused by the photodynamic effect. Our experiments have confirmed that the UCNPs mediated upconversion light stimulated ZnPC to produce ROS inside the Caco-2 cells and lead to cell death. On the other hand, UCNPs@TPGS-SH lacking the photosensitizer did not exhibit any cytotoxic effect regardless of irradiation. In addition, we examined the impact of different irradiation times on the photodynamic effect and found that 15 minutes of irradiation could not significantly improve the cytotoxicity; therefore, we selected 10 minutes as the optimum duration. Furthermore, it has been reported that continuous laser irradiation can cause some thermal effects, resulting in inaccurate results15. The cellular uptake experiment also confirmed that ZnPC-UCNPs@TPGS-SH could effectively enter into the cells. Our study further confirmed that the TPGS-SH modified UCNPs could enhance the particle distribution in enterocytes and decrease mucosal blocking, whereas the ZnPC-UCNPs@TPGS did not perform well in these aspects (figure 7). The reason for this is based on the mucus penetration ability of the thiomers, which has been discussed in previous reports36, 37. When the free thiol groups in thiomers encounter the cysteine-rich subdomains of mucosal fibers, a simple oxidation and thiol/disulfide exchange reaction occurs, resulting in the formation of reversible disulfide bonds that allow mucosal penetration. In contrast, ordinary nanoparticles could be easily buried and retarded by the mucus fibers.


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5. Conclusion In summary, we reported herein a mucosal penetrating bio-conjugate TPGS-SH coated upconverting nanoparticles which integrated biological tracking and PDT for gastrointestinal cancer treatment. Surface modification with TPGS-SH improved the hydrophilicity and stability of UCNPs for intestinal application and provided a hydrophobic reservoir for ZnPC loading. The modified UCNPs demonstrated uniform size distribution, good drug loading ratio and effective ROS production under irradiation at 980 nm. Due to the effective mucosal penetration and localization of ZnPC-UCNPs@TPGS-SH in Caco-2 cells and epithelial cells, the fluorescence emitted from UCNPs upon NIR light irradiation can easily be detected for biological imaging. Furthermore, since the wavelength of the emitted light corresponds to that of the excitation light of the photosensitizer ZnPC, it can also effectively produce ROS and kill the tumor cells. From all the advantages mentioned above, this mucosal penetrating TPGS-SH bio-conjugate coated upconverting nanoparticles could be a promising nanoplatform for simultaneous biological tracking and PDT for gastrointestinal cancer treatment.

Supporting Information. The synthesis process, 1H-NMR spectra and peak attribution of TPGS-SH conjugate were supplied in supporting information. The following files are available free of charge. Corresponding Author Dr. He Lian*


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Department of Biomedical Engineering, Faculty of Medical Instrumentation, Shenyang Pharmaceutical University, Shenyang, 110016, China Email: [email protected] ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (nos: 81503020, 31600764); the Scientific Research Project of Liaoning Provincial Department of Education (nos: 201610163L27, 2017LQN10); and the Innovation and Entrepreneurship Project for College Students of Liaoning Province (no: 1710163000092). ABBREVIATIONS PDT, photodynamic therapy; TPGS-SH, D-α-tocopherol polyethylene glycol 1000 succinatesuccinic

acid-mercapto-ethylamine;

UCNPs,

upconverting

nanoparticles;

ZnPC,

zinc

phthalocyanine; NIR, near infrared light; ROS, reactive oxygen singlet; SA, succinic anhydride; MEA·HCl, β-mercaptoethylamine hydrochloride; OA, oleic acid; PI, Propidium iodide; DAPI, 4, 6-diamidino-2-phenylindole;

DMAP,

4-dimethylaminopyridine;

HATU,

1-

[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate; DPBF, 1, 3-Diphenylisobenzofuran; DL, Drug loading; SGFsp, simulated gastric fluid; SIFsp, simulated intestinal fluid; SGF, simulated gastric fluid containing pepsin; SIF, simulated intestinal fluid containing trypsin; VES, vitamin E succinate; UCL, upconversion luminescence.

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For Table of Contents Use Only

A mucosal penetrating bio-conjugate coated upconverting nanoparticles that integrate biological tracking and photodynamic therapy for gastrointestinal cancer treatment

Zhaoxu Meng, Liping Zhang, Zhonggui He, He Lian*


35

Mucosal Penetrating Bioconjugate Coated Upconverting

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