Self-Assembly of a Pure Photosensitizer as a Versatile Theragnostic

Subscriber access provided by MEMORIAL UNIV

Biological and Medical Applications of Materials and Interfaces

Self-Assembly of Pure Photosensitizer as a Versatile Theranostic Nanoplatform for Imaging-Guided Antitumor Photothermal Therapy Xuanbo Zhang, Bingjun Sun, Shiyi Zuo, Qin Chen, Yanlin Gao, Hanqing Zhao, Mengchi Sun, Pengyu Chen, Han Yu, Wenjuan Zhang, Kaiyuan Wang, Ruoshi Zhang, Qiming Kan, Haotian Zhang, Zhonggui He, Cong Luo, and Jin Sun ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10421 • Publication Date (Web): 20 Aug 2018 Downloaded from http://pubs.acs.org on August 21, 2018

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 26 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 Applied Materials & Interfaces

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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 26

Self-Assembly of Pure Photosensitizer as a Versatile Theranostic

Nanoplatform

for

Imaging-Guided

Antitumor Photothermal Therapy †

†

†

‡

†

†

Xuanbo Zhang , Bingjun Sun , Shiyi Zuo , Qin Chen , Yanlin Gao , Hanqing Zhao , Mengchi Sun

†

, Pengyu Chen , Han Yu , Wenjuan Zhang , Kaiyuan Wang , Ruoshi Zhang , Qiming Kan⊥, §

†

†

†

†

Haotian Zhang⊥, Zhonggui He†, Cong Luo*,†, Jin Sun *,†

†

Department of Pharmaceutics, Wuya College of Innovation, Shenyang Pharmaceutical

University, Shenyang, Liaoning, 110016, P. R. China ‡

Department of Pharmacy, Cancer Hospital of China Medical University, Liaoning Cancer

Hospital & Institute, Shenyang 110042, P. R. China §

Key Laboratory of Structure-Based Drug Design and Discovery, Shenyang Pharmaceutical

University, Ministry of Education, Wenhua Road, No. 103, Shenyang 110016, China ⊥School

of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang,

Liaoning, 110016, P. R. China KEYWORDS: pure photosensitizer, DiR, self-assembly, theranostics, photothermal therapy


1

Page 3 of 26 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


ABSTRACT: Imaging-guided diagnosis and phototherapy has been emerging as promising theragnostic strategies for detection and treatment of cancer. 1,1′-dioctadecyl-3,3,3′,3′tetramethylindotricarbocyanine iodide (DiR) has been widely investigated for in vivo imaging and photothermal therapy (PTT). However, the tumor-homing ability and PTT efficiency of DiR is greatly limited by its extremely low water solubility and nonspecific distribution in off-target tissues. Herein, a facile nano-assembly of pure DiR is reported as a theragnostic nanocarrier platform for imaging-guided antitumor phototherapy. Self-assembly of DiR has almost no effect on its in vitro photothermal efficacy when compared with DiR solution. Interestingly, the PEGylated nanoassemblies of DiR showed distinct advantages over DiR solution and the nonPEGylated nanoassemblies in terms of systemic circulation and tumor-homing capability in vivo. As a result, PEGylated DiR nanoassemblies demonstrate potent photothermal tumor therapy in BALB/c mice bearing 4T1 xenograft tumors. Such a pure photosensitizer-based nano-assembly holds great potential as versatile platform for efficient imaging-guided cancer therapy.

1. INTRODUCTION Malignant tumor is still one of the leading threats to human health.1 The common therapeutic strategies include surgery, chemotherapy, radiotherapy and phototherapy.2 Among them, surgery is an effective therapeutic regimen for solid tumors, but the application of surgery is greatly limited in those patients with lately advanced and/or metastatic cancer.3-4 Despite the wide application in metastatic cancer patients, chemotherapy and radiotherapy have long been criticized for non-selective and off-target toxicity to normal tissues due to the narrow therapeutic index of chemotherapeutic agents.5-6 Distinctly distinguished from systemic treatment regimen of chemotherapy, imaging-guided phototherapy represents local and noninvasive treatment


2


Page 4 of 26

regimen.7 Under tumor-localizing light irradiation, photothermal therapy (PTT) and photodynamic therapy (PDT) could effectively kill tumor cells by generating localized heat or reactive oxygen species (ROS) in tumor site.8-10 Recently, great efforts have been made to explore the clinical applications of near-infrared (NIR) fluorescent dyes and imaging-guided PTT.11 Although PTT could effectively kill tumor cells by generating localized heat, its efficiency is usually limited by the inferior physicochemical and pharmacokinetic properties of most photosensitizers (PS).12 1,1′dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) is a hydrophobic fluorescent probe with strong absorption in the NIR region and negligible cytotoxicity without laser treatment, and it has been widely investigated for both in vivo optical imaging and PTT.13-14 However, DiR is a water-insoluble compound and will be quickly cleared from the body after intravenous administration, which greatly limits its application in clinical cancer therapy.15 With the burgeoning of nanoscience and biomaterials, nanoparticulate drug delivery systems (nano-DDS) have been intensively explored for anticancer drug delivery.16-17 Encapsulating therapeutic agents into nano-DDS could effectively address the challenges of poor physicochemical and pharmacokinetics properties. Moreover, making further modification on nanoparticles (NPs) could significantly influence their in vivo delivery fate and accelerate tumorspecific distribution through the enhanced permeability and retention (EPR) effect.18-20 DiRloaded nano-DDS was also widely investigated for tumor fluorescence imaging and PTT, especially inserting in the liposome membrane.21-22 However, low drug loading capacity of DiR in nano-DDS greatly restricted its application in PTT, and most DiR-loaded liposomes were used only for in vivo imaging purposes.21, 23 Therefore, it’s necessary to develop new drug delivery strategy to facilitate effective DiR delivery in photothermal cancer therapy.


3

Page 5 of 26 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


Figure 1. Schematic representation of self-assembly of DiR and efficient photothermal therapy under laser irradiation. The nanoassemblies were formed by pure DiR, and PEGylated DiR nanoassemblies was prepared using DSPE-PEG2k for PEGylation modification. After PEGylated DiR nanoassemblies were delivered into tumor site, efficient PTT could be realized under laser irradiation.

Herein, we reported a facile nano-assembly of pure photosensitizer for imaging-guided antitumor photothermal therapy (Figure 1). We found that DiR molecules demonstrated selfassembly characteristics, and the self-assembly mechanism of DiR was investigated by computational simulation. To increase the surface hydrophilicity of DiR nanoassemblies and to improve their pharmacokinetic behaviors, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2K) was used in the nano-formulation. The


4


Page 6 of 26

PEGylated DiR nanoassemblies showed comparable in vitro photothermal efficiency when compared with non-PEGylated DiR nanoassemblies and DiR solution, but demonstrated distinct advantages in terms of in vivo systemic circulation and tumor-homing accumulation. As expected, PEGylated DiR nanoassemblies demonstrated efficient photothermal tumor therapy in BALB/c mice bearing 4T1 xenograft tumors. Therefore, PEGylated DiR nanoassemblies demonstrated distinct drug delivery advantages: (i) improved colloidal stability and extended circulation time in blood; (ii) increased tumor-specific accumulation; and (iii) facilitated imaging-guided antitumor photothermal therapy. This is the first time that pure DiR was found to assemble into NPs without the assistance of carrier materials. Such a pure photosensitizer-driven nano-assembly holds promising application prospects for efficient imaging-guided cancer therapy.

2. EXPERIMENTAL SECTION 2.1 Materials. DiR were obtained from Meilun Biotech Co. Ltd. (Dalian, China). 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2K) was obtained from Shanghai Advanced Vehicle Technology Co. Ltd. Cell culture media, fetal bovine serum (FBS) penicillin and streptomycin were obtained from GIBCO, Invitrogen Corp. (Carlsbad, California, USA). Hoechst 33342 was obtained from BD Biosciences, USA. Trypsin and 3-(4,5-dimthyl-2-thiazolyl)-2,5-dipphenyl-2H-terazolium bromide (MTT) were obtained from Sigma-Aldrich, USA. Other chemical reagents mentioned in this article were of analytical grade. 2.2 Preparation of DiR Nanoassemblies. Self-assembly of DiR was performed by one-step nano-precipitation technique.18-20 In a typical procedure, 5 mg of DiR was dissolved in ethanol (1


5

Page 7 of 26 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


mL) to acquire the DiR ethanol solution (5 mg mL-1) , then 200 µL of the solution was added dropwise into aqueous solution (2 mL) under robust stirring (1400 rpm) for 5 min. After preparation, ethanol was removed from the colloidal solution in vacuum at room temperature. The final volume was regulated to 1 mL. In addition, DiR/DSPE-PEG2K NPs were prepared in the same procedure using a mixed ethanol solution of DiR and DSPE-PEG2K (20%, w/w). The prepared nanoassemblies were stored at 4 °C. 2.3 Characterization of DiR Nanoassemblies. The hydrodynamic diameter and zeta potential of DiR nanoassemblies were measured using a Zetasizer (Nano ZS, Malvern Co., UK). The particle size and zeta potential measurements were repeated in triplicate by diluting one prepared nanoassemblies into three different solutions. The morphology of nanoassemblies was observed using Transmission electron microscopy (TEM) (HITACHI, HT7700, Japan). Phosphotungstic acid (1%, w/v) were utilized to stain samples. 2.4 Ultraviolet and Fluorescence Spectra of DiR solution and DiR nanoassemblies. The fluorescence and ultraviolet spectra of DiR solution, non-PEGylated DiR nanoassemblies and DiR/DSPE-PEG2K NPs (0.1 mg mL−1 of DiR) was acquired using the Varioskan Flash multimode microreader (Thermo Scientific, USA). 2.5 Self-Assembly Simulation. The self-assembly mechanism and molecular interaction between DiR molecules were investigated using computational simulation.24 The 2-dimension structure of DiR molecule was built in Marvin sketch software (version: 16.4.25.0, Hungary), then a 3-dimension structure was produced by the Sybyl 6.9.1 software package (Tripos Associates: St. Louis, MO, 2003).24 The runtime environment and other method parameters were basically in line with our previous study. 24


6


Page 8 of 26

2.6 Stability. The stability of DiR nanoassemblies was studied. In a typical procedure, DiR nanoassemblies (50 µg mL-1 of DiR) were incubated in RPMI-1640 cell medium (10% FBS) for 24 h, and particle size and zeta potential was recorded at preset intervals. Moreover, the chemical stability of DiR in DiR solution, non-PEGylated DiR NPs and DiR/DSPE-PEG2K NPs incubated in mice serum was also determined using the Varioskan Flash multimode microreader (Thermo Scientific, USA). Additionally, long-term stability was performed with DiR nanoasseblies (1 mg mL-1) stored at 4 °C. 2.7 In Vitro Photothermal Efficiency. The photothermal efficiency of DiR nanoassemblies was investigated. In a typical procedure, PBS, DiR solutions (containing 10% ethanol), nonPEGylated DiR NPs, DiR/DSPE-PEG2K NPs with an equivalent DiR concentration of 0.1 mg mL-1 was added into centrifuge tube (1 mL). The above four samples were irradiated using a 808 nm laser (MDL-N-5W, Changchun New Industries, China) at 1.0 W cm-2 for 5 min, the temperature variations of these samples were measured by an infrared thermal imaging camera (Fotric 226). 2.8 Cellular Uptake and Cytotoxicity Evaluation. 4T1 cells were cultured in a humidified incubator (5% CO2) using RPMI-1640 cell medium containing 10% fetal bovine serum, penicillin-streptomycin (100 µg mL-1).18,24 To explore the cellular uptake efficiency of these formulations, 4T1 cells (5 × 104 cells) were seeded in 24-well plate and cultured for 24 h.24 Then the cells were incubated with DiR solutions, non-PEGylated DiR NPs, and DiR/DSPE-PEG2K NPs (250 ng mL-1 of DiR) for 0.5 h and 2 h. After removing the formulation containing medium, the cells were washed thrice using PBS for and incubated with Hoechst (Invitrogen) at 100 × 10−9 M for another 1 h and then washed thrice using PBS and fixed by 4% paraformaldehyde.


7

Page 9 of 26 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


Finally, cell imagings were obtained using confocal laser scanning microscopy (CLSM, TCS SP2/AOBS, LEICA, Germany). For quantitative determination, 4T1 cells (1 × 105 cells) were cultured in 12-well plates at 37 °C. Twenty-four hours later, the cells was incubated with DiR solutions, non-PEGylated DiR nanoassemblies and DiR/DSPE-PEG2k NPs (1 µg mL-1) for 0.5 h or 2 h. After that, the cells were washed, collected and redistributed in PBS. Intracellular fluorescence signals were recorded by FACSCalibur flow cytometer (n=3). To test the cell viability of DiR solution, DiR NPs, and DiR/DSPE-PEG2K NPs after irradiation, 4T1 cells (3 × 103 cells) were incubated in 96-well plate for 24 h. Then, the medium was replaced with various concentrations of DiR solutions, non-PEGylated DiR nanoassemblies and DiR/DSPE-PEG2k NPs. 4 h later, the cells were exposed to laser (808 nm, 4 W cm−2) for 3 min. MTT assay was utilized to investigate the cytotoxicity of DiR nanoassemblies. 2.9 Animal Studies. All the animal protocols were evaluated and approved by the Animal Laboratory Ethics Committee of Shenyang Pharmaceutical University.24 2.10 In Vivo Pharmacokinetics. To study the pharmacokinetics profiles, DiR Sol, nonPEGylated DiR NPs, and DiR/DSPE-PEG2K NPs (1 mg kg-1 of DiR) was i.v. injected to five Sprague-Dawley rats weighing 190-230 g. At preset time intervals, 500 µL blood was obtained from each rats and then centrifuged (1 × 104 rpm, 5 min) to obtain plasma. DiR was extracted from the plasma by protein precipitation. Then, the plasma concentration of DiR was measured using the Varioskan Flash multimode microreader(Thermo Scientific, USA). 2.11 Biodistribution. To explore the biodistribution of DiR nanoassemblies, BALB/c mice bearing 4T1 xenograft tumors were established.24 Briefly, 100 µL 4T1 cells (5 × 106 cells) were inoculated subcutaneously to female Balb/c mice. When the tumors reached to 300-400 mm3, the


8


Page 10 of 26

mice were i.v. injected with 200 µL of DiR Sol, non-PEGylated-DiR NPs, and DiR/DSPEPEG2K NPs (5 mg kg-1 of DiR). The images were obtained by a noninvasive optical imaging system (IVIS) to record the fluorescence signals of DiR at 4 and 12 h. The mice were sacrificed, and the tumors and main organs were taken out for imaging analysis. Then, the in vivo optical imaging of DiR Sol, non-PEGylated-DiR NPs, and DiR/DSPEPEG2K NPs was explored in 4T1 tumor-bearing Balb/c mice. After the animal model being successfully established, the mice were i.v. injected DiR Sol, non-PEGylated-DiR NPs, and DiR/DSPE-PEG2K NPs (3 mg kg-1 of DiR) via the tail vein. In vivo NIRF images were obtained by a noninvasive optical imaging system (IVIS) with a excitation wavelength of 748 nm at 1, 4, 12 and 24 h post-administration. 2.12 In Vivo Photothermal Efficacy. The in vivo photothermal efficiency of DiR nanoassemblies were studied using infrared thermal imaging camera (Fotric226). The BALB/c mice bearing 4T1 xenograft tumors were established as described above. DiR Sol, nonPEGylated DiR NPs, and DiR/DSPE-PEG2K NPs (1 mg kg-1 of DiR) were i.v. injected to mice, respectively. At 4 h (DiR Sol, non-PEGylated DiR NPs) and 12 h (DiR/DSPE-PEG2K NPs) after administration, tumors were exposed to laser (808 nm, 2 W cm−2) for 5 min. Mice with laser treatment only were performed as control. The infrared thermographic images and region temperature variations were recorded at preset time intervals. 2.13 Imaging-Guided Antitumor Phototherapy. The BALB/c mice bearing 4T1 xenograft tumors were utilized to carry out the imaging-guided antitumor phototherapy study. When the tumors growed up to 150 mm3, the mice were grouped in a random way: saline control, laser alone, DiR Sol/laser, non-PEGylated DiR NPs/laser, DiR/DSPE-PEG2K NPs/laser (n=5). These formulations (1 mg kg-1 of DiR) were i.v. administrated to the mice. At 4 h (DiR Sol and non-


9

Page 11 of 26 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


PEGylated DiR NPs ) and 12 h ( DiR/DSPE-PEG2K NPs ) after administration, the mice were exposed to laser (808 nm, 2 W cm−2) for 5 min. The mice with laser treatment only were used as control. The tumor volume and body weight change were recorded at preset time intervals. The mice were sacrificed after three days of the last treatment, then the blood was collected for hepatic function and renal function markers measurements. The tumors and main organs were excised, weighed, washed and fixed in the 4% paraformaldehyde, and stained with H&E to evaluate pathological change. 2.14 Statistical Analysis. Data were calculated and treated as mean value ± standard deviation. Statistic difference between different groups was analyzed with student’s T-test and one-way analysis of variance (ANOVA), and p values were less than 0.05 (p < 0.05) were deemed statistically significant differences.

3. RESULTS AND DISCUSSIONS 3.1 Self-Assembly of Pure DiR. One-step nanoprecipitation was utilized to perform the nanoassembly of DiR. Interestingly, when the ethanol solution of pure DiR was dropped into water, self-assembly of DiR molecules occurred without the help of any surfactant (Figure S1). Then, ethanol was removed via vacuum rotary evaporation. By contrast, when DiR powder was dispersed into water, it dispersed as flocculated precipitate due to its poor water solubility (Figure S1). As shown in Figure S2, DiR solution and nanoassemblies showed very similar ultraviolet spectra (Figure S2A), but the fluorescence intensity of DiR aqueous solution (ethanol/water = 10:90, v/v) and DiR nanoassemblies at 780 nm was very weak in water (Figure S2B). Which is in accordance with the previous study, and this phenomenon might be attributed to the interference of aqueous medium and the existence state of DiR molecules.23


10


Page 12 of 26

DiR has two long alkyl chains and aromatic heads, the specific chemical structure render it assembling into NPs. The self-assembly process of DiR molecules in aqueous medium was simulated.24 As show in Figure 2A, three DiR molecules quickly gathered together and formed a trimer cluster. Once the self-assembly was occurred, the structural conformation of DiR trimer was quite stable (Figure 2A). In the trimer, π-π stacking among the aromatic heads of DiR was observed, and the alkyl tails of DiR intertwined with each other. Therefore, during the selfassembly of the DiR molecules, the structural intertwining of alkyl chains and the intermolecular π-π stacking between aromatic heads would render thermodynamic stable state. A small amount of DSPE-PEG2k (20 wt%) was added to prepare PEGylated DiR nanoassemblies. As shown in Figure 2B and Table S1, the particle size of both the PEGylated and non-PEGylated DiR nanoassemblies was around 80 nm, and the transmission electron microscopy (TEM) images showed the spherical-shaped structure of DiR nanoassemblies. Notably, DiR molecules themselves constituted the main structure of nanoassemblies, the drug loading rate of the PEGylated DiR nanoassemblies was up to 80 wt%. As a result, the phototherapy efficiency and drug delivery safety of DiR could certainly be improved. The PEGylated DiR nanoassemblies demonstrated better colloidal stability than nonPEGylated nanoassemblies in cell culture medium (RPMI 1640 containing 10% FBS) for 24 h (Figure S3A), suggesting PEGylation modification could significantly improve the stability of nanoassemblies. Moreover, the zeta potential of DiR nanoassemblies was close to electroneutrality (about -5 mv) in cell culture medium (RPMI 1640 containing 10% FBS), probably due to the influence of negatively charged FBS (Figure S3B). Both the PEGylated and non-PEGylated DiR nanoassemblies showed good stability in the environment of 4 °C for 30 days (Figure S3C). Additionally, the chemical stability of DiR solution, non-PEGylated DiR


11

Page 13 of 26 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


nanoassemblies and PEGylated DiR nanoassemblies incubated in mouse serum was investigated. As shown in Figure S3D, DiR in the three formulations showed good chemical stability incubated in mouse serum, with no significant degradation observed after 24 h at 37 °C.

Figure 2. Self-assembly of DiR and in vitro photothermal efficiency of DiR nanoassemblies. (A) Self-assembly simulation of DiR in water (Sybyl 6.9.1 software package); (B) TEM images of PEGylated and non-PEGylated DiR nanoassemblies (scale bar represents 100 nm); (C) and (D) In vitro photothermal efficiency of DiR nanoassemblies (n=3).

3.2. In Vitro Photothermal Efficiency. To explore temperature elevation induced by laser treatment, PBS, DiR solution, non-PEGylated DiR nanoassemblies and PEGylated DiR nanoassemblies were treated using a continuous-wave fiber-coupled diode laser (808 nm, 1 W cm−2) for 5 min. The variation in temperature was detected by a digital thermometer every 20 s of samples under irradiation. As shown in Figure 2C-D, the temperature of DiR solution, non-


12


Page 14 of 26

PEGylated DiR nanoassemblies and PEGylated DiR nanoassemblies elevated increased over the exposure time, and there was no significant difference among them. By comparation, the temperature of PBS showed no significant change under laser treatment. For all these DiR formulations, the temperature could be readily heated up to about 45 °C under laser treatment for 5 min, tumor cells can be readily killed under such a condition.23 These results suggested that the nanoassembly of DiR had negligible impact on its in vitro photothermal efficiency. 3.3 Cellular Uptake. The cellular uptake of DiR solution, non-PEGylated DiR NPs and DiR/DSPE-PEG2k NPs was investigated by determining the intracellular fluorescence intensity after incubation with 4T1 cells. As shown in Figure 3A and S4, the intracellular fluorescence intensity of nanoassemblies was stronger than that of free DiR solution at both 0.5 h and 2 h, suggesting a higher cellular uptake efficiency of nanoassemblies. Moreover, no significant change was found between non-PEGylated DiR NPs and DiR/DSPE-PEG2k NPs. 3.4 In Vitro Photothermal Cytotoxicity. The cytotoxicity of DiR solution, non-PEGylated DiR NPs and DiR/DSPE-PEG2k NPs was studied by MTT assay. As shown in Figure 3B-C and Table S2, all these formulations demonstrated no obvious cytotoxicity against 4T1 cells. Under laser irradiation, cell apoptosis increased along with the increasement of DiR concentration. More importantly, DiR nanoassemblies showed higher cytotoxicity than DiR solution. Despite their similar in vitro photothermal efficiency (Figure 2C-D), the improved photothermal cytotoxicity could be attributed to the higher cellular uptake efficiency of DiR nanoassemblies (Figure 3A and S4).


13

Page 15 of 26 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


Figure 3. Cellular uptake and photothermal cytotoxicity of DiR nanoassemblies. (A) Cellular uptake of DiR nanoassemblies at 2 h (scale bar represents 25 µm); (B) Photothermal cytotoxicity of DiR Sol, non-PEGylated DiR nanoassemblies and PEGylated DiR nanoassemblies without laser treatment (n=3); (C) Photothermal cytotoxicity of DiR solution, non-PEGylated DiR nanoassemblies and PEGylated DiR nanoassemblies under laser treatment (808 nm, 4 W cm−2) for 3 min (n=3).

3.5 Pharmacokinetics. We expected that PEGylation modification on DiR nanoassemblies would prolong their systemic circulation in blood after intravenous administration. The pharmacokinetic profiles of DiR solution, non-PEGylated DiR NPs and DiR/DSPE-PEG2k NPs were explored in Sprague-Dawley (SD) rats. The concentration-time curves and the main pharmacokinetics parameters were showed in Figure 4 and Table S3, respectively. As shown in Figure 4 and Table S3, free DiR solution showed very short half-life in blood. Additionally, the


14


Page 16 of 26

non-PEGylated DiR nanoassemblies exhibited similar pharmacokinetics behavior with free DiR solution due to the hydrophobic surface of nanoassemblies and poor stability. By contrast, DiR/DSPE-PEG2k NPs significantly extended the circulation time in blood. Obviously, the hydrophilic PEG shell of DiR nanoassemblies played a key role in protecting NPs from rapid clearance.

Figure 4. In vivo pharmacokinetics studies of DiR solution, non-PEGylated DiR NPs, and DiR/DSPE-PEG2K NPs with a single i.v. injection of 1 mg kg−1 DiR (n=5).

3.6 In Vivo Biodistribution. DiR nanoassemblies, fabricated by a NIR fluorescent dye, could be used as a self-tracing nanoplatform for imaging-guided cancer phototherapy. In this section, the in vivo biodistribution of DiR solution, non-PEGylated DiR nanoassemblies and PEGylated DiR nanoassemblies was explored in 4T1 tumor-bearing BALB/c mice. As shown in Figure 5, all these formulations demonstrated similar biodistribution at 4 h, with high fluorescence intensity found in the blood-rich organs (liver, spleen and lung). Weak fluorescence signals could be


15

Page 17 of 26 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


found in the tumors at 4 h. Notably, PEGylated DiR nanoassemblies showed far higher tumorhoming ability when compared with DiR solution and non-PEGylated nanoassemblies at 12 h, with strong fluorescence signals were observed in tumors treated with PEGylated nanoassemblies. By contrast, the fluorescence intensity in tumors treated with DiR solution and non-PEGylated nanoassemblies were extremely weak at 12 h, even weaker than that at 4 h. Furthermore, in vivo optical imaging was carried out to observe the biodistribution of DiR solution, non-PEGylated DiR nanoassemblies and PEGylated DiR nanoassemblies. Similarly, PEGylated DiR nanoassemblies demonstrated much stronger fluorescence intensity in tumor site than DiR solution and non-PEGylated nanoassemblies at 12 h (Figure S5). These results were well in line with the pharmacokinetic results and suggested that PEGylation modification on DiR nanoassemblies could significantly extend the systemic circulation time, which in turn facilitated the accumulation of DiR in tumors via EPR effects.


16


Page 18 of 26

Figure 5. Biodistribution of DiR solution, non-PEGylated DiR nanoassemblies and PEGylated DiR nanoassemblies in BALB/c mice bearing 4T1 tumors. (A) Fluorescence pictures at 4 h; (B) Quantitative results at 4 h; (C) Fluorescence pictures at 12 h; (D) Quantitative results at 12 h. (n = 3)

3.7 Imaging-Guided Antitumor Phototherapy. Prolonged circulation time in blood and distinct tumor-homing capacity make PEGylated DiR nanoassemblies as promising candidate for evaluation of PTT efficiency in vivo. In this section, the antitumor activity was investigated in a 4T1 tumor xenograft. Saline, DiR solution, DiR NPs and DiR/DSPE-PEG2k NPs (5 mg kg−1 of


17

Page 19 of 26 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


DiR) were intravenously administrated to the mice for a total of 4 injections, respectively. According to the biodistribution results (Figure 5 and S5), DiR solution and non-PEGylated DiR nanoassemblies treated groups received light treatment (808 nm, 2.0 W cm-2) for 5 min at 4 h post-administration, and PEGylated DiR nanoassemblies treated groups received light treatment (808 nm, 2.0 W cm-2) for 5 min at 12 h post-administration. Saline treated mice without laser and laser treated mice without drug administration were used as control groups, respectively. As shown in Figure 6A-B, PEGylated DiR nanoassemblies demonstrated higher in vivo photothermal effect than DiR solution and non-PEGylated DiR nanoassemblies, with significantly elevated temperature in tumor site. The in vivo antitumor efficacy was further studied. As shown in Figure 6C-F, saline treated mice without laser and laser treated mice without drug administration demonstrated a rapid increase in tumor volume. DiR solution and non-PEGylated DiR nanoassemblies could partially suppress tumor growth to a certain degree, but the tumor volume still increased rapidly. Among them, PEGylated DiR nanoassemblies revealed distinct advantages over other formulations, with significantly delayed tumor growth. The poor antitumor efficacy of DiR solution and non-PEGylated DiR nanoassemblies could be contributed to their inferior drug delivery characteristics in vivo (Figure 4 and Figure 5), the less accumulation of DiR in tumors greatly compromised their PTT efficiency, even with tumorlocalizing light treatment. By contrast, the potent antitumor activity of PEGylated DiR nanoassemblies should be attributed to the improved cellular uptake, prolonged systemic circulation time (Figure 4) and accelerated biodistribution of DiR in tumors (Figure 5).


18


Page 20 of 26

Figure 6. Antitumor activity of DiR nanoassemblies in BALB/c mice bearing 4T1 tumors under laser treatment (808 nm, 2.0 W cm-2) (n = 5). (A) The corresponding temperature changing curves of photothermal imaging in tumor site; (B) In vivo photothermal imaging of tumors. (C)


19

Page 21 of 26 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


Tumor growth profiles after injected with different formulations (the arrows represent 4-time injections); (D) Images of tumors at 10 days post-administration; (E) Tumor burden; and (F) Body weight changes of BALB/c mice bearing 4T1 tumors after injected with different formulations.

Additionally, according to the hematological results, no significant change was found in all the groups (Figure S6), suggesting no significant hepatic and renal toxicity was caused. A notable weight loss was found in the group treated with DiR solution due to the ethanol used for dissolving DiR, but no obvious change in body weight was found in other groups (Figure 6F). Notably, the H&E staining results of liver revealed that some obvious metastatic lesions could be found in the animals treated with saline, laser, DiR solution and non-PEGylated DiR nanoassemblies. By contrast, there was no metastasis found in the animals treated with PEGylated DiR nanoassemblies (Fig. S7). These results suggested that PEGylated DiR nanoassemblies not only inhibited the growth of primary tumor, but also had anti-metastasis ability. Moreover, obvious apoptosis was found in the tumor sections of PEGylated DiR nanoassemblies (Fig. S7). Therefore, PEGylated DiR nanoassemblies, with distinct drug delivery advantages, demonstrated stronger antitumor activity than that of both DiR solution and nonPEGylated DiR nanoassemblies.

4. CONCLUSIONS. In summary, we found that pure DiR performed self-assembly characteristics without the assistance of any polymer or surfactant. In order to improve the in vivo drug delivery efficiency of DiR nanoassemblies, DSPE-PEG2k was used to prepared PEGylated nanoassemblies. The self-assembly mechanism was explored using computational


20


Page 22 of 26

dynamic simulation technology. Assembling into NPs demonstrated negligible influence on the in vitro photothermal efficiency of DiR, but DiR nanoassemblies revealed higher cellular uptake efficiency than DiR solution, resulting in significantly improved photothermal cytotoxicity. Moreover, PEGylated DiR nanoassemblies could significantly improve the in vivo drug delivery efficiency of DiR after intravenous injection, leading to accelerated tumor accumulation. In vivo, PEGylated DiR nanoassemblies showed distinct superiority over DiR solution and nonPEGylated DiR nanoassemblies. This is the first time pure DiR is found to perform selfassembly, and the PEGylated nanoassemblies demonstrated multiple drug delivery advantages. Such a self-delivering nanoplatform of DiR holds promising potential for clinical cancer imaging and therapy.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected]; Tel: +86-024-23986321; Fax: +86-024-23986321. * [email protected]; Tel: +86-024-23986325; Fax: +86-024-23986325. Notes There are no conflicts of interest to declare.


21

Page 23 of 26 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


ACKNOWLEDGMENT This work was supported by the National Basic Research Program of China (973 Program, no. 2015CB932100), the National Natural Science Foundation of China (no. 81703451), and the China Postdoctoral Science Foundation (no. 2017M611269 and 2018T110233).

REFERENCES (1) Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer Statistics, 2018. CA Cancer J. Clin. 2018, 68, 7-30. (2) Maman, S.; Witz, I. P. A History of Exploring Cancer in Context. Nat. Rev. Cancer 2018, 18, 359-376. (3) Steeg, P. S. Targeting Metastasis. Nat. Rev. Cancer 2016, 16, 201-218. (4) Tohme, S.; Simmons, R. L.; Tsung, A. Surgery for Cancer: A Trigger for Metastases. Cancer Res. 2017, 77, 1548-1552. (5) Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C. Cancer Nanomedicine: Progress, Challenges and Opportunities. Nat. Rev. Cancer 2017, 17, 20-37. (6) Dewhirst, M. W.; Secomb, T. W. Transport of Drugs from Blood Vessels to Tumour Tissue. Nat. Rev. Cancer 2017, 17, 738-750. (7) Liang, C.; Xu, L.; Song, G.; Liu, Z. Emerging Nanomedicine Approaches Fighting Tumor Metastasis:

Animal

Models,

Metastasis-Targeted

Drug

Delivery,

Phototherapy,

and

Immunotherapy. Chem. Soc. Rev. 2016, 45, 6250-6269.


22


Page 24 of 26

(8) Zhang, J.; Liang, Y. C.; Lin, X.; Zhu, X.; Yan, L.; Li, S.; Yang, X.; Zhu, G.; Rogach, A. L.; Yu, P. K.; Shi, P.; Tu, L. C.; Chang, C. C.; Zhang, X.; Chen, X.; Zhang, W.; Lee, C. S. SelfMonitoring and Self-Delivery of Photosensitizer-Doped Nanoparticles for Highly Effective Combination Cancer Therapy in Vitro and in Vivo. ACS Nano 2015, 9, 9741-9756. (9) Wen, Y.; Zhang, W.; Gong, N.; Wang, Y. F.; Guo, H. B.; Guo, W.; Wang, P. C.; Liang, X. J. Carrier-Free, Self-Assembled Pure Drug Nanorods Composed of 10-Hydroxycamptothecin and Chlorin E6 for Combinatorial Chemo-Photodynamic Antitumor Therapy In Vivo. Nanoscale 2017, 9, 14347-14356. (10) Vankayala, R.; Hwang, K. C. Near-Infrared-Light-Activatable Nanomaterial-Mediated Phototheranostic Nanomedicines: An Emerging Paradigm for Cancer Treatment. Adv. Mater. 2018, 30, e1706320. (11) Zheng, M.; Zhao, P.; Luo, Z.; Gong, P.; Zheng, C.; Zhang, P.; Yue, C.; Gao, D.; Ma, Y.; Cai, L. Robust ICG Theranostic Nanoparticles for Folate Targeted Cancer Imaging and Highly Effective Photothermal Therapy. ACS Appl. Mater. Interfaces 2014, 6, 6709-6716. (12) Zhao, P.; Zheng, M.; Yue, C.; Luo, Z.; Gong, P.; Gao, G.; Sheng, Z.; Zheng, C.; Cai, L. Improving Drug Accumulation and Photothermal Efficacy in Tumor Depending on Size of ICG Loaded Lipid-Polymer Nanoparticles. Biomaterials 2014, 35, 6037-6746. (13) Wang, J.; Guo, F.; Yu, M.; Liu, L.; Tan, F.; Yan, R.; Li, N. Rapamycin/Dir Loaded LipidPolyaniline Nanoparticles for Dual-Modal Imaging Guided Enhanced Photothermal and Antiangiogenic Combination Therapy. J. Control. Release 2016, 237, 23-34. (14) Han, X.; Chen, J.; Jiang, M.; Zhang, N.; Na, K.; Luo, C.; Zhang, R.; Sun, M.; Lin, G.; Zhang, R.; Ma, Y.; Liu, D.; Wang, Y. Paclitaxel-Paclitaxel Prodrug Nanoassembly as a Versatile


23

Page 25 of 26 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


Nanoplatform for Combinational Cancer Therapy. ACS Appl. Mater. Interfaces 2016, 8, 3350633513. (15) Liu, H.; Wu, D. C. In vivo Near-infrared Fluorescence Tumor Imaging Using DiR-loaded Nanocarriers. Curr. Drug Deliv. 2016, 13, 40-48. (16) Luo, C.; Sun, J.; Sun, B.; He, Z. Prodrug-Based Nanoparticulate Drug Delivery Strategies For Cancer Therapy. Trends Pharmacol. Sci. 2014, 35, 556-566. (17) Chen, Q.; Liu, G.; Liu, S.; Su, H.; Wang, Y.; Li, J.; Luo, C. Remodeling the Tumor Microenvironment with Emerging Nanotherapeutics. Trends Pharmacol. Sci. 2018, 39, 59-74. (18) Luo, C.; Sun, J.; Sun, B.; Liu, D.; Miao, L.; Goodwin, T. J.; Huang, L.; He, Z. Facile Fabrication of Tumor Redox-Sensitive Nanoassemblies of Small-Molecule Oleate Prodrug as Potent Chemotherapeutic Nanomedicine. Small 2016, 12, 6353-6362. (19) Luo, C.; Sun, J.; Liu, D.; Sun, B.; Miao, L.; Musetti, S.; Li, J.; Han, X.; Du, Y.; Li, L.; Huang, L.; He, Z. Self-Assembled Redox Dual-Responsive Prodrug-Nanosystem Formed by Single Thioether-Bridged Paclitaxel-Fatty Acid Conjugate for Cancer Chemotherapy. Nano Lett. 2016, 16, 5401-5408. (20) Sun, B.; Luo, C.; Yu, H.; Zhang, X.; Chen, Q.; Yang, W.; Wang, M.; Kan, Q.; Zhang, H.; Wang, Y.; He, Z.; Sun, J. Disulfide Bond-Driven Oxidation- and Reduction-Responsive Prodrug Nanoassemblies for Cancer Therapy. Nano Lett. 2018, 18, 3643-3650. (21) Cho, H.; Indig, G. L.; Weichert, J.; Shin, H. C.; Kwon, G. S. In Vivo Cancer Imaging By Poly(Ethylene Glycol)-b-Poly(Varepsilon-Caprolactone) Micelles Containing A Near-Infrared Probe. Nanomedicine 2012, 8, 228-236.


24


Page 26 of 26

(22) Feng, L.; Tao, D.; Dong, Z.; Chen, Q.; Chao, Y.; Liu, Z.; Chen, M. Near-Infrared Light Activation of Quenched Liposomal Ce6 for Synergistic Cancer Phototherapy with Effective Skin Protection. Biomaterials 2017, 127, 13-24. (23) He, X. Y.; Bao, X. Y.; Cao, H. Q.; Zhang, Z. W.; Yin, Q.; Gu, W. W.; Chen, L. L.; Yu, H. J.; Li, Y. P. Tumor-Penetrating Nanotherapeutics Loading a Near-Infrared Probe Inhibit Growth and Metastasis of Breast Cancer. Adv. Funct. Mater. 2015, 25, 2831-2839. (24) Zhang, S.; Guan, J.; Sun, M.; Zhang, D.; Zhang, H.; Sun, B.; Guo, W.; Lin, B.; Wang, Y.; He, Z.; Luo, C.; Sun, J. Self-Delivering Prodrug-Nanoassemblies Fabricated by Disulfide Bond Bridged Oleate Prodrug of Docetaxel for Breast Cancer Therapy. Drug deliv. 2017, 24, 14601469.


25

Page 27 of 26 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


Table of Contents Graphic


26

Self-Assembly of a Pure Photosensitizer as a Versatile Theragnostic

Recommend Documents