An NIR-Guided Aggregative and Self-Immolative Nanosystem for

Subscriber access provided by EKU Libraries

Article

An NIR guided aggregative and self-immolative nanosystem for efficient cancer targeting and combination anticancer therapy Qiang Zheng, Yun He, Qing Tang, Yanfang Wang, Ning Zhang, Jin Liu, Qiang Liu, Sheng Zhao, and Ping Hu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00599 • Publication Date (Web): 01 Oct 2018 Downloaded from http://pubs.acs.org on October 3, 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 24 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

Molecular Pharmaceutics

An NIR guided aggregative and self-immolative nanosystem

for efficient

cancer targeting

and

combination anticancer therapy

Qiang Zheng†#, Yun He†#, Qing Tang†, Yanfang Wang‡, Ning Zhang†, Jin Liu†, Qiang Liu†, Sheng Zhao†, Ping Hu†*,

†

School of Pharmaceutical Sciences and Chongqing Key Laboratory of Natural

Drug Research, Chongqing University, 55 South Daxuecheng Road, Chongqing 401331, China ‡

First Affiliated Hospital of the Medical College, Shihezi University, Xinjiang

832008, PR China

#: These authors contributed equally. Corresponding authors: Ping Hu: [email protected]

ACS Paragon Plus Environment

Molecular Pharmaceutics 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

Nanosized drug delivery systems based on polymeric structures have been proved to be promising approaches for cancer treatments. However, few was effective to selectively target cancer cells and release drug at desired tumor sites. Here, we report a “smart” polymeric nanoplatform which could actively accumulate at tumor sites and dissociate to release encapsulated cargos upon the irradiation of near infrared laser (NIR). This nanoplatform composed of a novel amphiphilic block copolymer poly(propylene

sulphide)-poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)

(PPS-P(NIPAM-co-DMAA)) formed spherical structures in aqueous solution and responded to both oxidants and elevated temperature. Upon laser irradiation at 808 nm, the NIR light was efficiently converted to local heat by the doxorubicin (DOX) and indocyanine green (ICG) co-loaded micelles for enhanced cell uptake and therapeutic efficacy. It showed that the micelles effectively accumulated at the tumor sites guided by the application of NIR laser in in vivo studies, exhibiting a 6-time higher and much faster targeting effect compared to the non-irradiation group. The effective tumor growth inhibition by the drug loaded micelles upon laser irradiation demonstrated significant tumor inhibition without regrowth in 16 days. This micellar nanoplatform for precise NIR guided cancer targeting and combination therapy provides a novel and robust strategy for cancer therapy.

Keywords: near infrared laser, photothermal therapy, oxidation-responsive, temperature-responsive, micelles, combination therapy


Page 2 of 24

Page 3 of 24 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


Introduction A major challenge remains in nanotechnology for cancer chemotherapy is the precise and highly efficient delivery of a therapeutic agent with nanocarriers to a tumor site to achieve excellent therapeutic efficacy1. In the past few decades, to overcome the the poor pharmacokinetics profiles and non-specifical distribution in the body of the most of the conventional chemotherapeutic agents2-4, much progress of using nanocarriers to realize cancer targeting therapy has been made5-7. Among these nanocarriers, polymer micelles, which are formed by the self-assembly of amphiphilic copolymers, are emerging as a class of the most promising nanocarriers for anti-cancer drug delivery 8-11. Polymeric micelles can be administered into human body and accumulated at the tumor site through the well-known enhanced permeation and retention (EPR) effect 12, 13

. However, this passive targeting effect is time-consuming and non-specific, often

compromised by the rapid removal of the micelles as foreign particles by the reticuloendothelial system (RES) and mononuclear phagocytic system (MPS)

14-16

.

The strategy of the modification of the surface of nanocarriers with targeting ligands to improve targeting, internalization, and retention in tumors was frequently adopted17, 18

. Nevertheless, these surface modifications require attaching specific ligands to the

nanocarrriers in order to bind the corresponding target tumor cells 19-21. Furthermore, one of the drawbacks of such strategy may restrict nanocarriers to reside on the vessel wall surface and prevent them from penetrating into the deeper regions of the tumor 22

. Here, we present a novel and efficient strategy to achieve active targeting of

nanocarriers to tumor sites by utilizing external NIR laser irradiation as the guide. The nanocarriers was prepared by the self-assembling of PPS-P(NIPAm-co-DMAA), a “smart” amphiphilic block copolymer responding to both higher temperature and oxidants

23

. The micelles prepared from the dually responsive polymer aggregate at

high temperature above its lower critical solution concentration (LCST) due to the loss of hydrogen bonding to water of P(NIPAm-co-DMAA) shell, meanwhile ACS Paragon Plus Environment


Page 4 of 24

dissociate upon oxidization of sulfur atoms in the PPS core under oxidative condition. In the current study24-26, this nanoplatform was composed of three functional units (Scheme 1A). The first unit is a PPS-P(NIPAm-co-DMAA) polymeric micelle for temperature-induced aggregation and oxidants-induced dissociation. The second component is a photosensitizer Indocyanine Green (ICG), an FDA approved imaging reagent, which has the intrinsic ability to convert near infrared (NIR) light to heat and release oxidative free radicals such as singlet oxygen as the source of oxidants

27-29

.

Under normal body temperature, these ICG and DOX were co-loaded into the micelles (DIMs) administered through systemic injection can circulate in the body. By applying NIR laser irradiation on tumor site, the DIMs absorb the light and raise the temperature of tumor site to kill cancer cells. More importantly, the elevated temperature triggered the conformational change of the micelles and led to rapid aggregation and accumulation of drug-loaded micelles at the tumor site, followed by the oxidation of PPS core with singlet oxygen to release DOX (Scheme 1B). The oxidized polymers could be transformed to a more hydrophilic form and excreted more easily from the body. Eventually the precisely released DOX and NIR induced photothermal effect could exert synergistic inhibition on tumor growth 30.


Page 5 of 24 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


Scheme

1.

A) Concepts

and schematics

of

DOX

and

ICG

co-loaded

PPS-P(NIPAm-co-DMAA) micelles and the mechanisms of their dual responsiveness towards oxidants and temperature. B) Schematic illustration of the aggregation of DIMs in vivo upon NIR laser irradiation. The aggregated micelles were internalized into tumor cells and dissociated to release encapsulated drugs.

Experimental Fabrication and Characterization of PPS-P(NIPAm-co-DMAA) Micelles. 10 mg of PPS-P(NIPAm-co-DMAA) was dissolved in 0.5 mL of THF and further added to 10 mL of deionized water. A rotary evaporator was used to slowly remove the THF and followed by filtration with a 0.25 µm PTFE filter to remove any solvent-insoluble species. A zetasizer based on dynamic laser light scattering was used to measure the hydrodynamic diameter of the preprared micelles (DLS; Malvern, Zetasizer Nano Series 90) at a concentration of approximately 1 mg/mL. The morphology of the micelle was characterized by Cryogenic transmission electron microscopy (Cryo-TEM, FEI, TF20, USA).

Thermo-responsive Behavior of Micelles. The scattered light intensity of the polymeric dispersion (0.5 mg/mL) under different temperature was measured by using Malvern Instruments Nano ZS9. The change of volume size was recorded at the same time. Temperature at which the scattered light intensity was half of the change was determined and calculated as the LCST value for the polymer solution. Temperature-dependent

1

HNMR spectra of PPS10-P(NIPAm50-co-DMAA10) (4

mg/mL) in 50 mM deuterated phosphate buffer were recorded at various temperatures from 25 °C to 43 °C.

Oxidation-responsive

Behavior

of

Micelles.

2

mg

of

PPS10-P(NIPAm50-co-DMAA10) was dissolved in 2 mL 1% H2O2 water solution and the scattered light intensity of the aqueous polymer solution was recorded per 10 min.

Temperature Measurements of ICG Loaded Micelles upon Laser Irradiation. 500 µL of 5 and 20 µg/mL free ICG solutions, DIMs (containing 5 and 20 µg/mL ICG), or water were added to 1 mL centrifuge tubes and irradiation with a NIR laser



(808 nm, 1.0 W/cm2, exposure for 15 min) was applied to these samples. Simultaneously, a data logger thermometer was used to record the solution temperature per 10s within 800s.

In vitro Controlled Drug Release Profiles of Micelles. Briefly, DIMs solutions (2 mL) including 0.6 mg DOX and 0.84 mg ICG were sealed in a dialysis bag with a MWCO of 2000 and incubated in 18 mL of DI water with or without 5 min NIR laser irradiation (1.5 W/cm2) on 0h, 4h, 8h. Periodically, the samples containing 2 mL of release media was withdrawn for measurement followed by replenishing an equal volume of fresh media. The released DOX was determined by the DOX calibration curve obtained from the absorbance. After the irradiation, the DIMs solution was taken out and then freeze-dried, and purified by silica column (DCM: MeOH=9:1) to remove ICG and DOX. The resulting solution was concentrated to remove the mix solvent and washed twice with diethyl ether. The obtained white powder was dissolved in 0.6 mL of CDCl3 containing a drop of D2O for 1HNMR measurement.

Cellular Uptake. A549 cells treated with different samples were observed with confocal laser scanning microscopy (CLSM). Briefly, A549 cells (1 × 10 5 cells per dish) were plated onto 24-well glass bottom Petri dishes, and then cultured in DMEM containing 10% FBS, penicillin (100 units/mL), and streptomycin (100 µg/mL) for 24 h at 37 °C in CO2 /air (5/95). After removing DMEM, 500µL fresh medium containing free DOX (5 µg/mL), free ICG (7 µg/mL), or DIMs (DOX: 5 µg/mL, ICG: 7 µg/mL) were added, followed by NIR laser irradiation for 4 min (1 W/cm2). After incubation for 6 h, A549 cells were washed, stained with DAPI and observed using CLSM.

Cytotoxicity Evaluation. MTT assays was employed to evaluate the samples against A549 cells. Briefly, a 96-well plates containing 100 µL of DMEM and 5 × 10 3 A549 cells was cultured at 37 °C under 5% CO2 condition. After 24 h, the medium was replaced with fresh culture medium containing free DOX, free ICG, IMs, DMS, or DIMs was added to the cells, followed by applying NIR laser irradiation for 5 min or without. The DOX and ICG equivalent concentration were fixed to three different concentrations: 0.5 µg/mL and 0.7 µg/mL, 1 µg/mL and 1.4 µg/mL, 2 µg/mL and 2.8 µg/mL, 5 µg/mL and 7 µg/mL, 10 µg/mL and 14 µg/mL. After incubation for 24 h, the ACS Paragon Plus Environment

Page 6 of 24

Page 7 of 24 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


MTT reagent was added to each well. After 2 h incubation, the medium in each well was replaced by 180 µL of DMSO. The absorbance of the plate was recorded at 490 nm using a microplate reader.

In vivo Fluorescence Imaging. The nude mice were tail vein injected with DiD and ICG loaded micelles at a dose of 2 mg/kg ICG. Then all the mice were randomly divided into three groups. Mice in group 1 without irradiation were set as control group. Group 2 and group 3 were laser treatment groups but in different part of body. The site of group 2 is in tumor site and the other is in leg. Images and fluorescence quantitative analysis of DiD were taken at 0 h, 0.5 h, 2 h, 4 h, and 6 h after injection. An in vivo imaging system with a 640 nm excitation wavelength and a 680 nm filter (PerkinElmer, IVIS Spectrum, USA) was used to collect the fluorescence signals of DiD.

Biodistribution. The nude mice were tail vein injected with DiD and ICG loaded micelles at a dose of 2 mg/kg ICG, and then treated with 5 min irradiation or without irradiation. At 6 h post dose administration, all of the mice were sacrificed and their livers, lungs, spleens, hearts, kidneys and tumors were collected for imaging and fluorescence recording.

In vivo Antitumor Efficacy. The mice were divided into four groups (five per group) that were vein tail injected with 150 µL of PBS, free DOX, free ICG, DIMs. The dose of DOX and ICG were 2 mg/kg and 2.8 mg/kg respectively. All the groups were irradiated by the 808 nm laser at 1 W/cm2 for 5 min. Region maximum temperature and infrared thermographic maps were obtained with an infrared thermal imaging camera (FLIR, E4, USA). After four days, the drug injection was carried out again. The tumor volumes and changes in body weight of each mouse were recorded every two days.

Histology. Major organs (liver, lung, kidney, spleen and heart) together with tumors were collected from the mice after 6 days when treated with twice injection. All the collected tissues were fixed in 4% formalin in PBS, processed into paraffin, sectioned to 5 mm and stained with hematoxylin and eosin (H&E). The samples were observed by upright metallurgical microscope (Olympus UIS2) under bright field. ACS Paragon Plus Environment


Statistical Analysis. Data were presented as mean ± standard deviation (SD). Statistical significance of the differences between treatments was evaluated by ANOVA analysis and we used 2-tailed hypothesis testing for multiple groups. It was considered to be significant when p < 0.05 (*) and very significant when p < 0.005 (***).

Results and Discussions Fabrication and Characterizations of Reactive Oxygen Species (ROS) and Temperature Responsive Micelles. The dually responsive diblock copolymer PPS10-P(NIPAm50-co-DMAA10) was successfully synthesized by the polymerization of PPS block and P(NIPAm-co-DMAA) block. The synthesis route was shown in Figure 1A. 1H-NMR in CDCl3, GPC and FT-IR were used to validate the formation of the PPS macroinitiator and amphiphilic block copolymers (Figure 1B and C, Figure S1, S2 and S3). The results showed the typical bands corresponding to both PPS and P(NIPAm-co-DMAA) segments (1H-NMR and FT-IR). Furthermore, the calculated molecular weight and narrow polydispersity index of the synthesized polymers (1.07, Table S1) further supported the successful synthesis of the polymer.


Page 8 of 24

Page 9 of 24 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. A) Synthesis route of PPS-P(NIPAm-co-DMAA) block copolymers. B) 1

H-NMR spectrum of PPS10-P(NIPAm50-co-DMAA10) copolymer. C) GPC results of

PPS

macroinitiator

and

copolymer.

D)

DLS

determinations

of

PPS10-P(NIPAm50-co-DMAA10) and DOX/ICG co-loaded micelles (DIMs). E) Cryo-TEM images of blank micelles and F) DIMs. The size distribution of the spherical micelles with a core−shell structure self-assembled by PPS-P(NIPAm-co-DMAA) was determined by dynamic light scattering (DLS). A peak around 70 nm was recorded, suggesting the formation of aggregates at a nanoscale (Figure 1D). Cryo-transmission electron microscopy (Cryo-TEM) was further employed to get insight of the self-assembly behavior (Figure 1E). As expected, well dispersed and uniform spherical structures were observed, which confirmed the formation of micellar structure. The temperature-responsiveness of the PPS-P(NIPAm-co-DMAA) micelles was investigated using a DLS method31. The LCST of the polymer was measured to be



39.1 °C (Figure. 2A). The average diameter (Dh) of micelles upon heating was also monitored: it remained almost identical from 25 to 37 °C under its LCST. When temperature reached 38 °C, the Dh slightly decreased from 68 nm to 57 nm and dramatically increased to 445 nm at 40 °C, exhibiting obvious phase separation and aggregation behaviour upon heating (Figure. 2B). Figure. 2D further shows the temperature responsiveness of NIPAm and DMAA units in NMR spectra (in D2O) characterized by the sharp decrease of the peaks upon heating, due to the dehydration and restricted mobility of the P(NIPAm-co-DMAA) block 32.

Figure 2. A) Temperature-dependent scattering intensity for micelles. B) The influence of the temperature on the size distributions of polymeric dispersion determined by DLS. C) The photothermal effect of ICG solutions and DIMs upon laser irradiation. D) The influence of the temperature on the 1H-NMR spectrum of polymeric dispersion in deuterated water. E) The changes of scattering intensity of micelles observed by DLS upon oxidation. F) DOX release from DIMs (laser


Page 10 of 24

Page 11 of 24 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


irradiation+/-). G) 1H-NMR spectra of the polymer from DIMs before and after laser irradiation. PPS-P(NIPAm-co-DMAA) micelles with PPS as the hydrophobic cores can be oxidized based on the fact that thioether structure can be oxidized by multiple kinds of ROS including hydrogen peroxide, hyperchloric acid and singlet oxygen

33, 34

. After

adding 1% hydrogen peroxide to the blank micellar dispersion, the scattering intensity of the micelles increases slightly at 15 min as monitored by DLS, followed by a drastic decrease in 2 hours, indicating that the oxidants can lead to the initial swelling of the PPS core and the fast decomposition of the spherical structures of the sample (Figure 2E). DOX and ICG co-loaded micelles (DIMs) were prepared and their dual responsiveness was systemically investigated. DIMs showed a slightly larger size distribution measured by DLS (Figure 1D) and uniform spherical structures observed by cryo-TEM (Figure 1F). Moderate drug loading and encapsulation efficiency were achieved when co-loading DOX and ICG into the micelles (Table S2). The stability of the ICG molecules in IM and DIM micelles was further investigated. The results showed that the encapsulated ICG in both micelles showed good stability even after 7 days in aqueous dispersion (Figure S5). The photothermal effect of DIMs was investigated and recorded within 800 s upon 808 nm NIR irradiation using water as control (Figure. 2C). Water sample demonstrated slight temperature increase to about 24 °C after 5 min NIR irradiation, whereas the temperature of DIMs sample increased obviously, especially the DIMs sample with higher concentration reached 43 °C. It was worth noting that the eventual temperature of the DIMs was higher than the free ICG solutions at the same concentrations, attributing to the increase of absorbance intensity for encapsulated ICG molecules at 808 nm35-37. The DOX release upon NIR irradiation was investigated using a dialysis bag method. The DIMs were dispersed into PBS solution followed by applying NIR irradiation for 5 minutes per 4 h to trigger the DOX release. As shown in Fig. 2F, applying NIR irradiation triggered obviously faster DOX release than that without,



recorded as more than 20 % released DOX with NIR irradiation by 12 h, comparing to only 2 % release for non-irradiated sample. The polymer in DIMs sample after irradiation was collected and tested by NMR (Figure 2G). The decrease of the signal of PPS block and appearance of hydrogen signals close to sulfone and sulfoxide confirmed the oxidation of the PPS block.

In Vitro Cell Uptake and Cytotoxicity Experiments. Confocal microscopy was used to observe the cellular uptake of free DOX, ICG and DIMs in A549 cells (Figure 3A). After incubation with cells for 6 h, free DOX was observed to localize in the nuclei, while ICG, previously reported actively bounding to intracellular glutathione S-transferase38, was observed to mainly localized in the cytoplasm. Significantly higher fluorescence intensity was observed for cells treated with DIMs with laser irradiation than the sample without. The phenomenon could be attributed to the hyperthermia induced by the NIR laser, which increased the hydrophobicity of the DIMs and thereby promoted the uptake of micelles into cancer cells39, 40. The results confirmed that the uptake of DIMs could be facilitated by the NIR laser irradiation. The cytotoxic effect of DIMs was investigated using MTT assay and the cytotoxicity was evaluated as a function of the concentration of DOX or ICG in the samples. Firstly, blank micelles showed insignificant difference of cell viability on A549 cells up to 500 µg/mL after incubation for 24 h (Figure S4), indicating the low toxicity of the blank micelles to cells. Moreover, different concentrations of free DOX, ICG, DOX loaded micelles (DMs), ICG loaded micelles (IMs) and DIMs (from 0.5-10 µg/mL in terms of DOX or ICG concentrations) were incubated with A549 cells. After adding samples, the cells treated with ICG and IMs and half of cells treated with DIMs were irradiated by NIR laser (2.0 W cm−2, 808 nm) followed by 24 h incubation. As shown in Figure 3B, micelles loaded with ICG and/or DOX showed insignificant toxicity to the cells at lower concentrations even with the laser irradiation. However, significant cytotoxicity was observed for cells with irradiation at higher concentrations. The cell viability of DIMs containing 5 µg/mL of encapsulated DOX with irradiation was about 32% and was further lowered to 8 % when the DOX


Page 12 of 24

Page 13 of 24 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


concentration reached 10 µg/mL, exhibiting comparable results to the free DOX groups and significantly lower than that of DIMs without NIR irradiation. The IC50 values of the samples were calculated and shown in Table 1, the results demonstrated that DIMs with the assistance of NIR irradiation demonstrated lower IC50 value compared to IMs and DMs groups, indicating the enhanced therapeutic efficacy by the combination of photothermal effect and chemotherapy.



Figure 3. A) Fluorescence images of free DOX, ICG with irradiation and DIMs with/without irradiation incubated with A549 cells for 6 h. Scale bar represent 25µm. B) Cytotoxicity of free DOX, ICG with irradiation and DIMs with/without irradiation against A549 cells by MTT assay. (Data represent mean ± SD, n = 6, Student’s t-test, ***P < 0.005).


Page 14 of 24

Page 15 of 24 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 1. IC50 of samples incubated with A549 cell lines IC50 (µg/mL)

DOX

ICG(+)

IMs(+)

DMs

DIMs

DIMs(+)

1.162

5.888

4.538

5.385

>10

4.017

NIR-Guided Tumor Imaging and Antitumor Efficacy of DIMs In Vivo. Biodistribution experiment of DIMs on tumor bearing mice was conducted to investigate the distribution of the micelles at tumor sites upon laser irradiation. Micelles co-loaded with a robust fluorescent reagent DiD and ICG were employed due to the rapid fluorescence quenching of ICG molecules after NIR irradiation in vivo 41. The mice xenografted with A549 tumors were administered with DiD/ICG co-loaded micelles by intravenous injection into the tail. The mice was immediately applied with NIR laser to the tumor site for 5 minutes intervals after adminstration and imaged at designed time. Figure 4A shows treatment with micelles and laser dramatically increased the accumulation rate and concentration of micelles at the tumor site, as evidenced by the fluorescence intensity of DiD. The fluorescence intensity of the tumor from the irradiation group could be clearly observed just after 0.5 h, much quicker than the non-irradiation group which showed very weak fluorescence after 2 h. Moreover, the average fluorescence intensity for the irradiation group was much stronger, exhibiting 6 times more than that from the non-irradiation group at 4 h (Figure 4B). In order to make a thorough study of the effect of NIR guided accumulation of the micelles, another group of tumor bearing mice were treated with micelles with the application of NIR laser on their right legs instead of tumors. As shown in Fig. 4A, the fluorescence intensity on the legs of the mice was obviously stronger than that of the tumor, calculated as twice much to that of the tumor site on the same mouse (Figure 4C). This phenomenon could occur as a result of the photothermal effect upon NIR irradiation and cause the phase transition of the micelles, leading to the aggregation and accumulation of the micelles at the body locations irradiated by the NIR laser. Such approach would allow the micelles to reach



and accumulate at desired site of body in a much faster fashion compared to the EPR passive targeting effect, as long as the NIR laser could apply to.

Figure 4. A) Time-dependent in vivo fluorescence images of BALB/c nude mice with


Page 16 of 24

Page 17 of 24 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


A549 xenografts. Images were taken at 0, 0.5, 2, 4 and 6h after the injection with 150 µL of DiD/ICG co-loaded micelles via the tail vein. B and C) Quantitative analysis of DiD fluorescence intensity acquired from tumors and legs at 0, 0.5, 2, 4 and 6h (n=5). D) Biodistribution of DiD in BALB/c nude mice bearing A549 at 6 h post-injection in different organs and tissue. E) Quantitative analysis of DiD fluorescence intensity acquired from heart, liver, spleen, lung, kidney, tumor. The major organs and tumor tissues of the mice in biodistribution experiment were collected and under fluorescent imaging. The result was shown in Figure 4D and 4E. The average fluorescence intensity of tumor in the NIR irradiated group was much stronger, exhibiting 5 times more than that of the non-irradiation group after 6 h treatment. Meanwhile the fluorescence intensity of the major organs (liver, lung, kidney, spleen and heart) almost keep in the same level. The result confirmed that the different distribution of the micelles in tumor tissues due to the NIR irradiation. We were encouraged to proceed to investigate the synergistic antitumor efficacy of DIMs on tumor-bearing mice. Four groups of Balb/c nude mice bearing A549 tumors were administered with PBS (Group1), free DOX (Group 2), ICG (Group 3) and DIMs (Group 4). 808 nm NIR Laser treatment was applied to the tumors immediately post administration. The same treatment was repeated at day 4 after first administration. Temperature change of the tumor during irradiation was monitored by an IR thermal camera (Figure. 5A and 5B). As expected, PBS and DOX groups showed very mild temperature increases, reaching about 42 °C after 5 minutes. In marked contrast, the temperature of tumors treated with ICG rapidly increased to

∼48 °C and the DIMs group reached even to ∼54 °C after laser irradiation42. The real photographs of tumor-bearing nude mice during the treatment were recorded (Figure. 5C). The tumor sizes and body weights were measured and recorded and the relative tumor volume and bodyweight was calculated and plotted in Figure 5D and 5E. Compared to the group treated with PBS, the DOX group showed significant tumor growth inhibition. Groups of free ICG plus NIR irradiation was observed to inhibit and delay tumor growth. On the other hand, combination therapy group (DIMs + laser



irradiation) was observed to completely eradicate tumor without regrowth in 16 days.

Figure 5. A) Infrared thermographic maps of mice administered with DIMs, ICG, DOX and PBS under laser irradiation. B) Temperature change of the tumor sites of groups administered with DIMs, ICG, DOX and PBS under laser irradiation. C) Photographs of A549 tumor-bearing mice and the tumors recorded during the studies (0, 2, 4, 8 and 16 days). D) Tumor growth inhibition profiles of PBS, DOX, ICG with irradiation and DIMs plus irradiation; the red arrows indicate the time points for treatment. E) Calculated relative body weight change of the animals during the studies. ACS Paragon Plus Environment

Page 18 of 24

Page 19 of 24 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


To further validate the antitumor efficacy of the DIMs, the major organs including heart, liver, spleen, lung and kidney and tumors of the tested mice were collected after 6 days treatment and examined by H&E staining. In Figure 6, the major organs of all the treatment groups showed normal histomorphology and no obvious pathological abnormality. In groups of tumors tissues, after treatment with DOX or ICG, small cavities and deformed tumor cells due to dissolved cell nuclei were found compared to the normal tumor tissue in PBS group. On the other hand, the tumor tissue from DIMs group showed multiple cavities and prominent necrosis of tumor cells. These results confirmed the efficacy and low toxicity of the DIMs treatment.

Figure 6. H&E stained main organs and tumor tissues of mice after 6 days with different treatments. Conclusions In conclusion, the combination of chemo-photothermal therapy was realized by the developed NIR-adsorbed theranostic agents on tumor in vitro and in vivo. The DIMs showed good biocompatibility and degradability, sensitive photothermal performance, NIR-guided tumoral distribution and excellent efficacy. These results strongly suggested the remarkable chemo-photothermal synergistic effect of DIMs under laser irradiation for cancer therapy. The photothermal effect from the pair of DIMs and NIR laser could not only directly destroy the tumor cells, but also be utilized to efficiently attract DIMs to accumulate at the tumor site. The oxidants generated during the irradiation could simultaneously oxidize the core of micelles and lead to the precise



Page 20 of 24

release of the encapsulated anti-cancer drugs to the tumor, therefore reducing the side effects while maximizing the benefits of the combination therapy. Overall, our study offers a convenient and efficient strategy for efficient tumor targeting and enhanced anticancer therapy. Supporting Information. Synthesis procedures, characterization data for PPS10-P(NIPAm50-co-DMAA10), other additional in vitro data and calculations are supplied as Supporting Information. Acknowledgements The authors acknowledge the National Natural Science Foundation of China (No. 21572027 and 21372267) for the financial support of this work.

References 1.

Peer, D.; Karp, J. M.; Hong, S.; FaroKhzad, O. C.; Margalit, R.; Langer, R.

Nanocarriers as an

emerging platform for cancer therapy. Nat. Nanotechnol. 2007, 2 (12), 751-760. 2.

Moghimi, S. M.; Hunter, A. C.; Murray, J. C.

Nanomedicine: Current status and future prospects.

FASEB J. 2005, 19 (3), 311-330. 3.

Galluzzi, L.; Buque, A.; Kepp, O.; Zitvogel, L.; Kroemer, G.

Immunological effects of

conventional chemotherapy and targeted anticancer agents. Cancer Cell 2015, 28 (6), 690-714. 4.

Danhier, F.; Feron, O.; Preat, V.

To exploit the tumor microenvironment: Passive and active

tumor targeting of nanocarriers for anti-cancer drug delivery. J. Controlled Release 2010, 148 (2), 135-46. 5.

Muthu, M. S.; Leong, D. T.; Mei, L.; Feng, S.-S.

Nanotheranostics - application and further

development of nanomedicine strategies for advanced theranostics. Theranostics 2014, 4 (6), 660-677. 6.

Raliya, R.; Chadha, T. S.; Haddad, K.; Biswas, P.

Perspective on nanoparticle technology for

biomedical use. Curr. Pharm. Des. 2016, 22 (17), 2481-2490. 7.

Biswas, S.; Torchilin, V. P.

Nanopreparations for organelle-specific delivery in cancer. Adv.

Drug Delivery Rev. 2014, 66, 26-41. 8.

Prabhu, R. H.; Patravale, V. B.; Joshi, M. D.

Polymeric nanoparticles for targeted treatment in

oncology: Current insights. Int. J. Nanomed. 2015, 10, 1001-1018. 9.

Cheng, R.; Meng, F.; Deng, C.; Zhong, Z.

Bioresponsive polymeric nanotherapeutics for

targeted cancer chemotherapy. Nano Today 2015, 10 (5), 656-670. 10. Cui, W.; Li, J.; Decher, G.

Self-assembled smart nanocarriers for targeted drug delivery. Adv.

Mater. 2016, 28 (6), 1302-1311. 11. Gothwal, A.; Khan, I.; Gupta, U.

Polymeric micelles: Recent advancements in the delivery of

anticancer drugs. Pharm. Res. 2016, 33 (1), 18-39. 12. Torchilin, V.

Tumor delivery of macromolecular drugs based on the epr effect. Adv. Drug

Delivery Rev. 2011, 63 (3), 131-135.


Page 21 of 24 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


13. Cabral, H.; Kataoka, K.

Progress of drug-loaded polymeric micelles into clinical studies. J.

Controlled Release 2014, 190, 465-476. 14. Prabhakar, U.; Maeda, H.; Jain, R. K.; Sevick-Muraca, E. M.; Zamboni, W.; Farokhzad, O. C.; Barry, S. T.; Gabizon, A.; Grodzinski, P.; Blakey, D. C.

Challenges and key considerations of the

enhanced permeability and retention effect for nanomedicine drug delivery in oncology. Cancer Res. 2013, 73 (8), 2412-2417. 15. Manzoor, A. A.; Lindner, L. H.; Landon, C. D.; Park, J.-Y.; Simnick, A. J.; Dreher, M. R.; Das, S.; Hanna, G.; Park, W.; Chilkoti, A.; Koning, G. A.; ten Hagen, T. L. M.; Needham, D.; Dewhirst, M. W. Overcoming limitations in nanoparticle drug delivery: Triggered, intravascular release to improve drug penetration into tumors. Cancer Res. 2012, 72 (21), 5566-5575. 16. Storm, G.; Belliot, S. O.; Daemen, T.; Lasic, D. D.

Surface modification of nanoparticles to

oppose uptake by the mononuclear phagocyte system. Adv. Drug Delivery Rev. 1995, 17 (1), 31-48. 17. Biju, V.

Chemical modifications and bioconjugate reactions of nanomaterials for sensing,

imaging, drug delivery and therapy. Chem. Soc. Rev. 2014, 43 (3), 744-764. 18. Feng, L.; Zhu, C.; Yuan, H.; Liu, L.; Lv, F.; Wang, S.

Conjugated polymer nanoparticles:

Preparation, properties, functionalization and biological applications. Chem. Soc. Rev. 2013, 42 (16), 6620-6633. 19. Ramzy, L.; Nasr, M.; Metwally, A. A.; Awad, G. A. S.

Cancer nanotheranostics: A review of the

role of conjugated ligands for overexpressed receptors. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences 2017, 104, 273-292. 20. Gao, W.; Xiang, B.; Meng, T.-T.; Liu, F.; Qi, X.-R.

Chemotherapeutic drug delivery to cancer

cells using a combination of folate targeting and tumor microenvironment-sensitive polypeptides. Biomaterials 2013, 34 (16), 4137-4149. 21. Bono, F.; De Smet, F.; Herbert, C.; De Bock, K.; Georgiadou, M.; Fons, P.; Tjwa, M.; Alcouffe, C.; Ny, A.; Bianciotto, M.; Jonckx, B.; Murakami, M.; Lanahan, A. A.; Michielsen, C.; Sibrac, D.; Dol-Gleizes, F.; Mazzone, M.; Zacchigna, S.; Herault, J.-P.; Fischer, C.; Rigon, P.; de Almodovar, C. R.; Claes, F.; Blanc, I.; Poesen, K.; Zhang, J.; Segura, I.; Gueguen, G.; Bordes, M.-F.; Lambrechts, D.; Broussy, R.; van de Wouwer, M.; Michaux, C.; Shimada, T.; Jean, I.; Blacher, S.; Noel, A.; Motte, P.; Rom, E.; Rakic, J.-M.; Katsuma, S.; Schaeffer, P.; Yayon, A.; Van Schepdael, A.; Schwalbe, H.; Luigi Gervasio, F.; Carmeliet, G.; Rozensky, J.; Dewerchin, M.; Simons, M.; Christopoulos, A.; Herbert, J.-M.; Carmeliet, P.

Inhibition of tumor angiogenesis and growth by a small-molecule multi-fgf

receptor blocker with allosteric properties. Cancer Cell 2013, 23 (4), 477-488. 22. Chen, F.; Hong, H.; Goel, S.; Graves, S. A.; Orbay, H.; Ehlerding, E. B.; Shi, S.; Theuer, C. P.; Nickles, R. J.; Cai, W.

In vivo tumor vasculature targeting of cus@msn based theranostic

nanomedicine. ACS Nano 2015, 9 (4), 3926-3934. 23. Zheng, Q.; Hu, P.; Tang, Q.; Tang, M.; Zang, Z.; Zhao, S.; Shao, P.; Wang, Z.; He, Y.

Dually

responsive amphiphilic block copolymer with oxidation-responsiveness and tuneable lcst behaviours. Mater. Lett. 2017, 201, 133-136. 24. Hu, P.; Tirelli, N.

Scavenging ros: Superoxide dismutase/catalase mimetics by the use of an

oxidation-sensitive nanocarrier/enzyme conjugate. Bioconj. Chem. 2012, 23 (3), 438-449. 25. Tang,

M.;

Zheng,

Q.;

Tirelli,

N.;

Hu,

P.;

Tang,

Q.;

Gu,

J.;

He,

Y.

Dual

thermo/oxidation-responsive block copolymers with self-assembly behaviour and synergistic release. React. Funct. Polym. 2017, 110, 55-61. 26. Tang, M.; Hu, P.; Zheng, Q.; Tirelli, N.; Yang, X.; Wang, Z.; Wang, Y.; Tang, Q.; He, Y.



Page 22 of 24

Polymeric micelles with dual thermal and reactive oxygen species (ros)-responsiveness for inflammatory cancer cell delivery. J. Nanobiotechnol 2017, 15. 27. Caesar, J.; Sherlock, S.; Shaldon, S.; Chiandussi, L.; Guevara, L.

Use of indocyanine green in

measurement of hepatic blood flow and as a test of hepatic function. Clin. Sci. 1961, 21 (1), 43-&. 28. Chen, W. R.; Adams, R. L.; Higgins, A. K.; Bartels, K. E.; Nordquist, R. E.

Photothermal effects

on murine mammary tumors using indocyanine green and an 808-nm diode laser: An in vivo efficacy study. Cancer Lett. 1996, 98 (2), 169-173. 29. Zheng, M.; Yue, C.; Ma, Y.; Gong, P.; Zhao, P.; Zheng, C.; Sheng, Z.; Zhang, P.; Wang, Z.; Cai, L. Single-step assembly of dox/icg loaded lipid-polymer

nanoparticles for highly effective

chemo-photothermal combination therapy. ACS Nano 2013, 7 (3), 2056-2067. 30. Vasdekis, A. E.; Scott, E. A.; O'Neil, C. P.; Psaltis, D.; Hubbell, J. A.

Precision intracellular

delivery based on optofluidic polymersome rupture. ACS Nano 2012, 6 (9), 7850-7857. 31. Wei, H.; Zhang, X. Z.; Zhou, Y.; Cheng, S. X.; Zhuo, R. X.

Self-assembled thermoresponsive

micelles of poly(n-isopropylacrylamide-b-methyl methacrylate). Biomaterials 2006, 27 (9), 2028-2034. 32. Yim, H.; Kent, M. S.; Huber, D. L.; Satija, S.; Majewski, J.; Smith, G. S.

Conformation of

end-tethered pnipam chains in water and in acetone by neutron reflectivity. Macromolecules 2003, 36 (14), 5244-5251. 33. Carampin, P.; Lallana, E.; Laliturai, J.; Carroccio, S. C.; Puglisi, C.; Tirelli, N. Oxidant-dependent redox responsiveness of polysulfides. Macromol. Chem. Phys. 2012, 213 (19), 2052-2061. 34. Dai, L. L.; Yu, Y. L.; Luo, Z.; Li, M. H.; Chen, W. Z.; Shen, X. K.; Chen, F.; Sun, Q.; Zhang, Q. F.; Gu, H.; Cai, K. Y.

Photosensitizer enhanced disassembly of amphiphilic micelle for ros-response

targeted tumor therapy in vivo. Biomaterials 2016, 104, 1-17. 35. Huang, M.; Li, H.; Ke, W.; Li, J.; Zhao, C.; Ge, Z.

Finely tuned thermo-responsive block

copolymer micelles for photothermal effect-triggered efficient cellular internalization. Macromol. Biosci. 2016, 16 (9), 1265-1272. 36. Zheng, X.; Xing, D.; Zhou, F.; Wu, B.; Chen, W. R.

Indocyanine green-containing nanostructure

as near infrared dual-functional targeting probes for optical imaging and photothermal therapy. Mol. Pharmaceutics 2011, 8 (2), 447-56. 37. Rodriguez, V. B.; Henry, S. M.; Hoffman, A. S.; Stayton, P. S.; Li, X.; Pun, S. H.

Encapsulation

and stabilization of indocyanine green within poly(styrene-alt-maleic anhydride) block-poly(styrene) micelles for near-infrared imaging. J. Biomed. Opt. 2008, 13 (1), 014025. 38. Tang, Y.; Lei, T.; Manchanda, R.; Nagesetti, A.; Fernandez-Fernandez, A.; Srinivasan, S.; McGoron, A. J.

Simultaneous delivery of chemotherapeutic and thermal-optical agents to cancer cells

by a polymeric (PLGA) nanocarrier: An in vitro study. Pharm. Res. 2010, 27 (10), 2242-2253. 39. Hiruta, Y.; Shimamura, M.; Matsuura, M.; Maekawa, Y.; Funatsu, T.; Suzuki, Y.; Ayano, E.; Okano, T.; Kanazawa, H.

Temperature-responsive fluorescence polymer probes with accurate

thermally controlled cellular uptakes. ACS Macro Letters 2014, 3 (3), 281–285. 40. Shen, Z.; Wei, W.; Zhao, Y.; Ma, G.; Dobashi, T.; Maki, Y.; Su, Z.; Wan, J.

Thermosensitive

polymer-conjugated albumin nanospheres as thermal targeting anti-cancer drug carrier. Eur. J. Pharm. Sci. 2008, 35 (4), 271-82. 41. Yan, J.; Estevez, M. C.; Smith, J. E.; Wang, K.; He, X.; Wang, L.; Tan, W.

Dye-doped

nanoparticles for bioanalysis. Nano Today 2007, 2 (3), 44-50. 42. Bucharskaya, A.; Maslyakova, G.; Terentyuk, G.; Yakunin, A.; Avetisyan, Y.; Bibikova, O.;


Page 23 of 24 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


Tuchina, E.; Khlebtsov, B.; Khlebtsov, N.; Tuchin, V.

Towards effective photothermal/photodynamic

treatment using plasmonic gold nanoparticles. Int. J. Mol. Sci. 2016, 17 (8).



For Table of Contents Use Only An NIR guided aggregative and self-immolative nanosystem for efficient cancer targeting and combination anticancer therapy Ping Hu 82x44mm (300 x 300 DPI)


Page 24 of 24

An NIR-Guided Aggregative and Self-Immolative Nanosystem for

Recommend Documents