From one to all: self-assembled theranostic nanoparticles for tumor-targeted imaging and programmed photoactive therapy
In recent years, multifunctional theranostic nanoparticles have been fabricated by integrating imaging and therapeutic moieties into one single nano-formulations. However, Complexity of production and safety issues limits their further application.
Herein, we demonstrated self-assembled nanoparticles with single structure as a “from one to all” theranostic platform for tumor-targeted dual-modal imaging and programmed photoactive therapy (PPAT). The nanoparticles were successfully developed through self-assembling of hyaluronic acid (HA)-cystamine-cholesterol (HSC) conjugate, in which IR780 was simultaneously incorporated (HSCI NPs). Due to the proper hydrodynamic size and intrinsic targeting ability of HA, the HSCI NPs could accumulate at the tumor site effectively after systemic administration. In the presence of incorporated IR780, in vivo biodistribution and accumulation behaviors of HSCI NPs could be monitored by photoacoustic imaging. After cellular uptake, the HSCI NPs would disintegrate resulting from cystamine reacting with over-expressed GSH. The released IR780 would induce fluorescence “turn-on” conversion, which could be used to image tumor sites effectively. Upon treatment with 808 nm laser irradiation, PPAT could be achieved in which generated reactive oxygen species (ROS) would produce photodynamic therapy (PDT), and subsequently the raised temperature would be beneficial to tumor photothermal therapy (PTT).
KeywordsFrom one to all Self-assembling Theranostic nanoparticles Targeted imaging Programmed photoactive therapy
hyaluronic acid-cystamine-cholesterol conjugate
- HSCI NPs
nanoparticles via self-assembling of hyaluronic acid-cystamine-cholesterol conjugate and IR780
programmed photoactive therapy
reactive oxygen species
magnetic resonance imaging
positron emission tomography
multispectral optoacoustic tomography
critical aggregation concentration
Blossom of smart and multifunctional theranostic nanoparticles [1, 2, 3, 4, 5, 6, 7, 8, 9], paralleled by advances in nanotechnology and other interdisciplinary sciences, is gradually provoking precision medicine into reality [10, 11, 12, 13, 14]. The theranostic nanoparticles integrating targeting, imaging and therapeutic abilities into one single nano-formulation could monitor drug accumulation in a real-time manner, allow precise disease diagnosis and evaluate treatment efficiency [15, 16]. However, in consideration of safety factors, complexity of production process and less than idea in vivo pharmacokinetics, most of theranostic nanomedicine is still at academic stage without further application in clinical translation [17, 18, 19]. Therefore, exploring theranostic nanoparticles constructed with safe materials and simple preparation method would be beneficial to further application.
Enormous molecular imaging strategies, including magnetic resonance imaging (MRI), positron emission tomography (PET), CT and fluorescence imaging have been widely applied in cancer theranostics [20, 21, 22, 23]. By virtue of the outstanding sensitivity, low cost and short acquisition time, fluorescence imaging is broadly employed in (pre)clinical research for disease diagnosis and monitoring the in vivo behaviors of nanoparticles. Nevertheless, poor spatial resolution of fluorescence hinders its further application [24, 25]. As a hybrid imaging modality, multispectral optoacoustic tomography (MSOT) could conquer the optical diffusion limitation via combining the spectral selectivity of molecular excitation with the high resolution of ultrasound detection, which is based on the photoacoustic (PA) effect [26, 27]. On the one hand, outstanding sensitivity of fluorescence could be used to image and track the in vivo behaviors of nanoparticles. On the other hand, high spatial resolution of MSOT could be used to noninvasively monitor drug accumulation behaviors in vivo. Therefore, integrating fluorescence and MSOT imaging into one single nanoplatform represent effective means to boosting its further application and development [28, 29].
Light-triggered photoactive therapy utilizing photo-conversion of photosensitizer could produce reactive oxygen species (ROS) via singlet-to-triplet for photodynamic therapy (PDT) [30, 31] or high temperature for photothermal therapy (PTT) [32, 33, 34]. Photoactive therapy has many advantages including noninvasiveness, selective local treatment, negligible drug resistance and minimized side effects. More importantly, incorporating PDT and PTT together can achieve more effective therapeutic effects [35, 36]. Therefore, realizing programed photoactive therapy (PPAT) together with PDT and PTT on one domain would be a superior strategy for cancer treatment. However, combining multiple moieties with different functions would bring about more uncertainties in drug absorption, distribution, metabolism, excretion and toxicity. Therefore, realizing multiple functions in one single platform would be crucial.
Results and discussion
Materials preparation and characterization
Programmed photoactive therapy in vitro
Next, we evaluated the cell cytotoxicity effect of HSCI NPs with different patterns of laser treatment (Fig. 2f). In the cells treated with short time of laser (2 min), the inhibition efficiency of MDA-MB-231 cells reached to 48.59%. As demonstrated before, during this time the temperature was not high enough to kill cancer cells, which demonstrated that the cell cytotoxicity was mainly due to ROS production. At subsequent laser irradiation (> 2 min), we found that the cell viability was remarkably reduced. Under the same time for laser irradiation for 6 min, the cell viability of HSCI NPs treatment (26.17%) was lower than free IR780 treatment (54.82%). According to our above discussion, as a type of small molecule, free IR780 entered cells mainly depending on the concentration gradient, which illustrated that IR780 concentration inside cells would not exceed the concentration in the medium. However, the cellular uptake way of NPs was through endocytosis-mediated pattern. As active uptake pathway, more IR780 could be delivered into cells by nanoparticles and IR780 in the cells could exhibit higher concentration, thus presenting well PTT effect compared with free IR780.
Photoacoustic and fluorescence targeting imaging in vivo
In vivo programmed photoactive therapy efficacy evaluation
Taken together, we constructed “from one to all” theranostic nanoparticles by self-assembling of hyaluronic acid-cystamine-cholesterol conjugate, in which IR780 was simultaneously incorporated. The HSCI NPs could realize specific accumulation at the tumor site due to the active targeting specificity of hyaluronic acid for cancer cells over-expressing CD44 receptor. In addition, the in vivo biodistribution and accumulation behaviors could be monitored and tracked through the fluorescence and photoacoustic imaging abilities of loaded IR780. NIR laser of 808 nm with penetrating deeply tissue was conducted for programmed photoactive therapy (PPAT, PDT followed by PTT), which could inhibit tumor growth efficiently. In addition, the HSCI NPs possessed excellent biosafety. The overall data demonstrated that the constructed HSCI NPs have a great potential to be used as a tumor-targeted dual-modal imaging platform and exhibited programed photoactive therapy (PPAT) pattern with good biocompability.
Method and materials
Hyaluronic acid with molecular weight 10 kDa was obtained from Freda Biochem company.11-Chloro-1,1′-di-n-propyl-3,3,3′,3′-tetramethyl-10,12-trimethyleneindatricarbocyanine iodide (IR780) and Cholesteryl chloroformate were purchased from Alfa Aesar Co. Cystamine dihydrochloride, N-hydroxysuccinimide (NHS) and 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Sigma-Aldrich (St. Lousi, MO, USA). Glitathione (GSH) was purchased from Aladdin Reagent Database Inc. All other chemicals were analytical grade and used without further purification. Ultrapure water (deionized (DI) water) was supplied by a Milli-Q water system (Millipore, USA).
Preparation and characterization of HA-SS-Chol (HSC) conjugates
The amphiphilic HSC conjugates were firstly synthesized according to the method reported previously with some modifications (Additional file 1: Figure S1) . Cystamine modified cholesterol (Chol-SS) was synthesized by coupling cholesterol chloroformate with primary amine group of cystamine. In brief, cysteamine dihydrochloride (1.35 g, 6 mmol) was dissolved in mixed solution (180 mL, volume ratio = 10:5:4) of acetonitrile (CH3CN), dichloromethane (CH2Cl2) and triethylamine(TEA). After stirring for 3 h at 4 °C and another 1 h at room temperature, cholesterol chloroformate (0.27 g, 0.6 mmol) dissolved in CH2Cl2 (10 mL) was dropwise added into the mixed solution. After stirring for another 24 h, the reaction mixture was washed with DI water. The mixture was then moved to rotary evaporation following freeze-drying. Afterwards, the backbone of HA was chemically modified with the hydrophobic Chol-SS by help of EDC and NHS. HA (100 mg, 264 µmol) and Chol-SS (30 mg, 52.8 µmol) were added into H2O/THF (50 mL, volume ratio = 1:1) and dissolved via ultrasound about 30 min. EDC (50.424 mg, 264 µmol) and NHS (30.36 mg, 264 µmol) were dropwise added into the mixed solution. After stirring for 24 h, the reaction was dialyzed with DI water and freeze-dried gaining HA-SS-Chol conjugates. Its chemical structure was characterized by using infrared spectrometer (Spectrum One, Perkin Elmer Instruments Co. Ltd. USA).
Critical aggregation concentration of HSC
Pyrene was used as the fluorescent probe to assess the critical aggregation concentration of HSC conjugates. The solutions ranging from 1.0 × 10−7 to 2.0 mg/mL were added respectively to pyrene solution (6 × 10−7 M). The above mixture was sonicated over 30 min and kept for 2 h to trigger HSC self-assembling into nanoparticles. Then, the fluorescent spectrum of mixture was conducted on a fluorescence spectrophotometer (F-4500, Hitachi). The fluorescent ratio of I338/I333 was calculated as a function of logarithm HSC concentration.
Preparation and characterization of HSC-IR780 nanoparticles (HSCI NPs)
HSC NPs were prepared as the same protocol without joining IR780 into CH3CN. Dynamic light scattering (DLS) measurements (Nano-ZS, Malvern instruments, UK) was used to measure hydrodynamic size and polydispersity (PDI). Measurement temperature were set value of 25 °C. Transmission electron microscopy (TEM, Tecnai G2 20 S-TWIN, Perkin Elmer Instruments Co.Ltd. USA) was conducted for further evaluation of morphology and size.
Cell viability assays
MDA-MB-231 were incubated in DMEM media containing 10% FBS and at 37 °C in a moist atmosphere with 5% CO2. MDA-MB-231 cells were transfered into 96-well plates with a density of 5 × 103 cells/well. After incubation overnight, 100 µL of medium containing free IR780 or HSCI NPs (10 µg/mL of IR780) were added into plates replacing the origin medium. After incubation for 4 h, the cells were washed with fresh medium twice and laser (808 nm, 0.8 W/cm2, 0 ~ 6 min) was conducted on cells for programed photoactive therapy. After 12 h incubation, CCK-8 assays were used to assess cell viability. For blocking assay, HA was added into the plates ahead of 2 h.
In vitro cellular uptake evaluation
MDA-MB-231 cells (2 × 104/well) were seeded on the chambered cover-glass (Lab-Tek, Nunc, USA). After incubation overnight, new medium containing IR780 (10 µg/mL), HSCI NPs (10 µg/mL of IR780 concentration) or HSCI NPs co-incubation with HA (10 mg/mL, HSCI NPs + HA) were respectively added. After incubation 30 min at 37 °C, the cells were washed thrice with PBS and stained with lysosome-green for 15 min. Then, the cells were washed thrice with PBS. Cellular uptake evaluation was conducted by confocal laser scanning microscope (Perkin Elmer, Ultra View Vox system, USA).
Female BALB/c nude mice (6–8 weeks) were provided with Vital River Laboratory Animal Technology Co. Ltd. All relevant experiments were carried out following the ethical rules enacted by Experimental Animal Ethics Committee in Beijing. MDA-MB-231 cells (5 × 106) were injected on the right flank of the mice to establish tumor-bearing mice. When the volume of tumors reached to 100–200 mm3, further studies including imaging and therapy experiments were conducted.
Tumor-targeting imaging analysis in vivo
The mice were divided into two groups, IR780-treated mice (0.7 mg/kg of IR780) and HSCI NPs-treated mice (0.7 mg/kg of IR780). After tail vein injection, images with multi-spectral fluorescence (Cri-M2, CRI USA) and photoacoustic (M-128, iThera GER) living animal imaging system were taken at different time-points. Then major organs and tumor tissue were dissected for fluorescence imaging ex vivo by the Maestro imaging system.
In vivo tumor-inhibition assessment
All mice were randomly separated into five groups. The mice were injected with different formulations through tail vein injection, including saline, IR780 (1.4 mg/kg), HSCI NPs (1.4 mg/kg of IR780), IR780 + Laser (1.4 mg/kg and 0.8 W/cm2 10 min), HSCI NPs + Laser (1.4 kg/mg equivalent for nanoparticles and 0.8 W/cm2 10 min). After the injection for 12 h, the temperature profiles of tumor during laser irradiation was recorded by thermal imager (Ti400EN, FLUKE USA). Mice body weights and tumor volume were recorded every 3 days. The duration experimental time is 3 weeks. And the formula of tumor volume is “length × width2/2”.
XL and XW contributed to the majority of experimental work and the writing of the manuscript. YW, XD and ML directed the research, designed and coordinated the project, and provided advice and revised the manuscript. CZ performed in vivo NIR fluorescence imaging assay. LS and JiL helped perform photoacoustic imaging measurements. YT helped perform flow analysis, LC and XC addressed the confocal scanning microscope (CLSM) experiments. HS helped conduct the FT-IR analysis and JuL performed the H&E staining of tissues. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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Consent for publication
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Ethics approval and consent to participate
All relevant experiments involving animals were carried out following the ethical rules enacted by Experimental Animal Ethics Committee in Beijing.
This study was supported by National Natural Science Foundation of China (Grant Nos. 81472850 and 81773185) and the National Key Research and Development Program of China (Grant No. 2016YFA0200902).
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