{"id":40917,"date":"2023-02-17T02:55:40","date_gmt":"2023-02-17T03:55:40","guid":{"rendered":"https:\/\/peymantaeidi.net\/stem-cell\/?p=40917"},"modified":"2023-02-17T04:36:00","modified_gmt":"2023-02-17T04:36:00","slug":"combinatorial-polydopamine-liposome-nanoformulation-as-an-effective-anti-breast-cancer-therapy","status":"publish","type":"post","link":"https:\/\/peymantaeidi.net\/stem-cell\/2023\/02\/17\/combinatorial-polydopamine-liposome-nanoformulation-as-an-effective-anti-breast-cancer-therapy\/","title":{"rendered":"Combinatorial Polydopamine-Liposome Nanoformulation as an Effective Anti-Breast Cancer Therapy"},"content":{"rendered":"
\n

Introduction<\/h2>\n

According to the global cancer statistics 2018, breast cancer has the highest global incidence (28.9%) and mortality rates (23.0%) in women.1<\/a><\/sup> Chemotherapy, as a mainstay in clinical breast cancer treatment, is limited by severe side-effects resulting from the nonspecific drug distribution in the human body.2\u20134<\/a><\/sup> What is worse, a single therapeutic modality alone has shown restricted therapeutic efficacy during clinical applications.5<\/a><\/sup> Phototherapy,6<\/a><\/sup> including photodynamic therapy (PDT)7<\/a>,8<\/a><\/sup> and photothermal therapy (PTT),9\u201311<\/a><\/sup> has been widely accepted as a potent therapy modality for cancer treatment, due to the merits of excellent controllability, non-invasive property, minimal side effects, and so on.12\u201314<\/a><\/sup> PDT significantly increases the reactive oxygen species (ROS) levels in tumor cells by light-activated photosensitizers (PSs).15\u201317<\/a><\/sup> PDT relies on locally produced cytotoxic ROS to kill cancer cells, leaving normal tissue without light irradiation be free from damage.17\u201320<\/a><\/sup> PTT takes use of near infrared (NIR) light-absorbing agents to transfer light energy into hyperthermia, eventually resulting in thermal ablation of cancer cells, and the heat generated can be monitored by real-time photothermal imaging.21\u201323<\/a><\/sup> It is worth noting that the combination of PTT and PDT can achieve a more synergistic therapeutic effect.24<\/a><\/sup> On the one hand, thermal energy induced by PTT can promote cells to produce ROS to improve PDT efficacy.25<\/a>,26<\/a><\/sup> On the other hand, tumor cells are more sensitive to the hyperthermia produced by PTT due to the promotion of ROS.27<\/a><\/sup> Therefore, phototherapy can be combined with chemotherapy to achieve greater tumor destruction or even thoroughly ablate tumor by different mechanisms.28\u201330<\/a><\/sup> Indocyanine green (ICG) as the FDA-approved PS and photothermal agent in clinical application,31<\/a><\/sup> which also acts as a NIR fluorescence imaging (FI) and photoacoustic imaging (PAI) dye, can induce hyperthermia for PTT and produce cytotoxic ROS for PDT simultaneously.32<\/a><\/sup> Nevertheless, the actual applications of ICG have been hindered seriously by its poor stability, quick elimination and weak targeting ability.33<\/a><\/sup><\/p>\n

In recent decades, multifunctional nanomaterial-based combination treatments have drawn much attention because of great efficacy and low side effects.34<\/a><\/sup> Various nanocarriers, including liposomes,35<\/a>,36<\/a><\/sup> polymer materials,37<\/a>,38<\/a><\/sup> inorganic nanoparticles, etc.,39<\/a>,40<\/a><\/sup> have been explored to encapsulate ICG for avoiding these problems of free ICG molecules. Compared to other nanocarriers, it is proverbial that lipid-based nanoparticles with low toxicity and outstanding biocompatibility have been proven to be the most clinically established drug delivery systems.41<\/a>,42<\/a><\/sup> Doxil\u00ae<\/sup>, doxorubicin (DOX) liposome, is one of the earliest clinical examples for cancer therapy.43<\/a>,44<\/a><\/sup> However, liposomes have been greatly limited due to their premature drug leakage caused by inherent instability and the payload loss or undesired mixing caused by falling surface tension.45<\/a><\/sup> Fortunately, these problems can be overcome by the modification of the surface of liposomes with suitable polymers.46<\/a>,47<\/a><\/sup> Polydopamine (PDA)-based organic nanoparticles have been reported to be superior to other polymers due to its reactive functional groups, simple modification technologies, non-cytotoxicity and excellent biocompatibility.48\u201350<\/a><\/sup> Due to its superior adhesion, PDA can be easily and strongly deposited on either organic or inorganic solid substrates with nucleophilic groups, such as, liposomes,51<\/a>,52<\/a><\/sup> graphene oxide,53<\/a>,54<\/a><\/sup> and cerium oxide55<\/a><\/sup> to improve haemocompatibility significantly of pristine carriers.56<\/a><\/sup> It is noteworthy that PDA-based nanoparticles (NPs) are pH-sensitive, which means that the release behaviors of loaded drug molecules can be accelerated at acidic conditions (pH < 6.0) of tumor tissues57<\/a>,58<\/a><\/sup>. In addition, PDA has been demonstrated as exceptional PTT agents with strong photothermal conversion efficiency (>40%) under 808 nm laser irradiation.59<\/a><\/sup> NIR laser-controlled drug release from the PDA-based NPs can be achieved by decreased hydrogen bonding interaction between PDA and drugs due to the increasable heat of PTT effect.60<\/a><\/sup><\/p>\n

In this study, ICG and DOX were co-incorporated into the lipid bilayer, which were further coated with PDA by a facile two-step method to construct PDA-liposome NPs ([email protected]<\/a>\/DOX\/ICG) (Figure 1<\/a>). PDA layer not only prevented undesired release of ICG and DOX encapsulated in liposomes to minimize the side effects but also enhances the photothermal effect with 808 nm laser irradiation. Via intravenous injection, [email protected]<\/a>\/DOX\/ICG was passively targeted in tumor sites, which could be monitored by the fluorescence (only ICG) and photoacoustic dual-modal imaging of ICG and PDA. ICG not only produced ROS to participate in PDT but also converted the NIR light energy into local hyperthermia for PTT, which was significantly enhanced by the synergistic effect of PDA. Acidic conditions of tumor sites and NIR laser irradiation significantly increased the release of DOX from the PDA-based NPs for subsequent chemotherapy. Above all, we constructed a multifunctional nanoplatform based on PDA-coated liposomes for accurate and efficient combinatorial tumor therapy of chemotherapy and laser-induced PDT\/PTT.\n<\/p>\n\n\n\n
\"\"<\/a><\/td>\n\n

Figure 1<\/strong> Schematic illustration of combination treatment and imaging of [email protected]<\/a>\/DOX\/ICG.<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Methods<\/h2>\n

The Synthesis of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

To prepare [email protected]<\/a>\/DOX\/ICG, 32 mg HPSC, 10 mg cholesterol, 10 mg DSPE-mPEG2000, 4 mg DOX, and 2 mg ICG were co-dissolved in 2 mL methanol solvent. The organic phase was dropwisely added into 10\u2009mL ultrapure water under 80 rpm stirring at 55 \u00b0C. After removing the methanol solvent by evaporation, the obtained solution was centrifuged (4000 rpm, 5 min) to remove unloaded DOX and ICG, further sonicated by an ultrasonic cell disruption system (120 W, 3 s, on, 3 s, off, 3 min), and then passed through 0.45 \u03bcm water-based filter to obtain homogeneous liposomes (Lipo\/DOX\/ICG).<\/p>\n

0.5 mg\/mL of DA\u00b7HCl was dissolved in 10 mL Tris-HCl (10 mM) at pH 8.5 and gradually polymerized under stirring at room temperature. After continuously stirring for 24 h, the synthesized PDA solution was then washed twice with ultrapure water by repeated centrifugation at 12,000 rpm for 10 min and stored at 4 \u00b0C in the dark for use. Afterwards, methanol solvent including 4 mg DOX and 2 mg ICG was added dropwise into 10 mL reacted PDA solution, further stirring for 12 h at 40 \u00b0C. PDA nanoparticles (NPs) loaded with drugs (PDA\/DOX\/ICG) were obtained following purification by dialysis.<\/p>\n

Four-milliliter Lipo\/DOX\/ICG was immersed into 3 mL Tris-HCl buffer (10 mM) under a weakly alkaline environment (pH 8.5), which contained 1.5 mg of DA\u00b7HCl, to polymerize a PDA layer on the surface of Lipo\/DOX\/ICG. Subsequently, the suspension was continuously stirred (80 rpm) for 12 h at room temperature until the solution darkened, which demonstrated [email protected]<\/a>\/DOX\/ICG was obtained.<\/p>\n

Drug-free Liposomes (Lipo), PDA, and [email protected]<\/a> ([email protected]<\/a>) NPs were prepared by the same method above, but no drugs were added during the process. All samples were sealed at 4 \u00b0C for further study.<\/p>\n

The Characterization of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

The surface morphology of [email protected]<\/a>\/DOX\/ICG, PDA\/DOX\/ICG, and Lipo\/DOX\/ICG was characterized by transmission electron microscopy (TEM, JEOL JEM1200EX, Japan). Malvern Zetasizer Nano Series instrument (Zetasizer Nano-ZS, Malvern Instruments, UK) was utilized to measure their sizes, polydispersity indexes (PDIs) and Zeta potentials. The aforementioned formulations were monitored by UV-Vis-NIR spectrometer (UV-2600, Japan) and the Fourier transform infrared (FTIR) spectrometer (Bruker, Germany). After ultrafiltration centrifugation and methanol extraction, the encapsulation efficiency (EE) and drug loading capacity (LC) of DOX in the NPs were quantified and calculated using high-performance liquid chromatography (HPLC) analysis (Agilent, Santa Clara, USA). In detail, 200 \u03bcL NPs in ultrafiltration centrifuge tube was centrifuged at 4000g for 15 min. The upper filter residue was concentrated NPs, and the free drugs were located in the filtrate. The total DOX in the NPs was determined by disrupting the mixture with methanol. The mobile phase was 0.01 mol\/L potassium dihydrogen phosphate solution\/acetonitrile (72:28, v\/v), and the total flow rate was 1.0 mL\/min. The detection wavelength was set at 254 nm. EE and LC were calculated as follows:<\/p>\n

\"image\"<\/p>\n

\"image\"<\/p>\n

Dialysis method was adopted to compare DOX release behaviours from [email protected]<\/a>\/DOX\/ICG, PDA\/DOX\/ICG, or Lipo\/DOX\/ICG. In brief, solutions of different NPs (1.0 mL) in dialysis bags (MwCO = 3500) were placed into 30 mL PBS (pH 7.4) and oscillated (100 rpm, 37 \u00b0C). At certain time points (0.5, 1, 2, 4, 6, 8, and 12 h), 200 \u03bcL PBS solution was collected for DOX quantification by HPLC, and replaced with fresh PBS solution. The DOX release behaviours of [email protected]<\/a>\/DOX\/ICG were investigated in different pH release media (pH = 7.4, 5.0) with or without laser irradiation at 0.5 h (2.0 W\/cm2<\/sup>, 5 min). Each experiment was determined in triplicate.<\/p>\n

The Stability of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

To test the stability of NPs in different physiological solutions, 2 mL [email protected]<\/a>\/DOX\/ICG, PDA\/DOX\/ICG, or Lipo\/DOX\/ICG were put into ultrapure water or FBS (NPs: media = 1:1, v\/v), respectively, and incubated at 4 \u00b0C. Samples were taken and analyzed for size distribution and content at certain time periods (1, 2, 5, 10, and 15 d).<\/p>\n

The in vitro Singlet Oxygen (1<\/sup>O2<\/sub>) Detection and Photothermal Effect of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

DPBF, with a characteristic absorption peak at 417 nm, was used to evaluate the in vitro 1<\/sup>O2<\/sub> generation capability of [email protected]<\/a>\/DOX\/ICG.61<\/a>,62<\/a><\/sup> Briefly, 3 mL of PBS, free ICG and [email protected]<\/a>\/DOX\/ICG containing 5 \u03bcM DPBF were irradiated by 808 nm laser (VA-I-DC, Beijing Viasho Technology Co., Ltd.) with the power intensity of 1.0 W\/cm2<\/sup>, and the absorbance of DPBF was measured every 1 min by a UV-vis-NIR spectrometer.<\/p>\n

To compare the photothermal performance of different formulations, 1 mL H2O, blank carrier PDA, Lipo\/DOX\/ICG, and [email protected]<\/a>\/DOX\/ICG were irradiated by 808 nm laser (2.0 W\/cm2<\/sup>). The temperature was monitored every 1 min for ten cycles by an infrared thermal imaging camera (FLIR E50, Estonia). Photothermal effect of [email protected]<\/a>\/DOX\/ICG was further analyzed with different power densities (0.5, 1.0 and 2.0 W\/cm2<\/sup>), and the laser irradiation time varied from 1 to 10 min.<\/p>\n

Cell Culture<\/h3>\n

The human breast cancer cells MDA-MB-231 and normal human embryonic kidney cells HEK293 were obtained from XiangYa Central Experiment Laboratory (Hunan, China), which were cultured in Dulbecco\u2019s Modified Eagle\u2019s Medium (DMEM) supplemented with 10% (v\/v) fetal bovine serum (FBS) and 1% (v\/v) penicillin-streptomycin, in a humidity atmosphere containing 5% CO2<\/sub> at 37 \u00b0C.<\/p>\n

The Cellular Uptake of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

The cellular uptake of [email protected]<\/a>\/DOX\/ICG was qualitatively visualized by inverted fluorescence microscopy (Olympus, Germany). MDA-MB-231 cells were incubated overnight in 12-well plates (1 \u00d7 105<\/sup> cells per well). Then, [email protected]<\/a>\/DOX\/ICG was added with DOX concentration of 10 \u03bcg\/mL and further incubated for different time periods (0, 2, 6, and 12 h). Subsequently, the cells were rinsed twice with PBS, fixed with 4% paraformaldehyde for 10 min. Finally, cells were stained with Hoechst 33342 (10 \u03bcM) for 10 min and thoroughly washed prior to FI.<\/p>\n

To analyze the fluorescence intensity quantitatively, flow cytometry (FACS Verse, USA) was carried out, and the previous operation was performed in the same manner as above. After incubating for different time periods (1, 2, 6, 12, and 24 h), the cells were collected and analyzed using a flow cytometer.<\/p>\n

The Intracellular ROS Detection of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

Intracellular ROS detection of [email protected]<\/a>\/DOX\/ICG was measured using DCFH-DA fluorescent probe, which can be rapidly oxidized into DCF with green fluorescent in the presence of ROS.23<\/a><\/sup> Briefly, MDA-MB-231 cells were seeded in 6-well plates (5 \u00d7 105<\/sup> cells per well) and incubated overnight. ROS inhibitor NaN3<\/sub> was added and incubated for another 12 h, depleting the ROS produced by MDA-MB-231 cells themselves. Then, two different concentrations of [email protected]<\/a>\/DOX\/ICG (10 \u03bcg\/mL, 20 \u03bcg\/mL) were added for 2 h incubation. After washing thrice with PBS, the cells were cultured with 2 mL of DCFH-DA (10 \u03bcM) for 30 min. Finally, after being irradiated for 2 min (1.0 W\/cm2<\/sup>), the cells were trypsinized and resuspended in 1.0 mL PBS, followed by flow cytometry analysis with 488 nm excitation.<\/p>\n

The in vitro Cytotoxicity of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

The in vitro cytotoxicity studies were assessed on HEK293 and MDA-MB-231 cells by MTT method. To verify the safety of blank carriers, HEK293 cells (1 \u00d7 104<\/sup> cells per well) were cultured in 96-well plates for 24 h. Then, the cells were treated with PDA, PDA + NIR, Lipo, Lipo + NIR, [email protected]<\/a>, and [email protected]<\/a> + NIR at different concentrations in media, and 808 nm laser irradiation (1.0 W\/cm2<\/sup>, 2 min) was given after 8 h incubation in laser-irradiated groups. Twenty-four hours later, cells were treated with 20 \u03bcL MTT solution (5 mg\/mL) and incubated continued for 4 h. Finally, cell viability was evaluated using ELISA plate reader (TECAN, Austria) after reacting with 150 \u03bcL DMSO under mild shaking at 37 \u00b0C.<\/p>\n

For the cytotoxicity assay of NPs, MDA-MB-231 cells were divided into five groups, including free DOX\u00b7HCl, Lipo\/DOX\/ICG, Lipo\/DOX\/ICG + NIR, [email protected]<\/a>\/DOX\/ICG, and [email protected]<\/a>\/DOX\/ICG + NIR, and tested using the above-mentioned procedure. The cell inhibition rates of five groups were compared at a range of concentrations.<\/p>\n

To visualize the combination therapy cytotoxicity of [email protected]<\/a>\/DOX\/ICG, we used Calcein-AM and 0.4% trypan blue staining live and dead cells separately to imaging as the qualitative method. Specifically, MDA-MB-231 cells were seeded in 24-well culture plate (1 \u00d7 105<\/sup> cells per wall) for 24 h incubation and treated with different concentrations of [email protected]<\/a>\/DOX\/ICG (5 \u03bcg\/mL, 10 \u03bcg\/mL, 30 \u03bcg\/mL) for another 8 h incubation. Afterwards, the cells were irradiated for 2 min (1.0 W\/cm2<\/sup>) and continued to incubate for 12 h. Finally, the cells were stained with Calcein-AM and 0.4% trypan blue, and rinsed with PBS thoroughly to observe with Inverted fluorescence microscope.<\/p>\n

Tumor Models<\/h3>\n

BALB\/c nude mice (female, 4\u20135 weeks) were conducted under a protocol approved by the Animal Ethics Committee of Central South University in compliance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and were acclimated to standard diet at 25 \u00b0C for 1 week prior to the study. MDA-MB-231 cells (2 \u00d7 106<\/sup>) were injected subcutaneously in the right forelimb of the mice to establish breast cancer tumor-bearing models. Tumor volume was calculated as follows: tumor volume = length \u00d7 (width)2<\/sup>\/2.<\/p>\n

The in vivo FI and PAI of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

First, tumor-bearing mice with a volume of about 200 mm3<\/sup> were randomly selected. Experimental group mice were injected with 200 \u03bcL of [email protected]<\/a>\/DOX\/ICG nanoparticle solution via tail vein, and the control group mice were injected with an equal amount of free ICG as a control. After 1 h and 24 h administration, respectively, the mice were anesthetized with isoflurane and then placed in IVIS Spectrum imaging system (Perkin Elmer, USA) for comparison and observation (Ex: 745 nm, Em: 820 nm). Finally, the mice were sacrificed, and their tumor tissues and main organs (heart, liver, spleen, lung, and kidney) were obtained for the ex vivo imaging. For the PAI, photoacoustic signals of tumor-bearing mice were monitored using a VEVO LASER PAI system (VEVO 2100, FUJIFILM Visual Sonics, INC, USA) at different time points.<\/p>\n

The in vivo Antitumor Efficiency of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

When the tumor volumes reached about 100 mm3<\/sup>, the tumor-bearing mice were randomly divided into seven groups (n = 5): (1) PBS; (2) PBS + NIR; (3) free DOX\u00b7HCl; (4) Lipo\/DOX\/ICG; (5) Lipo\/DOX\/ICG + NIR; (6) [email protected]<\/a>\/DOX\/ICG; (7) [email protected]<\/a>\/DOX\/ICG + NIR (DOX equivalent of 2.5 mg\/kg). Then, the treatment was carried out by intravenous injection, and the three laser groups were irradiated for 24 h after injection at a power intensity of 1 W\/cm2<\/sup> for 2 min. The temperature changes in tumors during irradiation were monitored using an infrared thermal imaging camera.<\/p>\n

DOX\u00b7HCl group was injected every 3 days for a total of 5 administrations, and other groups were injected only once during the treatment cycle. The tumor sizes and body weights of mice in the groups were recorded every 3 days. Twenty-one days later, all the mice were sacrificed, and their tumors and major organs were resected, weighed (only tumors), and fixed in formalin for paraffin embedding to hematoxylin and eosin (H&E) analysis and Tunel staining (only tumors). Inhibition rates of tumor growth (IRT) were calculated as follows: IRT = (mean tumor weight of the PBS group \u2013 mean tumor weight of the experimental group)\/mean tumor weight of the PBS group \u00d7 100%.<\/p>\n

Statistical Analysis<\/h3>\n

The statistical analysis (t<\/em>-test) was carried out by OriginPro 9.0 (OriginLab, MA, USA). All statistical analyses were shown as mean \u00b1 standard deviation (SD), *p < 0.05 or less was considered statistically significant (NS, no significance, *p\u2009<\u20090.05, **p\u2009<\u20090.01, ***p\u2009<\u20090.001, ****p\u2009<\u20090.0001).<\/p>\n

Results and Discussion<\/h2>\n

The Synthesis and Characterizations of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

Lipo\/DOX\/ICG NPs were firstly prepared through a methanol injection method, and wherefore DOX\/ICG was encapsulated in liposomes. A PDA-coating was developed on the surface of Lipo\/DOX\/ICG NPs to form [email protected]<\/a>\/DOX\/ICG NPs in an alkaline condition (pH = 8.5). The whole synthesis method was universal and straightforward, and all it needed was a brief stirring under alkaline conditions to obtain PDA coated liposomes. To compare the difference between drug delivery systems, PDA\/DOX\/ICG NPs were also prepared by an oxidative polymerization reaction.52<\/a><\/sup><\/p>\n

FTIR spectra were used to investigate the disappearance, movement or broadening of the characteristic peaks of PDA coated liposomes, which can indicate the interaction between different materials.63<\/a><\/sup> As shown in Figure 2A<\/a>, PDA, Lipo, and [email protected]<\/a> all exhibit obvious characteristic peaks from 3615 cm\u22121<\/sup> to 3100 cm\u22121<\/sup>, which might be caused by the stretching vibration of the water hydroxyl O-H in the water-soluble system.64<\/a><\/sup> In the meantime, the two characteristic peaks located at 2960 cm\u22121<\/sup> and 2870 cm\u22121<\/sup> are corresponded to the stretching vibration of C-H, which prove that the structures of three blank carriers all contain methyl groups. In addition, the characteristic peaks of C=O (1750 cm\u22121<\/sup>) and phosphate ester (1250 cm\u22121<\/sup>) are attributed to blank Lipo, while the characteristic peak at 1650 cm\u22121<\/sup> is assigned to the C=C skeleton stretching vibration of benzene rings in PDA. Further comparison shows that [email protected]<\/a> has both the characteristic absorption peaks of PDA (C=C on the benzene ring) and Lipo (carbonyl and phosphate), indicating the successful conjugation of [email protected]<\/a>\n<\/p>\n

The morphologies of NPs were measured by TEM and shown in Figure 2B<\/a> and Supplementary Figure S1a and b<\/a><\/u>, which all present almost spherical shapes. However, Lipo\/DOX\/ICG NPs vary in size and had adhesion, indicating that there might be a fusion phenomenon (Supplementary Figure S1a<\/a><\/u>); PDA\/DOX\/ICG also displays partial aggregation (Supplementary Figure S1b<\/a><\/u>). In contrast, [email protected]<\/a>\/DOX\/ICG NPs (Figure 2B<\/a>) are more dispersed, and there is a clear visible core-shell structure, which indicates the successful deposition of PDA film coating on the liposomes.<\/p>\n

The sizes and PDIs of NPs are shown in Figure 2B<\/a>, Supplementary Figure S1a and b<\/a><\/u>, and Table 1<\/a>. PDA\/DOX\/ICG NPs have a mean particle size of 285.4 nm, Lipo\/DOX\/ICG NPs are measured as 99.8 nm, while that of [email protected]<\/a>\/DOX\/ICG NPs are 167.1 nm. Thus, the thickness of the coated PDA film is approximately 30 nm. Compared to the size obtained from TEM, the sizes measured by Malvern Zetasizer Nano Series instrument are relatively larger, which might be caused by the potential slight clustering and hydration effect.5<\/a><\/sup> This phenomenon also proves that the PDA film was successfully formed on the surface of Lipo\/DOX\/ICG. The small size of [email protected]<\/a>\/DOX\/ICG means that it is easy to permeate into tumor tissue through the EPR effect.65<\/a><\/sup> The particle size peak is unimodal, symmetrical and concentrated, indicating that this system is relatively stable, and the PDI <0.2 indicates a homogenous dispersion. In contrast, the PDI value of Lipo\/DOX\/ICG and PDA\/DOX\/ICG is more than 0.2, and the particle size peak of Lipo\/DOX\/ICG is bimodal, indicating that the particle distribution is poor and might be agglomerated Zeta potential is an important indicator of the stability of the dispersion system. A higher absolute value indicates larger mutual repulsion between the NPs, and a more stable dispersion system. The results of Zeta potential are displayed in Table 1<\/a>. The Zeta potential of the blank liposomes is positive and small, while the Zeta potential of [email protected]<\/a> is changed to negative after coating by PDA since PDA is a negative polyelectrolyte which could increase the negative charge of the entire system.66<\/a><\/sup> Similarly, the absolute value of [email protected]<\/a>\/DOX\/ICG was much higher than that of Lipo\/DOX\/ICG, which increased the mutual repulsion between the NPs, making the entire system more stable. The characteristic peaks of UV absorption for free DOX and ICG appear at 490 nm and 750\u2013800 nm, respectively.67<\/a>,68<\/a><\/sup> To verify whether DOX and ICG were successfully encapsulated into [email protected]<\/a>\/DOX\/ICG, we characterized the formulation by UV-vis-NIR spectroscopy, and the characteristic peaks of free DOX and ICG were observed in [email protected]<\/a>\/DOX\/ICG (Figure 2C<\/a>). The blank carrier [email protected]<\/a> has broad-spectrum absorption characteristics in the range of 350\u2013850 nm, and its intensity is proportional to the concentration (Supplementary Figure S1c<\/a><\/u>). Notably, there is a slight red-shift from 780 nm to 815 nm in the absorption spectra of [email protected]<\/a>\/DOX\/ICG compared to that of free ICG, which can be ascribed to the close-packed ICG in the NPs or the aggregation of ICG induced by the addition of DOX, and the aggregated ICG absorbs at a longer wavelength compared to a single ICG molecule.68\u201370<\/a><\/sup>\n<\/p>\n\n\n\n
\"\"<\/a><\/td>\n\n

Table 1<\/strong> Characterization (Sizes, PDIs, Zeta Potentials, Drug Loading and Contents of DOX) of Different NPs<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

The LC, EE and content of DOX are also illustrated in Table 1<\/a>. Since PDA is water-soluble and difficult to load fat-soluble drugs, the EE of PDA\/DOX\/ICG is only 23.5%. Both the EE (85.4%) and LC (3.4%) of Lipo\/DOX\/ICG are low, which might be related to the drug leakage of liposomes. After the PDA coating, the EE of [email protected]<\/a>\/DOX\/ICG is close to 100%, and the LC (5.7%) is higher than that of Lipo\/DOX\/ICG, which indicates the PDA coating could reduce the leakage of free DOX. However, the added volume of PDA also diluted the concentration of DOX resulting in the content of [email protected]<\/a>\/DOX\/ICG lower than that of Lipo\/DOX\/ICG.<\/p>\n

To explore the drug release process, the formulations were sealed in dialysis bags under different pH release media (pH = 7.4, 5.0) with or without irradiation, and the release profiles of DOX were recorded by monitoring the drug concentration outside the bag using HPLC. As observed in Supplementary Figure S1d<\/a><\/u>, PDA\/DOX\/ICG and Lipo\/DOX\/ICG show a rapid release (>30%) within 0.5 h, while the release rate of [email protected]<\/a>\/DOX\/ICG (Figure 2D<\/a>) is delayed. This property might attribute to the package of PDA film, which is dissolved and degraded slowly. Compared with in PBS (pH = 7.4), the cumulative release of the three NPs both has a certain increase at pH 5.5, which could be attributed to the protonated amino group of DOX to provide a positive charge to improve the solubility in acidic solutions, thereby induces the release of DOX.50<\/a><\/sup> Moreover, whether at pH 7.4 or pH 5.5, irradiation added could improve the total amount of released DOX from [email protected]<\/a>\/DOX\/ICG significantly, from 63.67% to 72.13% and from 78.32% to 92.31% at pH 7.4 and pH 5.5, respectively. The promotion of DOX release is attributed to the photothermal effects of PDA and ICG in [email protected]<\/a>\/DOX\/ICG. On the one hand, the heat could destroy the PDA film to promote the release of DOX; on the other hand, the high temperature accelerates the diffusion of DOX molecules.<\/p>\n

The Stability of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

In order to compare the stability of the different prepared NPs, the sizes were monitored. As shown in Supplementary Figure S2a<\/a><\/u>, PDA\/DOX\/ICG had a significant increase in particle sizes in the two physiological solutions. Especially, the size of NPs in FBS exceeded 1500 nm after only one day, and exceeded 3000 nm after 15 days. Combined with Supplementary Figure S2b<\/a><\/u>, black precipitates of PDA\/DOX\/ICG in water and FBS solutions could be seen clearly, which show that PDA\/DOX\/ICG is very unstable with poor dispersibility. Therefore, PDA\/DOX\/ICG has not been studied in subsequent cell and animal experiments. Lipo\/DOX\/ICG also occurred aggregation 15 days later. In contrast, size of [email protected]<\/a>\/DOX\/ICG had no obvious change 15 days later (Figure 3A<\/a>), and no obvious aggregation phenomenon of [email protected]<\/a>\/DOX\/ICG could be observed in both aqueous solution and FBS (Figure 3B<\/a>), indicating that the stability of [email protected]<\/a>\/DOX\/ICG is superior to PDA\/DOX\/ICG and Lipo\/DOX\/ICG.\n<\/p>\n

The drug contents of the NPs in water and FBS over time were further compared. The results show that the contents of the three kinds of NPs decreased gradually with time, and the contents of NPs in FBS decreased relatively faster (Figure 3C<\/a> and Supplementary Figure S2c<\/a><\/u>). After 15 days, DOX contents in both PDA\/DOX\/ICG and Lipo\/DOX\/ICG decreased by more than 50% (Supplementary Figure S2c<\/a><\/u>), while DOX levels in [email protected]<\/a>\/DOX\/ICG remained stable (Figure 3C<\/a>).<\/p>\n

The in vitro 1<\/sup>O2<\/sub> Detection and Photothermal Effect of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

To detect the PDT property, DPBF was used as a capture agent for 1<\/sup>O2<\/sub>, which can quickly react with 1<\/sup>O2<\/sub> to reduce its absorption intensity at 417 nm, and the greater the decrease in absorbance, the higher the 1<\/sup>O2<\/sub> generation.71<\/a><\/sup> The absorption intensity at 417 nm of free DPBF hardly changed within 10 minutes, indicating that there was basically no generation of 1<\/sup>O2<\/sub> (Supplementary Figure S3a<\/a><\/u>), but the free ICG (Supplementary Figure S3b<\/a><\/u>) and [email protected]<\/a>\/DOX\/ICG NPs (Figure 4A<\/a>) resulted in a decreased absorption intensity of DPBF at 417 nm gradually with the increase of irradiation time, implying the strong 1<\/sup>O2<\/sub> generation ability and great PDT property of [email protected]<\/a>\/DOX\/ICG NPs.\n<\/p>\n

The photothermal performances of H2<\/sub>O, blank carrier PDA, Lipo\/DOX\/ICG, and [email protected]<\/a>\/DOX\/ICG were measured by infrared imager under the irradiation of an 808 nm laser (2.0 W\/cm2<\/sup>, 10 min). As shown in Figure 4B<\/a>, the temperature of water varies from 23.4 \u00b0C to 26.7\u00b0C, while the temperature increases of PDA, Lipo\/DOX\/ICG, and [email protected]<\/a>\/DOX\/ICG are 21.9 \u00b0C, 17.3 \u00b0C, and 48.6 \u00b0C, respectively. Interestingly, [email protected]<\/a>\/DOX\/ICG showed better photothermal effect than PDA\/DOX\/ICG, the reason might be that the ICG loading capacity of water-soluble PDA was weak, and lower concentration of ICG resulted in limited photothermal property. [email protected]<\/a>\/DOX\/ICG exceeds 50 \u00b0C in less than 4 minutes and reaches 69.0 \u00b0C after 10 minutes irradiation, which performs obvious time-dependent photothermal characteristics. Because the photothermal effect of [email protected]<\/a>\/DOX\/ICG comes from PDA and ICG, it is stronger than that of single carrier PDA or Lipo\/DOX\/ICG.<\/p>\n

In addition, the relationship between thermal performance and irradiation power for [email protected]<\/a>\/DOX\/ICG was examined. As shown in Figure 4C<\/a> and D<\/a>, by improving the power density of irradiation, the temperature is significantly increased. Especially at 2.0 W\/cm2<\/sup>, the temperature increases closed to 70 \u00b0C, indicating the exceptional PTT effect of the optimal formulation. Considering that too high temperature may exceed the skin tolerance and cause damage to normal cells, 1.0 W\/cm2<\/sup> was used in the subsequent cell and animal experiments.<\/p>\n

The Cellular Uptake of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

The cellular uptake of [email protected]<\/a>\/DOX\/ICG was observed by inverted fluorescence microscopy (Figure 5A<\/a>). After incubation for 2 h, red fluorescence of DOX was found in the cells. The fluorescence intensity was enhanced by prolonging the incubation time. The cells showed obvious red fluorescence at 12 h, indicating that [email protected]<\/a>\/DOX\/ICG NPs could successfully deliver DOX into cells. The quantitative determination of uptake ability of [email protected]<\/a>\/DOX\/ICG NPs in cells was also studied using flow cytometer. As seen in Figure 5B<\/a>, the fluorescence intensity of DOX is directly proportional to the incubation time in a certain time period. After 12 h, the fluorescence intensity does not increase obviously, indicating that the cellular uptake has reached saturation.\n<\/p>\n\n\n\n
\"\"<\/a><\/td>\n\n

Figure 5<\/strong> (A<\/strong>) Fluorescence images of MDA-MB-231 cells incubated with [email protected]<\/a>\/[email protected]<\/a> for 2, 6 or 12 h (The scale bar is 50 \u03bcm). (B<\/strong>) Flow cytometry analysis of MDA-MB-231 cells incubated with [email protected]<\/a>\/[email protected]<\/a> for different incubation periods (Ex: 505 nm, Em: 550 nm). (C<\/strong>) Flow cytometry analysis of intracellular ROS generation of [email protected]<\/a>\/[email protected]<\/a> with 1.0 W\/cm2<\/sup> laser for different ICG concentrations (0, 10, and 20 \u03bcg\/mL) inside MDA-MB-231 cells (ROS-sensitive probe DCFH-DA, Ex: 488 nm, Em: 525 nm).<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

The Intracellular ROS Detection of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

Pretreated with NaN3<\/sub> to scavenge endogenous 1<\/sup>O2<\/sub> of MDA-MB-231 cells, the intracellular ROS produced by [email protected]<\/a>\/DOX\/ICG with irradiation was detected using an ROS-sensitive probe DCFH-DA. As shown in Figure 5C<\/a>, the fluorescence intensity of PBS groups is basically unchanged with or without irradiation. As for the [email protected]<\/a>\/DOX\/ICG groups, the fluorescence intensity of irradiation groups is significantly higher than that of no irradiation groups, meaning that [email protected]<\/a>\/DOX\/ICG has produced an additional high level of ROS inside tumor cells by irradiation, which plays an indispensable role in PDT. Moreover, with the increase of drug concentration, the fluorescence intensity becomes stronger after irradiation, suggesting that ROS production is directly proportional to the cellular concentration of [email protected]<\/a>\/DOX\/ICG.<\/p>\n

The in vitro Cytotoxicity Assay of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

At first, the cytotoxicity of three carrier materials (PDA, Lipo, and [email protected]<\/a>) towards HEK293 cells was assessed using MTT assay. When the concentration of PDA was in the range of 10\u2013200 \u03bcg\/mL, the cell survival rates of the group without irradiation was more than 90%, indicating that PDA had biocompatibility and was basically non-toxic to HEK293 cells (Supplementary Figure S4a<\/a><\/u>). As for Lipo and [email protected]<\/a> (Supplementary Figure S4b<\/a><\/u> and Figure 6A<\/a>), the cell survival rate also exceeded 80% as the concentration increased from 50 \u03bcg\/mL to 1000 \u03bcg\/mL, showing good biological safety. Moreover, after 1.0 W\/cm2<\/sup> irradiation for 2 min, PDA and [email protected]<\/a> showed significant cytotoxicity to cells with the increase of PDA concentration, while Lipo remained basically unchanged after irradiation. The results show the biosafety of PDA and Lipo and the photothermal effect of PDA.\n<\/p>\n\n\n\n
\"\"<\/a><\/td>\n\n

Figure 6<\/strong> (A<\/strong>) MTT assays of blank carrier [email protected]<\/a> over 24 h in MDA-MB-231 cells with\/without irradiation (1.0 W\/cm2<\/sup>, 2 min). (B<\/strong>) MTT assays of DOX\u00b7HCl, Lipo\/[email protected]<\/a>, and [email protected]<\/a>\/[email protected]<\/a> over 24 h in MDA-MB-231 cells with\/without irradiation (1.0 W\/cm2<\/sup>, 2 min). (C<\/strong>) Fluorescence images of Calcein-AM costained MDA-MB-231 cells (live cells) and bright field images of trypan blue costained MDA-MB-231 cells (dead cells) treated with different concentrations of [email protected]<\/a>\/[email protected]<\/a> (+\/\u2013 indicated with or without irradiation. The scale bar is 100 \u03bcm). (The data are shown as mean \u00b1 SD, n = 3 per group, NS, no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Similarly, the inhibitory rate of different NPs towards MDA-MB-231 cells was also measured by MTT assay. As shown in Figure 6B<\/a>, the cell inhibitory rates of all NPs gradually increase as the concentration increases, implying a concentration-dependent cytotoxic effect. Basically, both Lipo\/DOX\/ICG and [email protected]<\/a>\/DOX\/ICG NPs show a stronger cytotoxicity than free DOX\u00b7HCl at each corresponding concentration, suggesting that NPs can be better taken up by MDA-MB-231 cells than free DOX\u00b7HCl. For Lipo\/DOX\/ICG and [email protected]<\/a>\/DOX\/ICG, when the DOX concentrations are higher than 5 \u03bcg\/mL, the inhibition rates of the irradiation groups are significantly higher than those of the no irradiation groups. In particular, the cell inhibition rate of [email protected]<\/a>\/DOX\/ICG + NIR group could reach 68.2% at a concentration of 10 \u03bcg\/mL, while the cell inhibition rate of no irradiation group is less than 40%, indicating that combination of chemotherapy and phototherapy could achieve better antitumor effect at relatively low doses.<\/p>\n

The induced apoptosis ability of different concentrations of [email protected]<\/a>\/DOX\/ICG was further visualized by staining dead cells with trypan blue and live cells with Calcein-AM. As shown in Figure 6C<\/a>, both PBS group and PBS + NIR group exhibit intense green fluorescence and few staining blue, indicating almost all cells were intact and normal. In contrast, green fluorescent numbers are obviously reduced by increasing the DOX concentration in [email protected]<\/a>\/DOX\/ICG groups. This phenomenon is more obvious in [email protected]<\/a>\/DOX\/ICG + NIR groups, and only a few cells survived when the concentration of DOX was 10 \u03bcg\/mL. When the concentration reached 30 \u03bcg\/mL, almost all cells with no clear outline and complete morphology are stained by trypan blue. In vitro experiments, including cellular uptake, intracellular ROS detection, and cytotoxicity, confirm that [email protected]<\/a>\/DOX\/ICG could not only convert NIR light energy into local hyperthermia for PTT but also produce an additional high level of ROS to participate in PDT, indicating that the NPs have certain phototherapeutic effect. Moreover, the synergistic therapy efficacy of phototherapy and chemotherapy is far superior to that of chemotherapy alone.<\/p>\n

The in vivo FI and PAI of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

Both ICG and PDA have been widely explored as fluorescence (only ICG) and\/or photoacoustic contrast agents because of their strong optical absorbance in the NIR region. Both FI and PAI are nonradioactive imaging methods with intrinsic advantages. FI could visualize the dynamic distribution of NPs throughout the whole body.72<\/a>,73<\/a><\/sup> At the same time, PAI could monitor the accumulation of NPs within the deep regions of tumor with improved imaging resolution.74<\/a><\/sup> By injecting intravenously free ICG and [email protected]<\/a>\/DOX\/ICG, the in vivo FI of tumor and the major organs (heart, liver, spleen, lung, and kidney) were performed. Two hours later, the mice in both [email protected]<\/a>\/DOX\/ICG and ICG groups had obvious fluorescence signal all over the body (Figure 7A<\/a>). After 24 h, fluorescence signals were obviously accumulated at the tumor sites in [email protected]<\/a>\/DOX\/ICG group. Due to its unique EPR effect, the fluorescence intensity was significantly stronger than that of other organs. On the contrary, free ICG was mainly distributed in the liver, and no obvious fluorescence signal was observed in the tumor.\n<\/p>\n\n\n\n
\"\"<\/a><\/td>\n\n

Figure 7<\/strong> (A<\/strong>) Fluorescence images of mice bearing MDA-MB-231 tumors after intravenous injection of free ICG and [email protected]<\/a>\/[email protected]<\/a> at 2 and 24 h. (B<\/strong>) Ex vivo FL images of major organs and tumors dissected from mice 24 h post injection of different treatment groups. (C<\/strong>) Semiquantitative biodistribution of free ICG and [email protected]<\/a>\/[email protected]<\/a> in mice determined by the averaged Fluorescence intensities of organs and tumor. (D<\/strong>) PA images of tumor sites on MDA-MB-231-tumor-bearing mice post injection of free ICG and [email protected]<\/a>\/[email protected]<\/a> at 2, 12, and 24 h. (E<\/strong>) The corresponding PA signals at the tumor sites of MDA-MB-231-tumor-bearing mice post injection of free ICG and [email protected]<\/a>\/[email protected]<\/a> at different time points. ****p < 0.0001.<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

To confirm tumor accumulation of the NPs through EPR effect, the tumor bearing mice were sacrificed 24 h post injection, and the tumors and major organs were collected for FI. Ex vivo imaging shows bright fluorescence at the tumor regions of mice treated with [email protected]<\/a>\/DOX\/ICG (Figure 7B<\/a>). However, the free ICG is mainly distributed in the liver, and almost no fluorescence signal occurred in the tumor regions. As shown in Figure 7C<\/a>, the fluorescence intensity of the tumor regions in [email protected]<\/a>\/DOX\/ICG group is approximately 10.6-fold higher than that in free ICG group, and also much higher than that of other organs within the group.<\/p>\n

Subsequently, the in vivo PAI of [email protected]<\/a>\/DOX\/ICG was evaluated using a Vevo LAZR system.75<\/a>,76<\/a><\/sup> After intravenous injection, the photoacoustic images of the mice in these groups were obtained at different time periods (Figure 7D<\/a> and E<\/a>). Owing to the rapid in vivo clearance of free ICG, no obvious signal was observed in tumor sites of free ICG group at any time points (Figure 7D<\/a> and E<\/a>). On the contrary, the signal intensity of [email protected]<\/a>\/DOX\/ICG in tumor sites gradually increased over time and reached the peak value at 24 h post injection, indicating deep accumulation of [email protected]<\/a>\/DOX\/ICG within the tumor.<\/p>\n

The in vivo Antitumor Activity of [email protected]<\/a>\/DOX\/ICG<\/h3>\n

Owing to the excellent imaging ability, photothermal performance of [email protected]<\/a>\/DOX\/ICG and the imaging-guided antitumor therapy were subsequently investigated in vivo. Mice bearing MDA-MB-231 tumors were intravenously injected with [email protected]<\/a>\/DOX\/ICG or other control formulations, and their tumors were irradiated with 808 nm laser (1.0 W\/cm2<\/sup>) at 24 h post injection. In vivo thermal images were recorded using an infrared thermal imaging camera at different time periods (Figure 8A<\/a>). The thermal images show that the temperature of tumor regions in [email protected]<\/a>\/DOX\/ICG + NIR group rapidly reaches about 48.4 \u00b0C. Compared to PBS + NIR group and Lipo\/DOX\/ICG + NIR group, [email protected]<\/a>\/DOX\/ICG + NIR group shows prominent ability to increase temperature within a short time, demonstrating that [email protected]<\/a>\/DOX\/ICG not only could accumulate in tumor sites by passive targeting but also is capable of excellent photothermal conversion ability to kill tumor cells in vivo.\n<\/p>\n\n\n\n
\"\"<\/a><\/td>\n\n

Figure 8<\/strong> (A<\/strong>) Thermal images of MDA-MB-231-tumor-bearing mice from different treating groups (PBS + NIR, Lipo\/DOX\/ICG + NIR, and [email protected]<\/a>\/[email protected]<\/a> + NIR) under 808 nm laser irradiation for different irradiation periods. (B<\/strong>) Tumor volume curves of different groups after various treatments including: PBS, PBS + NIR, DOX\u00b7HCl, Lipo\/DOX\/ICG, [email protected]<\/a>\/[email protected]<\/a>, Lipo\/DOX\/ICG + NIR, and [email protected]<\/a>\/[email protected]<\/a> + NIR. (C<\/strong>) Tumor weights of each group at the end of experiment and IRT. (D<\/strong>) Photographs of tumor tissues removed from groups treated with different formulations after 21 d. The red circles indicated disappeared tumors. (E<\/strong>) Body weight curves of MDA-MB-231 tumor-bearing mice for each group. (F<\/strong>) Representative H&E and Tunel stain images of tumor samples from the treated mice. (G<\/strong>) Representative H&E stain images of heart samples from the treated mice. (The data are shown as mean \u00b1 SD, n = 5 per group, NS, no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

In order to prove the in vivo synergistic phototherapy and chemotherapy of [email protected]<\/a>\/DOX\/ICG, the antitumor effect was evaluated by the tumor volumes and body weight changes of tumor-bearing mice, and the visualized photographs of tumor bearing mice were shown in Supplementary Figure S5<\/a><\/u>. During the treatment, compared with PBS and PBS\u2009+\u2009NIR groups, tumor growth inhibition was displayed in all drug-treated groups (Figure 8B<\/a>). However, the tumors could only be partly inhibited by free DOX\u00b7HCl, Lipo\/DOX\/ICG, [email protected]<\/a>\/DOX\/ICG, and Lipo\/DOX\/ICG + NIR groups because of their different deficiencies. In addition, [email protected]<\/a>\/DOX\/ICG and Lipo\/DOX\/ICG + NIR had similar inhibitory effects on tumor volume growth, and both enhanced the antitumor activity than that of free DOX\u00b7HCl (Figure 8C<\/a>). What\u2019s more, the tumor growth of [email protected]<\/a>\/DOX\/ICG + NIR was completely inhibited and thus no tumors were removed (Figure 8D<\/a>). The tumors did not recur during the treatment, and the burnt wounds on the skin surface gradually returned to normal, which indicated the best tumor suppression effect. Meanwhile, mice body weight change curves were used to explore the therapeutic efficacy and toxicity. Compared with other six groups, the body weight in free DOX\u00b7HCl group was significantly reduced (Figure 8E<\/a>).<\/p>\n

Beyond that, the in vivo toxicity and antitumor efficacy were further evaluated with histological study, in which the tumors and major organs were stained with H&E and Tunel. The results of H&E staining (Figure 8F<\/a>) show that there were regularly and tightly packed spherical tumor cells in the PBS and PBS + NIR groups, while drug-treated groups exhibited significantly decreased cellularity, tissue fibrosis, and nucleus shrinkage, fragmentation and dissolution, which are typical apoptotic and necrotic characteristics. According to the Tunel staining results, the apoptotic cells of drug-treated groups significantly increased, which are consistent with the results of the in vitro and in vivo antitumor activity experiments (Figures 6<\/a> and 8C<\/a>). In addition, Figure 8G<\/a> shows the H&E staining images of hearts and there are apparent necrosis of myocardial cells only in free DOX\u00b7HCl group because of its cardiotoxicity. Meanwhile, no obvious damage of major organs is observed in each group (Supplementary Figure S6<\/a><\/u>). The histological studies, together with curves of weight change in mice, demonstrate negligible cardiotoxicity and no treatment-induced side effects of [email protected]<\/a>\/DOX\/ICG.<\/p>\n

Conclusions<\/h2>\n

In this study, a multifunctional nanoplatform ([email protected]<\/a>\/DOX\/ICG) based on PDA-coated liposomes was successfully fabricated, which was loaded with doxorubicin and indocyanine green for combinatorial therapy of breast cancer. At first, polydopamine coating could improve the stability and drug loading of liposomes, and avoid premature drug leakage and unwanted fusion of liposomes. Moreover, surface polydopamine modification can markedly enhance the photothermal conversion efficiency of indocyanine green, and the generated heat could promote the permeation of nanoparticles into tumor cells and the release of doxorubicin. Owing to the synergistic effect of phototherapy and chemotherapy, [email protected]<\/a>\/DOX\/ICG displayed a remarkably improved cytotoxicity in MDA-MB-231 cells at relatively low doses. Finally, both indocyanine green and polydopamine can be used as contrast agents for fluorescence imaging (only indocyanine green) and\/or photoacoustic imaging and the accumulation of [email protected]<\/a>\/DOX\/ICG with obvious signals throughout the treatment could be monitored by the fluorescence imaging and photoacoustic imaging dual-modal imaging.77<\/a><\/sup> According to our experimental results, [email protected]<\/a>\/DOX\/ICG efficiently suppressed the proliferation of MDA-MB-231 cells and even thoroughly ablated tumors, which was far superior to the theranostic outcomes of Lipo\/DOX\/ICG or doxorubicin hydrochloride groups. Meanwhile, [email protected]<\/a>\/DOX\/ICG avoided the cardiotoxicity of doxorubicin hydrochloride groups and no other adverse effects were observed. In summary, this novel nano-system would be a very promising nanoplatform for multi-drug delivery, imaging-guided and multimodal treatments for superior antitumor efficacy.<\/p>\n<\/div>\n

\n

Abbreviations<\/h2>\n

DA\u00b7HCl, Dopamine hydrochloride; DCFH-DA, 2\u2032, 7\u2032-dichlorofluorescin diacetate; DDSs, Drug delivery systems; DOX, Doxorubicin; DOX\u00b7HCl, Doxorubicin hydrochloride; DPBF, 1,3-diphenylisobenzofuran; DSPE-mPEG2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; EE, Encapsulation efficiency; FBS, Fetal bovine serum; FI, Fluorescence imaging; FTIR, Fourier transform infrared; H&E, Hematoxylin and eosin; HPLC, High-performance liquid chromatography; HSPC, Hydrogenated soybean phosphatidylcholine; ICG, Indocyanine green; IRT, Inhibition rates of tumor growth; LC, Loading capacity; Lipo, Liposomes; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NIR, Near infrared; NPs, Nanoparticles; PAI, Photoacoustic imaging; PDA, Polydopamine; PDIs, Polydispersity indexes; PDT, Photodynamic therapy; PSs, Photosensitizers; PTT, Photothermal therapy; ROS, Reactive oxygen species; SD, Standard deviation.<\/p>\n

Data Sharing Statement<\/h2>\n

All data generated or analyzed during this study are included in this published article and its Supplementary Information Files<\/a><\/u>.<\/p>\n

Ethics Approval and Informed Consent<\/h2>\n

All animal experiments were approved by the Animal Ethics Committee of Central South University and were conducted in compliance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978), as well as the internationally recognized `3Rs\u201d principles of animal experiments.<\/p>\n

Consent for Publication<\/h2>\n

All authors agreed to publish this manuscript.<\/p>\n

Author Contributions<\/h2>\n

All authors made significant contributions to the manuscript in all areas of conceptualization, study design, data, and analysis. All authors have participated in drafting or writing, or have substantially revised or critically reviewed the manuscript. All authors have read and approved the final manuscript prior to submission. All authors have agreed on the journal to which the manuscript will be submitted. All authors agree to take responsibility and are accountable for the content of the manuscript.<\/p>\n

Funding<\/h2>\n

This research was supported by National Natural Science Foundation of China (Grant No. 82073932).<\/p>\n

Disclosure<\/h2>\n

The authors declare that they have no competing interests.<\/p>\n

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