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Controlled Release and Delivery Systems

DOX/ICG Coencapsulated Liposome Coated Thermosensitive Nanogels for NIR-triggered Simultaneous Drug Release and Photothermal Effect Lixia Yu, Anjie Dong, Ruiwei Guo, Muyang Yang, Liandong Deng, and Jianhua Zhang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b00379 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 15, 2018

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ACS Biomaterials Science & Engineering

DOX/ICG

Coencapsulated

Thermosensitive

Nanogels

Liposome for

Coated

NIR-triggered

Simultaneous Drug Release and Photothermal Effect Lixia Yu,† Anjie Dong, †‡ Ruiwei Guo,† Muyang Yang, † Liandong Deng, † and Jianhua Zhang*, †,§ †

Department of Polymer Science and Engineering, Key Laboratory of Systems

Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China ‡

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300072, China §

Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin

University, Tianjin 300072, China

Corresponding author: Jianhua Zhang Email [email protected]

ACS Paragon Plus Environment

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ABSTRACT: Chemo-photothermal therapy has shown enormous potentials in treating cancer. To achieve the chemo-photothermal synergistic effect, an efficient nanoparticulate system with the ability of simultaneous co-delivery of chemotherapeutic drug and photothermal agent as well as photothermal-triggered drug release is highly desirable. Herein, an in situ polymerization within

liposome

template

was

designed

to

prepare

liposome

coated

poly(N-

isopropylacrylamide-co-acrylamide) (P(NIPAM-co-AAM)) nanogels, which can efficiently coencapsulate a NIR dye indocyanine green (ICG) and high amount of doxorubicin hydrochloride (DOX). The DOX/ICG coloaded hybrid nanogels, denoted as DI-NGs@lipo, integrated the desirable functions of PEGylated liposomes and thermosensitive nanogels. The PEGylated liposomes shell provided excellent storage stability, hemodynamic stability and fluorescence stability. Meanwhile, the thermosensitive P(NIPAM-co-AAM) nanogels core endowed DI-NGs@lipo with volume phase transition temperature (VPTT) at about 40 °C, allowing for thermo-controlled transformation and drug release. The significant photothermal effect of DI-NGs@lipo and the simultaneous hyperthermia-triggered DOX release were observed under NIR light irradiation. The DI-NGs@lipo was demonstrated to be uptaken by 4T1 murine breast cancer cells via endocytosis, enhancing the distribution of DOX in the cell nucleus. Compared with chemo or photothermal treatment alone, the combination treatment of DI-NGs@lipo with NIR light irradiation induced significantly higher cytotoxicity to 4T1 cells, demonstrating the chemo-photothermal synergistic therapeutic effects on tumor cells. In a word, the strategy provided here offers a facile approach to develop a multifunctional nanoplatform for co-delivery of DOX and ICG, which can synergistically improve the cancer cell killing efficiency, demonstrating great potential in chemo-photothermal therapy.

KEYWORDS: liposomes; template polymerization; temperature-sensitive nanogels; active loading; photothermal effect


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INTRODUCTION Cancer has presently become one of the deadliest diseases around the world. Among cancer therapeutics, chemotherapy has been widely applied. However, it usually confronts some limits, especially with respect to the non-specific distribution and thus severe systemic toxicity serious.1-2 Nanoparticulate drug delivery systems open a new avenue toward the diagnosis and treatment of a variety of cancers.3-6 In particular, the environmentally sensitive nanocarriers that can trigger drug release at the tumor site in response to endogenous stimuli (e.g. pH, redox, and enzyme) or exogenous stimuli (e.g. temperature, light, magnetic field, ultrasound intensity or electric pulses) have been developed to enable tumor-specific release and thus improve chemotherapeutic efficacy.7-11 However, due to the uncontrollable specific microenvironments and individual differences in the human body, the accurate control and release of drugs triggered by endogenous stimuli often achieve limited efficiency.8 Therefore, the exogenous stimuli-responsive drug delivery systems might be more favourable for tunable and specific anticancer drug delivery.8, 11 Owing to its non-invasiveness, high biocompatibility, ease of application reversible and the remote spatiotemporal control, utilization of light as an external stimulus to trigger drug release or induce local hyperthermia has generated considerable interest. The near-infrared (NIR) light with wavelengths in the range of about 700 to 900 nm has been considered to be the most promising optical stimulus, due to its minimal attenuation and deep tissue penetration as well as less damage to tissues. Especially, the photothermal therapy (PTT) based on photosensitizers, such as indocyanine green (ICG), that strongly absorb NIR light to generate heat for tumor treatment, has been demonstrated as a highly efficient therapeutic technique and exhibited several unique advantages in cancer therapy, such as minimal invasiveness, high specificity, and precise spatiotemporal selectivity.12-14 More importantly, hyperthermia induced by NIR light not only can directly cause thermal ablation of cancer cells and increase the sensitivity of


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tumor cells to chemotherapy drugs, but also can improve the permeability of tumor vessels due to the enhancement of blood flow in the hyperthermia area.15-17 Therefore, the photothermal therapies based on NIR-induced hyperthermia have been widely combined with chemotherapy to enhance the anticancer efficacy.15, 18 Especially, the integration of photothermal therapy and chemotherapy into a single nanoplatform has shown therapeutic efficiencies superior to those achieved by a single treatment.15, 19-22 However, to achieve the synergistic effects of chemophotothermal combination therapy, it is expected that the high doses of chemotherapy drug and photosensitizer can be simultaneously delivered to the same tumor cells and exert chemophotothermal therapeutic functions in a synergistic manner. Apparently, the synergistic codelivery of chemotherapeutic agent and photosensitizer will necessarily require further development of multifunctional nanocarriers with the ability of efficient encapsulation, hemodynamic stability and NIR-triggered drug release by the photothermal-induced nanostructural transition.14, 23 With the development of nanocarriers, versatile nanogels have attracted great attentions.19, 24 In particular, stimuli-responsive nanogels that can respond to environmental stimuli, such as pH, redox, temperature, are one of the most promising nanoparticulate platforms in the area of drug delivery.7,

25-27

Thermosensitive nanogels based on poly(N-isopropylacrylamide)

(PNIPAM) that can change their particle volume in response to the environmental temperature have been widely used as thermosensitive drug delivery systems.28-30 More importantly, the VPTT of PNIPAM, generally at around 32 °C, can be simply tuned by controlling the hydrophilic/hydrophobic balance of the polymer via copolymerization of another hydrophilic monomer.31-33 For example, the introduction of good hydrophilic acrylamide (AAM) component could increase the VPTT of PNIPAM, which may be suitable for the application of photothermal-induced drug release.29 By now, a variety of PNIPAM-based nanogels have been reported and successfully used to improve drug delivery efficacy.28, 34-36 However, only very


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few studies have employed PNIPAM-based nanogels in the field of photothermal-induced drug release, because their pharmaceutical applications still suffer from extremely low drug loading, relatively rapid drug release and thus high premature leakage, low batch-to-batch reproducibility and difficulty in scalable morphology-controlled fabrication .8, 24, 37-38 Recently, the liposomes coated nanogels, featuring an inner polymeric hydrogels core and an outer liposomes shell, integrate the desirable features of both nanogels and liposomes in a single nanocarrier.39-44 The cross-linked hydrogels cores not only can markedly improve the mechanical stability, but also bring new functionality, e.g. rapid stimuli-responsiveness, to the liposomal formulations. Meanwhile, the liposomal shell not only can improve the biocompatibility and hemodynamic stability of nanogels, but also decrease premature drug leakage from nanogels.39-40, 45 Apparently, these beneficial features make the liposome-coated nanogels a promising platform for theranostic and pharmaceutical applications.40-41, 46-47 In this study, an in situ polymerization within liposome template was designed to prepare liposome coated poly(N-isopropylacrylamide-co-acrylamide) (P(NIPAM-co-AAM)) nanogels, which were used as a robust drug delivery platform to efficiently co-encapsulate and simultaneously co-deliver a NIR dye indocyanine green (ICG) and high amount of doxorubicin hydrochloride (DOX), denoted as DI-NGs@lipo. As shown in Scheme 1, after the formation of liposomes, the liposomes coated PNIPAM nanogels (NGs@lipo) can be obtained by initiating the intravesicular copolymerization of N-isopropylacrylamide (NIPAM), acrylamide (AAM) and N,N-methylene-bis-acrylamide (MBA as crosslinker) in the presence of 2,2'Azobis (2-methylpropionamide) dihydrochloride (V-50) as a widely used water-soluble initiator for free radical polymerization and ICG as well as ammonium sulfate. To prevent macroscopic hydrogel formation outside liposomes, TEMPO-PEG-TEMPO as a membraneimpermeable macromolecular inhibitor has been proven to be able to selectively and effectively inhibit the extravesicular polymerization.48-49 Subsequently, DI-NGs@lipo was obtained after


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efficiently encapsulating a large number of DOX was into the resultant nanogels by ammonium sulfate gradient method.16, 50-52 The obtained DI-NGs@lipo combined the desirable features of both PNIPAM nanogels and PEGylated liposomes. The PNIPAM nanogels as core bring mechanical stability and fast stimuli responsiveness to the final formulation, which are expected to be able to respond to the NIR irradiation-induced hyperthermia and thus achieve quick release of DOX in tumor cells. In addition, the PEGylated liposome as polymerization template not only can obtain NGs@lipo with controlled size, but also can improve their biocompatibility, hemodynamic stability and prevent premature drug leakage. The structure characters of DI-NGs@lipo, the stability and photothermal effect of the entrapped ICG, and light-triggered drug rapid release were evaluated. The results demonstrated the great potential of DI-NGs@lipo in chemo-photothermal combination therapy.

Scheme 1. Schematic illustration of DI-NGs@lipo prepared by in situ polymerization within liposome template (A) and NIR irradiation-induced hyperthermia triggered intracellular DOX release for synergistic chemo-photothermal therapy (B).


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EXPERIMENT SECTION Materials Distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG), egg phosphatidylcholine (egg PC) with the purity above 99.0% were obtained from Aladdin Biochemical Technology Company CO, Ltd (shanghai, China). Indocyanine green (ICG) was purchased from Sigma-Aldrich (St Louis, MO, U.S.A.). Doxorubicin hydrochloride (DOX) with the purity > 98.0% was purchased from Beijing Huafeng United Technology Co., Ltd. (Beijing, China). N-isopropylacrylamide (NIPAM) with the purity > 98.0%, acrylamide (AAM), ammonium sulfate, 2,2'-Azobis (2-methylpropionamide) dihydrochloride (V-50), cholesterol and N,N-methylene-bis-acrylamide (MBA) were purchased from TCI Chemicals Pvt. Ltd. (Shanghai, China). According to our previous study,48 TEMPO-PEG-TEMPO as a membrane-impermeable macromolecular inhibitor was synthesized by conjugating 4-amino2,2,6,6-tetramethylpiperidine-1-oxyl (4-aimno-TEMPO) and O,O'-Bis [2-(N-Succinimidylsuccinylamino)ethyl] polyethylene glycol 3000 in tetrahydrofuran (THF) with triethylamine as catalyst. 4' 6-diamidino-2-phenylindole (DAPI) and Lysotracker green and Live/Dead viability/cytotoxicity kit were obtained from Invitrogen Corp. (Carlsbad, CA, USA). Amicon ultra-4 Ultrafiltration tube with a molecular weight cutoff of 100 kDa was bought from Millipore (USA). All other chemicals reagents used were commercially available with analytical grade in this study. Preparation and Characterization of NGs@lipo, I-NGs@lipo and DI-NGs@lipo The liposomes as polymerization templates with size of about 100 nm composed of Egg PC, cholesterol and DSPE-PEG of 75:13:12 in a mass ratio were prepared by the hydration method. Briefly, a mixture (about 56 mg) of egg PC, cholesterol and DSPE-PEG was dissolved in chloroform and a transferred to a round-bottom flask. And the lipid film was formed after


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evaporating the solvents with a rotary evaporator at 80 rpm in a 35 °C water bath. The resulting lipid thin film was further dried overnight under vacuum to remove the residual solvent. To prepare drug-free liposomes coated nanogels (NGs@lipo), an in situ polymerization within liposome template was designed by using hydrogel precursor solution. Briefly, the resulting dried lipid film was rehydrated by sonication in an ice-water bath using 5 mL of hydrogel precursor solution including the calculated amount of NIPAM, AAM, V-50 and MBA with the corresponding feeding molar ratio of 89.2:9.6:0.4:0.8 at the monomer concentration of about 4.5 wt%. In order to acquire nanoscale and homogeneous liposomes, the abovementioned solution was sequentially extruded through 100 nm polycarbonate films for ten cycles using an Avanti® Mini-Extruder (Avanti Polar Lipids, Inc., Alabaster, AL, USA). The membrane-impermeable macromolecular inhibitor TEMPO-PEG-TEMPO (30mg) was added to the obtained liposome solution to prevent the polymerization of monomers outside of the liposomes vesicles. The polymerization within the liposome was initiated under nitrogen atmosphere at 30 °C for 2 h to obtain the lipid-coated nanogels (NGs@lipo). The unreacted monomers and TEMPO-PEG-TEMPO were removed through dialysis in 1000 mL of 0.9% NaCl solution for 12 h by changing the dialysis solution every 4 hours. To prepare ICG-loaded liposomes coated nanogels (I-NGs@lipo), an in situ polymerization within liposome template was used by using hydrogel precursor solution with ICG by the same procedure except that indocyanine green (ICG) (2 mg) was added into the same hydrogel precursor solution. To prepare DOX and ICG co-loaded liposomes coated nanogels (DI-NGs@lipo) by active loading, an in situ polymerization within liposome template was used by using hydrogel precursor solution with ICG and ammonium sulfate ((NH4)2SO4) by the same procedure except that indocyanine green (ICG) (2 mg) and 150 mM (NH4)2SO4 were added into the same hydrogel precursor solution. After removing the unencapsulated ICG, (NH4)2SO4, unreacted monomers and TEMPO-PEG-TEMPO, DOX solution of 6 mg/mL at a drug-to-lipid ratio of 1:8 (weight)


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was dropwise added to the above (NH4)2SO4-encapsulated I-NGs@lipo solution.50 And the solution was incubated at 30 °C for 30 min to allow more doxorubicin enter into nanogels through the ammonium sulfate gradient method. The obtained DI-NGs@lipo solution was stored at 4 °C for later experiments. To prepare DI-NGs@lipo by passive loading, the INGs@lipo prepared above was incubated in the DOX solution (6 mg/mL) for 10 h and then the DI-NGs@lipo was obtained after ultrafiltration using Amicon ultra centrifugal filter. DOX loaded liposomes were prepared according to a previous study.52 To determine the drug loading capacity and encapsulation efficiency, a sample was taken and ultra-filtrated using Amicon ultra centrifugal filter with a molecular weight cutoff of 100 kDa. The amounts of DOX in the filtrate was determined by UV–vis spectrophotometry (TU1810, Purkinje General Instrument Co, China) at 480 nm using a standard curve method. The amount of entrapped DOX into DI-NGs@lipo was calculated by subtracting the unloaded amount of drug from the total amount used for drug loading. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated from the following equations: weight of loaded drug ×100% weight of lipids

[1]

weight of loaded drug ×100% weight of drug in feed

[2]

DLC(%)= DLE(%)=

The hydrodynamic diameter, polydispersity index (PDI) and zeta-potential of NGs@lipo and DI-NGs@lipo were measured at 25 °C by dynamic light scattering (DLS) using a Zeta Sizer Nano series Nano-ZS (Malvern. U.K). The results were averaged over triplicate measurements. The morphologies of NGs@lipo and DI-NGs@lipo were observed using a JEM100CXII transmission electron microscope (TEM, JEOL, Japan) with 100KV acceleration voltage. UVvis spectrometer (TU-1810, Purkinje General Instrument Co, China) fitted with temperature and stirring controller was used to monitor the absorbtion of NGs@lipo at different temperatures at 330 nm. The incident light passes through the center of sample cell fitted with


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a thermometer. About 4 mL (1.0 mg/mL) NGs@lipo solution tuned by PBS at pH = 7.4 was added to a UV cell. The VPTT was defined when the absorbtion of the NGs@lipo solution sharply changed. The VPTT of NGs@lipo was also measured by a STA449F3 simultaneous thermal analyzer (STA, NETZSCH, Germany). The UV absorption spectrum of free DOX, free ICG, DOX-loaded DI-NGs@lipo by active and passive loading methods were measured by a UV–vis spectrometer. In Vitro Stability The in vitro stability of DI-NGs@lipo was tested in 5% bull serum albumin (BSA) in PBS by monitoring the hydrodynamic diameter at 37 °C for different incubation time periods on a dynamic light scattering (DLS) equipment. The fluorescence stability of ICG in DI-NGs@lipo was measured by FluoroMax-4 spectrofluorometer (Horiba Jobin Yvon, USA) at different time, with free ICG as a contrast. In Vitro Photothermal Effect A NIR laser (808 nm, MDL-N-808, Changchun New Industry Optoelectronic Technology Co., Ltd. China) was used in the experiments. PBS buffer solution, solution of free ICG, INGs@lipo and DI-NGs@lipo at the final ICG concentration of 25 µg/mL were put into at 1.5 mL centrifuge tubes, respectively, and irradiated by the 808 nm laser with a power density of 1.5 W/cm2 for 6 min. Real-time imaging was monitored and infrared thermographic maps were obtained by the infrared thermal imaging camera (FLIR A5, FLIR Systems, USA). The size of DI-NGs@lipo at different time points under the irradiation of 808 nm laser was characterized by dynamic light scattering (DLS). In Vitro Drug Release In vitro release of DOX from DI-NGs@lipo was determined by adding 1.5 mL of DINGs@lipo solution into a dialysis tube with a molecular weight cut-off of 8000Da (Slide-ALyzer, Thermo Scientific, USA). The dialysis tube was immersed into 10 mL of PBS at pH 7.4


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and shaken at different temperatures (37 °C and 42 °C) in thermostatic shaking bed (SHZ-82, Champion Instruments, Changzhou, China). The drug release triggered by NIR light were investigated according to the similar procedure except that the samples were irradiated for 5 min at 37 °C using NIR laser (808 nm, 1.5 W/cm2). At predetermined time intervals, 5.0 mL of the release medium was withdrawn followed by replacing with the equal volume of fresh PBS to continue further study. The release amount of DOX was quantified by UV-Vis spectrophotometry. The procedure about drug release from DOX-loaded liposomes was the same as above. All the results were the mean of three test runs, and all data were presented as the mean ± SD. The cumulative drug release percentage was calculated using the following equation: Vt ∑n-1 1 Ci +V0 Cn Er (%) = ×100% mDOX

[3]

Where Er is the cumulative release amount, the mDOX represents the amounts of DOX in the DI-NGs@lipo of dialysis bag, Ve is the volume of replaced medium (5 mL) V0 is the total volume of the released media (V0=10 mL), Ci and Cn represent the concentration of DOX in the sample. In Vitro Cytotoxicity MTT assay was utilized in appraising the cellular toxicity of the designed formulations. 4T1 cells (5000 per well) were seeded into a 96-well plate cultured in 100 µL of complete DMEM and incubated at 37 °C with 5% CO2. After 24 h, the supernatants were discarded, and the cells were washed twice with PBS (pH 7.4). Firstly, we studied the cytotoxicity of NGs@lipo and I-NGs@lipo at a lipid concentration of 0.5, 50, 100, 250, 500, 1000 µg/mL. And Free DOX, DI-NGs@lipo were also put in the wells for 24 h at a DOX concentration of 0.125, 1.25, 2.5, 5, 10, 20, 40 µg/mL, respectively. In different control groups, the concentrations of DOX and ICG were 10 µg/mL and 8.5 µg/mL, separately. The presence of laser of 808 nm was 1.5 W/cm2 NIR irradiation for 5 min. The culture medium PBS was put in the wells for control groups.


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Then 20 μL of MTT solution (5 mg/mL) was added. The cells were incubated for 4 h at 37 °C, and the medium was replaced by 150 µL of DMSO to dissolve the resulting purple crystals. The absorbtion was recorded at 570 nm using a microplate reader (Thermo Scientific Varioskan Flash, Waltham, MA, USA). The experiments were conducted in triple and the results were presented as the average ±standard deviation. Cell viability rate (%) was calculated as the following Eq. (4).

Cell Viability (%) =

I0 -I1 ×100% I0

[4]

where I0 was the absorbance of the cells incubated with the culture medium, and I1 was the absorbance of the cells incubated with different formulations. In Vitro Cellular Uptake The cellular uptake and intracellular drug release were evaluated on 4T1 murine breast cancer cells lines. Confocal laser scanning microscope (CLSM) was used to qualitatively observe the cellular uptake of free DOX and DI-NGs@lipo by 4T1 cells. Intracellular release of DOX from DI-NGs@lipo was followed with fluorescence microscope using 4T1 cells. The cells were cultured on specified wells at 5000 cells per well, incubated at 37 °C for 24 h, then culture medium was replaced by fresh medium containing the free DOX (10 µg/mL) or DI-NGs@lipo (including 10 µg/mL DOX and 8.5 µg/mL ICG) at pH 7.4. After incubation for 2 h at 37 °C, the center region in the 12 well plate of DI-NGs@lipo was or not exposed to 808 nm wavelength laser irradiation (1.5 W/cm2) for 5 min. In order to observe the cell uptake at 42 °C, some cells treated with DI-NGs@lipo were incubated at 42 °C for 30 min. Continuing to incubation for 2 h, the cells were washed with PBS (10 mM, pH 7.4). Thereafter, the cell nuclei were stained with DAPI (blue) and the lysosomes were stained with Lyso-Tracker Deep red (green), then these cells were observed by CLSM (Olympus, Tokyo, Japan). The fluorescence signal was imaged at λEx (485 nm) for DOX.


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Hemolysis Assay Red blood cells (RBCs) were isolated from 600 µL of fresh whole blood by centrifuging at 2000 rpm for 5 min and washed five times with PBS pH 7.4. Then the RBCs were re-suspended in 6 mL sterile PBS. 0.2 mL of the RBCs solution was added to 0.8 mL NGs@lipo solution in PBS at a concentration of 100 µg/mL. The positive reference (100% lysis) was a blood/deionized water mixture, and the negative reference (0% lysis) was a blood/PBS mixture. Three parallel samples were made in each group, incubated at 37 °C for 4 h, and the mixture was centrifuged at 2000 rpm for 5 min. The optical density (OD) of the supernatant was read at 545 nm using a UV-vis spectrophotometer. The hemolytic ratios of the samples were calculated as follows: Hemolytic ratio(%)=

sample absorbance -negative control ×100% positive control -negative control


[5]

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RESULTS AND DISCUSSION Synthesis and Characterization of NGs@lipo Liposomes coated nanogels, consisted of a hydrogel core and a PEGylated liposomal shell, represent an intriguing nanoplatform for drug delivery.40, 43-44 In this study, the liposomes coated PNIPAM nanogels, i.e. NGs@lipo, were prepared through in situ liposome-template polymerization. The liposomal vesicles as a nanoreactor was used to encapsulate the hydrogel formation mixture including monomers, crosslinkers, and initiators with or without ICG and ammonium sulfate. The size of drug-free NGs@lipo was simply controlled by extrusion through a nanopore polycarbonate filter with 100nm size. After the formation of liposomes by sonication in an ice-water bath and nanopore-extrusion, the polymerization of NIPAM and AAM in the liposomes was initiated under nitrogen atmosphere at 30 °C for 2 h in the presence of macromolecular inhibitor (TEMPO-PEG-TEMPO). The TEMPO-PEG-TEMPO as a membrane-impermeable macromolecular inhibitor has been proven to be able to selectively inhibit the extravesicular polymerization reaction while maintaining the intravesicular reaction alive.48 As shown in Figure S1, TEMPO-PEG-TEMPO was also found to be able to completely inhibit the polymerization of hydrogel forming solution at 30 °C for 3 h or even longer time. The DOX drug was loaded into the NGs@lipo by ammonium sulfate gradient active loading method. The drug-free NGs@lipo and drug-loaded DI-NGs@lipo derived from in situ liposome-template polymerization were visualized with TEM and characterized by DLS, as shown in Figure 1A and 1B, respectively. Both the NGs@lipo and DI-NGs@lipo in the dry state generally exhibit spherical morphology with good monodispersity. The particles sizes of both NGs@lipo and DI-NGs@lipo in TEM images range from about 103 ±10 nm. Figure S2 shows the TEM images of blank liposomes. The circular structures reflect the presence of hollow vesicles. Compared with the TEM images of liposomes, the TEM images of NGs@lipo and DI-NGs@lipo are more spherical and solid, which may confirm the formation of nanogels


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in the liposomal intravesicular space. The TEM photo of DI-NGs@lipo (Figure 1B) exhibited a more compact and spherical morphology, which may be due to the presence of complexes of DOX and ammonium sulfate. The double-layered lipid shell outside the nanogels were not observed, which may be due to the collapse of polymeric nanogels core during the process of TEM sample preparation. The hydrodynamic sizes and thermo-responsiveness of NGs@lipo were confirmed by monitoring the size change of incubation at different temperature using dynamic light scattering technique, as shown in Figure 1C. The hydrodynamic diameter of NGs@lipo was measured at 106 ± 7.3 nm at 25 °C, but decreased to 72 ± 3.6 nm when temperature increased to 42 °C. The particle dispersion index (PDI) changed from 0.191 to 0.090. Therefore, there was a decrease of size and volume of NGs@lipo when temperature increased from 25 °C to 42 °C. The zeta potential of NGs@lipo and DI-NGs@lipo in diluted PBS solution at pH 7.4 was tested and shown in Figure 1D. The zeta potential of blank NGs@lipo was -4.23 ±0.3 mV, which was close to neutral. The zeta potential of DI-NGs@lipo was -10.6 ±0.65 mV. It can be found that the drug loading process has little effect on the zeta potential of NGs@lipo. To further confirm the temperature sensitivity of NGs@lipo, we measured the UV absorbtion of NGs@lipo at different temperature, as shown in Figure 1E. It can be observed that the absorbtion sharply increased at near 40 °C, which was contributed to that the hydrophilic PNIPAM chains transferred into hydrophobic chains and thus the NGs@lipo dispersion solution changed from transparent to turbid at this temperature. In addition, simultaneous thermal analyzer (STA) was employed to study the thermo-sensitivity of NGs@lipo in aqueous solution. As shown in Figure 1F, it was apparent that NGs@lipo presented thermo-sensitivity with VPTT about 40 °C due to phase transition of the P(NIPAMco-AAM) nanogels. Consequently, the results demonstrated that the VPTT of NGs@lipo was close to 40 °C, which was higher than human body temperature and thus provided an effective approach to control drug release by temperature mediation.


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Figure 1. Characterizations of NGs@lipo and DI-NGs@lipo. TEM image of NGs@lipo (A) and DI-NGs@lipo (B); Hydrodynamic diameter and thermo-responsiveness of NGs@lipo (C); Zeta potentials of NGs@lipo and DI-NGs@lipo at pH 7.4 (D); Temperature dependence of UV absorbtion at 330nm of NGs@lipo in aqueous solutions (E); Simultaneous thermal analyzer curve of NGs@lipo (scanning rate 2 °C /min, aluminium pans) (F). In Vitro Stability of DI-NGs@lipo Stability is one of the critical factors in ensuring long shelf-life, high safety and clinical efficacy of drug formulations, especially for lipid-based colloidal suspensions.53 In our study, a size stability assay of DI-NGs@lipo was evaluated in PBS containing 5% BSA for 5 days at 37 °C (Figure 2A). The particle size of DI-NGs@lipo almost remained the original size without precipitation or phase separation in PBS containing 5% BSA for 5 days. The polydispersity index (PDI) was between 0.17-0.20. The good stability of DI-NGs@lipo can be ascribed to the excellent steric stabilization of PEGylated liposome shell to prevent colloidal agglomeration, and the inside three-dimensional cross-linking network to stabilize liposome. ICG was proved to an effective photosensitive reagent by laser-excitation heat for photothermal therapy.54 However, the application of the free ICG is limited by its numerous defects,


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including poor aqueous stability, concentration-dependent aggregation, rapid elimination from the body.55 In this study, we also investigated the fluorescence stability of ICG in DI-NGs@lipo. As shown in Figure 2B, the fluorescence measurement illustrated that fluorescence intensity of ICG in DI-NGs@lipo remained nearly 97% for 1 days and above 85% after 7 days, while fluorescence intensity of free ICG decreased to 66% of initially intensity after 1 day and degraded to 2% after 7 days. These results suggested that fluorescence stability of ICG was significantly improved by encapsulation of NGs@lipo. The higher fluorescence stability of ICG in DI-NGs@lipo may be due to the ICG entrapped in nanogels was isolated form the surrounding environment.55-56 The Figure 2C revealed that the obtained DI-NGs@lipo showed a strong DOX absorbtion at 485 nm and ICG absorbtion at 800 nm, which certificated that DOX and ICG were successfully loaded. The spectra also showed that the content of DOX by active loading was higher than the passive loading. A large number of studies have confirmed that the ammonium sulfate gradient active loading method can significantly increase the DOX loading content in liposomal nanocarriers.16, 50-52, 57 The drug loading content (DLC) of DINGs@lipo by passive loading and active loading were about 2.5‰ and 6.6%, respectively, which was calculated on the basis of lipids weight. The drug loading efficiency (DLE) were about 29.6‰ and 80.2%, respectively. The apparent photographs of NGs@lipo before and after ICG and DOX loading were shown in the Figure 2D. The significant changes of color of NGs@lipo further indicated that the ICG and DOX were encapsulated into the NGs@lipo. Moreover, as shown in Figure 2E and 2F, it can be found that DOX and ICG presented independent fluorescence and concentration-dependent fluorescence intensity when they were co-loaded in DI-NGs@lipo, which may be advantageous to track in vitro and in vivo.


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Figure 2. Physicochemical properties of DI-NGs@lipo. Size stability of DI-NGs@lipo in PBS containing 5% BSA at 37 °C (A); Fluorescence stability of free ICG and ICG in DI-NGs@lipo (B); The UV-vis spectra of free ICG, free DOX, DOX-loaded DI-NGs@lipo by active loading and passive loading methods (C); Photographs of NGs@lipo (I), I-NGs@lipo (II) and DINGs@lipo (III) (D); Fluorescence emission spectra of DI-NGs@lipo at different concentration. Excitation wavelength at 480 nm related to DOX (E) and excitation wavelength at 780 nm related to ICG (F). In Vitro Photothermal Effects To investigate the photothermal property, DI-NGs@lipo solution was exposed to an NIR laser (808 nm, 1.5 W/cm2) for 6 min and the temperature was simultaneously recorded per 30 s using an infrared thermal imaging camera. As shown in Figure 3A and B, only 1.5 °C increase on temperature was observed for PBS after 6 min irradiation. By contrast, obvious temperature increase was found for I-NGs@lipo and DI-NGs@lipo under NIR irradiation. The solution temperatures of both I-NGs@lipo and DI-NGs@lipo were increased to about 52 °C after 6 min irradiation, which were appreciably higher than that of free ICG solution (about 49 °C). The temperature increase of free ICG was slightly lower than that of I-NGs@lipo and


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DI-NGs@lipo. This may be due to the improved stability of ICG in NGs@lipo, as demonstrated above. Moreover, the ICG encapsulation in the I-NGs@lipo or DI-NGs@lipo possessed a highly condensed concentration over that of free ICG, and the enclosure of NGs@lipo entrapped the photothermal radiation, resulting in the lower heat dissipation and higher energy efficiency in the I-NGs@lipo or DI-NGs@lipo after laser irradiation.55-56 Considering the thermo-sensitivity of NGs@lipo, the size change of DI-NGs@lipo under NIR irradiation was also investigated. As shown in Figure 3C, the hydrodynamic size of DINGs@lipo significantly decreased from 108 to 74 nm under 808 nm irradiation for 6 min. The significant decrease in DI-NGs@lipo size can be ascribed to the shrinkage of nanogels from the hydrophilic-to-hydrophobic transition of PNIPAM chains. These results not only further confirmed the thermo-sensitivity of DI-NGs@lipo, but also indicated the great potential of DINGs@lipo in NIR-triggered drug release by the photothermal-induced nanostructural transition.

Figure 3. Photothermal effect of DI-NGs@lipo under NIR irradiation of 808 nm laser (1.5 W/cm2, 6 min). Peak temperature maps of PBS, free ICG, I-NGs@lipo and DI-NGs@lipo


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recorded with an infrared thermal imaging camera (A); Temperature profiles of PBS, free ICG, I-NGs@lipo and DI-NGs@lipo dispersions (B); Hydrodynamic size of DI-NGs@lipo at different time points under NIR irradiation (C). In Vitro Drug Release To demonstrate that hyperthermia-trigged drug release from DI-NGs@lipo, the drug release from the DOX-loaded liposomes and DI-NGs@lipo were comparatively investigated in 37 °C and 42 °C. The DOX release results from liposome were shown in Figure 4A. There was almost no difference in the release of DOX from the DOX-loaded liposomes at 37 °C and 42 °C, with a cumulative release of 15% within 8 h. However, the DI-NGs@lipo exhibited a temperature-dependent release profile (Figure 4B). The DOX release from DI-NGs@lipo at 37 °C was very low. Only about 11% of the total encapsulated DOX was released from DINGs@lipo incubated in the release medium of PBS within 8h. When DI-NGs@lipo was incubated with PBS at 42 °C, the release of DOX was significantly accelerated, reaching a cumulative release of about 12.7% of total encapsulated drug in 1 h and 34.2% in 8 h. This may be due to that the cross-linked nanogels cores in liposome at 37 °C could efficiently store drugs and thus delay drug release. The temperature increased to 42 °C, which was higher than VPTT of the DI-NGs@lipo (40 °C), and thus the collapse and shrinkage of nanogels in liposomes will occur due to the hydrophilic-to-hydrophobic transition of PNIPAM chains. The collapse and shrinkage of nanogels core not only can break the liposomes shells, facilitating the drug release, but also can squeeze out the loaded drug, thus leading to an increase in drug release under 42 °C. We further investigated the release of DOX from DI-NGs@lipo under NIR irradiation (808 nm, 1.5 W/cm2, 5 min), as shown in Figure 4C. In a short time of 30 min, nearly no DOX release from DI-NGs@lipo at 37 °C without NIR irradiation was observed. However, after NIR irradiation of 5 min, drug release was accelerated significantly, and the total release of DOX increased to 22.3% at 30 min, indicating that NIR laser can trigger drug


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release from DI-NGs@lipo eﬀectively. It was well known that the intracellular-triggered drug release is not only an efficient strategy to increase therapy efficacy, but also an important method to reduce the side-effects and suppress drug resistance. The DI-NGs@lipo presented a temperature-dependent drug release profile, which should be very beneficial for cancer treatment. Most of the loaded DOX will remain in DI-NGs@lipo for a considerable length of time when the DI-NGs@lipo stay in the extracellular space under normal physiological conditions. However, the encapsulated DOX will be quickly released when the DI-NGs@lipo is taken up by the tumor cells under NIR irradiation due to hyperthermia induced nanostructural transition. Therefore, the efficacy of therapy can be significantly improved due to high and sustained local drug concentrations in the tumor cells.

Figure 4. Drug release profiles of DOX-loaded liposomes and DI-NGs@lipo. Cumulative release of DOX from DOX-loaded liposomes (A) and DI-NGs@lipo (B) at 37 °C and 42 °C; Cumulative release of DOX from DI-NGs@lipo with and without NIR irradiation for 5 min (C). In Vitro Cell Uptake Efficient cellular uptake plays a key prerequisite for drug treatment efficacy. To demonstrate whether NIR light-induced hyperthermia affected the internalization, the cellular uptake of free DOX and DI-NGs@lipo in 4T1 cells following with or without NIR irradiation was determined. The cellular uptake at 42 °C was performed as a control. The subcellular localization of DOX was investigated by confocal laser scanning microscopy (CLSM). As shown in Figure 5, free


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DOX mainly distributed in cell nuclei in 4T1 cells after 4 h incubation, indicating the entering of free DOX by a passive diffusion mechanism, which corresponded with the previous report.5859

In addition, the remarkably brighter red fluorescence of DOX and most of DOX distributed

in nuclei of the 4T1 cells treated by DI-NGs@lipo + laser were observed. However, the 4T1 cells treated only by DI-NGs@lipo without laser showed weaker red fluorescence. The low DOX distribution in the nucleus of DI-NGs@lipo was attributed to the difficult release of the DOX tightly wrapped by nanogels. In comparison, higher DOX distribution was observed at nucleus in the cells treated by DI-NGs@lipo + laser, which revealed that the hyperthermia caused by laser make the collapse of nanogels and facilitate the DOX release. In addition, the similar phenomenon to the cells treated by DI-NGs@lipo at 42 °C was observed, further verifying temperature-dependent release profile of DI-NGs@lipo. Consequently, the results demonstrated that DI-NGs@lipo with temperature-responsive nanogels cores had a high potential as the carrier for intracellular drug delivery.


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Figure 5. CLSM images of 4T1 cells incubated with free DOX, DI-NGs@lipo, DI-NGs@lipo at 42 °C for 30 min, DI-NGs@lipo with NIR irradiation for 5 min, respectively. Nuclei was stained in blue, DOX was in red (Scale bar: 10 μm.) In Vitro Cytotoxicity and Biocompatibility of NGs@lipo, I-NGs@lipo and DI-NGs@lipo The cytotoxicities of drug-free NGs@lipo and I-NGs@lipo were evaluated for the cell compatibility of the drug delivery system. The cell viability kept 90% even after treatment with DOX-free carriers at a high concentration of 1000 µg/mL (Figure 6A), manifesting that NGs@lipo and I-NGs@lipo were safe and biocompatibility for drug delivery. Meanwhile, the free DOX and DI-NGs@lipo (contain different concentrations of DOX) exhibited similar toxicity to the 4T1 cell lines after 24 h of incubation without irradiation (Figure 6B). Compared to cells only treated with I-NGs@lipo containing 8.5 µg/mL ICG with viability of 97.7%, an obviously enhanced therapeutic effect was observed for 4T1 cell treated with I-NGs@lipo


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under NIR irradiation, which can be ascribed to NIR irradiation-induced heat toxicity. On the other hand, in comparison with the groups of I-NGs@lipo and I-NGs@lipo + laser, the DINGs@lipo under NIR irradiation had obvious anticancer activity (Figure 6C). Furthermore, it can be seen that the cell treated with the same concentration of free DOX drug (10 µg/mL) still survived 50%. The cells treated with NGs@lipo under NIR irradiation only maintained about 24% cell viability, which was two times as low as that of the cells treated with DI-NGs@lipo without NIR irradiation and free DOX. These results indicated the synergetic effect of NGs@lipo under NIR irradiation, which was caused by the combination of laser-induced hyperthermia effect and the NIR-induced hyperthermia triggered DOX release. In addition, hyperthermia enhanced cells permeability and increased the sensitivity of tumor cells to chemotherapy drugs.19-20, 23 Conclusively, DI-NGs@lipo designed herein as nanoplatform for co-delivery of DOX and ICG exhibited very promising properties, which can synergistically improve the tumor cell killing efficiency and thus has great potential in chemo-photothermal therapy. Hemocompatibility is an important criterion for biocompatibility of nanoparticles drug delivery systems. The biocompatibility of NGs@lipo was examined by hemolysis assay using PBS and deionized water as negative and positive control, respectively, as shown in Figure 6D. After incubating the NGs@lipo with RBCs in PBS for 4 h at 37 °C at a concentration of 100 µg/mL, the NGs@lipo dispersions did not exhibit any marked hemolytic activities in the RBCs. The hemolytic percentage of NGs@lipo was lower than 2% at the tested concentration, exhibiting a very high hemocompatibility. These results indicated that the PEGylated liposomes-coated nanogels have excellent hemocompatibility, which may enable them to be safe for various biomedical applications.


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Figure 6. In vitro cytotoxicity and biocompatibility. Cell viability of 4T1 cells treated with NGs@lipo and I-NGs@lipo (A) and treated with free DOX and DI-NGs@lipo containing various DOX concentrations (B); Cell viability of 4T1 cells treated with NGs@lipo, NIR laser, Free DOX, I-NGs@lipo and DI-NGs@lipo with or without NIR irradiation. Each bar represents the mean ±SD. of six experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 (C). Photograph of hemolysis of erythrocytes incubated with NGs@lipo at concentration of 100 μg/mL for 4 h and hemolytic ratio measured by UV-vis spectrophotometry at 545 nm. Deionized water and PBS are used as positive and negative control, respectively (D).


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CONCLUSIONS In this study, an in situ liposome-template polymerization was used to prepare liposomes coated poly(N-isopropylacrymide-co-acrylamide)(P(NIPAM-co-AAM)) nanogels, which can efficiently co-encapsulate ICG and DOX by an ammonium sulfate gradient active method. The DOX/ICG coloaded hybrid nanogels with uniform and controlled nanoscale size (denoted as DI-NGs@lipo) integrated the desirable functions of PEGylated liposomes and thermosensitive nanogels. The PEGylated liposomes shell provided excellent storage stability, hemodynamic stability and fluorescence stability. Meanwhile, the thermosensitive nanogels cores endowed DI-NGs@lipo with VPTT at about 40 °C, allowing for NIR-induced hyperthermia controlled transformation and thus quick DOX release. As a result, the concurrent chemo-photothermal therapy was achieved by NIR light, as NIR not only induced hyperthermia but also simultaneously triggered quick DOX release by thermo-triggered nanogels transformation. Notedly, the DI-NGs@lipo combined desirable multifunctionality for chemo-photothermal therapies and can synergistically improve the cancer cell killing efficiency, demonstrating great potential in photoinduced cancer therapy.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publication website. Additional details including inhibition effect of TEMPO-PEG-TEMPO on polymerization of hydrogel forming solution; TEM image of liposomes (PDF). AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by National Natural Science Foundation of China (31470925, 31671021 and 31470963) and Tianjin Research Program of Application Foundation and Advanced Technology (15JCQNJC03000).


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For Table of Contents Use only：

DOX/ICG

Coencapsulated

Thermosensitive

Nanogels

Liposome for

Coated

NIR-triggered

Simultaneous Drug Release and Photothermal Effect Lixia Yu,† Anjie Dong, †‡ Ruiwei Guo,† Muyang Yang, † Liandong Deng, † and Jianhua Zhang*, †,§


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ICG Coencapsulated Liposome Coated ... - ACS Publications

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