Thermo-sensitive Lipid Bilayer Coated Mesoporous Carbon

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Biological and Medical Applications of Materials and Interfaces

Thermo-sensitive Lipid Bilayer Coated Mesoporous Carbon Nanoparticles for Synergistic Thermo-chemotherapy of Tumor Xian Li, Xiudan Wang, Luping Sha, Da Wang, Wei Shi, Qinfu Zhao, and Siling Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 25, 2018

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ACS Applied Materials & Interfaces

Thermo-sensitive Lipid Bilayer Coated Mesoporous Carbon Nanoparticles for Synergistic Thermo-chemotherapy of Tumor Xian Lia, Xiudan Wanga, Luping Shaa, Da Wanga, Wei Shib, Qinfu Zhaoa,*, and Siling Wanga,* a School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, China b Shenyang No.2 High School, 6 Wuai Street, Shenyang, China

Corresponding authors Qinfu Zhao Phone: +86 024 23986346 E-mail: [email protected]

Siling Wang Phone: +86 024 43520555 E-mail: [email protected]

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Abstract Thermo-chemotherapy exhibits a synergistic therapeutic efficiency for cancer, and the sensitivity of cancer cells to chemical drugs could be increased to a large extent at elevated temperature. In this work, a biocompatible nanocomposite TSMCN was prepared through covering a liposome on mesoporous carbon nanoparticles (MCN). The TSMCN had good photothermal efficiency and photostability. The DOX loaded TSMCN (DOX/TSMCN) showed a slower release than DOX loaded MCN-COOH (DOX/MCN-COOH) both in simulated tumor environment and physiological environment. And release curves of DOX/TSMCN exposed to NIR laser exhibited the fast release property. The CLSM results illustrated that cellular uptake of DOX for DOX/TSMCN can be enhanced by NIR laser. The temperature of the tumor site reached up to 51.9oC within 3 min after exposure to laser at 1.25 W cm-2 power density, which is above the phase transition temperature (Tm) of liposome (40.7oC). The biodistribution of DOX in vivo indicated that NIR laser can prolong retardation time of DOX in tumor site. The results of both MTT and antitumor efficiency elucidated that the DOX/TSMCN under NIR irradiation had a synergistic therapeutic effect for cancer. Thus the TSMCN could be explored as a powerful nanoplatform which shows great prospect in thermo-chemotherapy of tumor therapy.

Keywords: mesoporous carbon; thermo-sensitive; liposome; photothermal therapy; chemotherapy; synergistic


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Introduction Cancer has become the leading killer threatening human health around the world, which overwhelmed other types of diseases1. Chemotherapy, a common clinical cancer therapy strategy, sometimes may not make the tumors ablated completely at a low safe dosage and has systemic side effects due to lack of specificity for tumor. Besides, many patients undergoing chemotherapy could develop multidrug resistance and eventually caused poor treatment results2. Nowadays, phototherapy, as a non-invasive therapeutic modality mediated by light, has recently elicited widespread interest thanks to its particular superiorities such as local controllability, minimal invasiveness and high efficiency3-5. Photothermal therapy (PTT) is a major category of phototherapy, in which the photothermal agents can convert the absorbed near infrared (NIR) laser (650-900 nm) energy into heat to kill the tumor6-9. Recently, it is reported that the cellular uptake of anti-cancer drug could be enhanced by the mild photothermal heating effect.10 The thermo-chemotherapy, combined photothermal and chemotherapy, was proved to be a useful strategy for cancer treatment in a synergistic manner11-13. The elevated temperature can facilitate the release of chemotherapeutic drugs in tumor site, increase the sensitivity of tumor to chemical drugs and overcome the chemotherapy resistance14, thus producing the synergistic therapeutic effects. The most important issue of thermo-chemotherapy is to design a multifunctional drug delivery system with good photothermal conversion efficiency as well as the high payload for chemical drugs. Among all photothermal agents such as graphene15-16, gold nanomaterials17-18,



conjugated polymers19-20 and a few other types21, mesoporous carbon nanoparticles (MCN) exhibit great potential in thermo-chemotherapy. To begin with, MCN have wide absorption in the wavelength ranging from 650 to 900 nm and are suitable to be used as photothermal agents. In addition, MCN have the easily functionalized property and high pore volume for drug loading. But the application of naked MCN in cancer treatment is far from enough, since the MCN can be recognized by reticulo endothelial systems (RES) as foreign invaders and cleared rapidly from blood circulation. Additionally, the aggregation and hemolysis properties of MCN under physiological environment will inevitably restrict their biomedical applications22. Recently, the lipid bilayer coating technique has been proved to be a promising strategy to prolong blood retention and avoid nonspecific macrophage uptake of the nanoparticles. Zhang et al. had successfully constructed a liposome coated mesoporous silica for co-delivering of paclitaxel and gemcitabine, exhibiting a synergistic therapeutic efficiency for pancreatic cancer23. Han et al. also introduced the application of lipid bilayer coated MSNs for intravenous drug delivery, in which the lipid bilayer improved the dispersion stability, biocompatibility and cell uptake efficiency of the nanoparticles24. So far, the researches about liposome coated MCN for drug delivery have rarely been reported. Zhang et al. prepared the lipid bilayer coated MCN for improving oral administration of the insoluble drug25. And Mandlmeier et al. synthesized lipid bilayer coated bimodal MCN for controlling the release of calcein26. However, as is known to us, the study of lipid bilayer coated MCN acted as photothermal agents for PTT has not been reported yet.


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As a consequence, considering the good heat generating capacity of MCN, herein, we subtly exploited and fabricated a novel nanocomposite (TSMCN) by introducing a biocompatible liposome on the surface of the MCN. The liposome consisting of DSPE-PEG2000, MSPC and DPPC can be a preferred alternative since it entered clinical trials (ThermoDox®)27. The transformation temperature (Tm) of liposome approximates to the temperature of tumor ablation (45oC)1 and the elevated temperature induced by PTT can accelerate the drug release, thus generating a synergistic therapeutic effect on cancer. In detail, the liposome was introduced to coat the surface of the MCN by co-incubation the MCN and liposome at a temperature above the Tm of liposome. The liposome was first adhered to the surface of the MCN, then gradually deformed, finally ruptured and spread, forming a continuous coverage on MCN. After confirming the successful construction of TSMCN, the photothermal effect was investigated. And then the influences of NIR laser and liposome on the drug release property of DOX were discussed. Moreover, the effects of liposome coating and NIR irradiation on the drug biodistribution were elucidated. The synergistic therapeutic efficiency of DOX/TSMCN was fully demonstrated and presented in Scheme 1.



Scheme 1 (A) Loading procedure and NIR-promoted DOX release from DOX/TSMCN. (B) The combined photothermal therapy and chemotherapy for tumor. 2. Experimental Section 2.1 Chemistry Tetraethoxysilane (TEOS), resorcinol, sulfuric acid (98%), hexadecyl trimethyl ammonium chloride (CTAC, ≥99%), formaldehyde solution and ammonium persulfate trypsin and MTT were all provided by Aladdin Chemical Inc. (Shanghai, China). The DSPE-PEG2000, MSPC and DPPC were all offered by A.V.T (Shang Hai, China). Fetal bovine serum (FBS), RPMI 1640 and fluorescent Hoechst 33258, propidium iodide (PI), anticancer drug doxorubicin hydrochloride (DOX) and calcein AM were all attained from Dalian Meilune Biotech Com. Ltd. (Dalian, China). All analytical reagents were not purified.


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2.2 Preparation of MCN and MCN-COOH MCN were fabricated according to the preceding method with some modification28. Typically, 25 wt% CTAC solution was mixed with a solution that contains 4 mL absolute ethanol, 19 mL deionized water and 0.1 mL ammonia water (25-28 wt%). And these substances were mixed well via stir for half an hour at ambient temperature. Then 0.36 mL TEOS were dropwise added into the above reaction solution and followed by adding 0.28 mL formaldehyde solution, further stirring for one day at 30oC. The as-prepared nanoparticles were centrifuged and isolated for further use. To obtain the C-Si compounds, the above solid powder were calcinated at 700oC for 2 h with the heat-up rate of 2oC per min under the environment of nitrogen. The final products (MCN) were obtained by soaking the above products in 10 wt% hydrogen fluoride (HF) solution for one day to erode the silica embed in the carbon. The carboxylated MCN (denoted as MCN-COOH) were fabricated according to the following steps. Briefly speaking, 0.1 g MCN were added to the mixture of 6 mL ammonium peroxodisulfate (APS) solution (114 mg/mL) and 639 µL concentrated sulfuric acid and reacted at 60oC for 3 h via reflux operation. The reaction products were attained after centrifugation and rinsed for three times using deionized water. 2.3 Preparation of TSMCN The liposomes were fabricated successfully according to the reported literature29. To begin with, the mixture including DSPE-PEG2000/MSPC/DPPC (10:5:63 by mass) were dissolved in a certain amount of chloroform. Next, the above mixture was put in



a boiling flask with round bottom and rotated for 20 min at 60oC to form lipid membrane by rotary evaporation. Then, the lipid membrane was hydrated at concentration of 10 mg/mL using pH 7.4 PBS at 60oC and the as-prepared liposomes with uniform size were obtained through ultrasonication and extrusion and stored at 4oC for no more than a week for further experiment. The TSMCN were fabricated by mixing 0.02 g MCN carriers with distilled water firstly. Next, 2 mL liposomes as prepared in section 2.3 were put into the above MCN suspensions. The above mixed suspensions were heated to 60oC and immediately vortexed for 3 min and were sat for 1 h at 60oC and thus MCN could be fused by the liposome completely. The TSMCN were obtained by centrifugation to remove the residual substance in reaction mixture. Finally, the reaction product was re-dispersed in PBS buffer for future use. 2.4 Loading procedure and NIR-promoted drug release in vitro The DOX loading was performed before the lipid bilayer covering. In detail, 20 mg MCN-COOH nanoparticles and 20 mg DOX were dispersed in 4 mL distilled water and stirred overnight to make the MCN-COOH fully absorb the drug. The DOX loaded TSMCN (denoted as DOX/TSMCN) was obtained by centrifuging and then fusing with lipid vesicles according to the procedure as depicted in Section 2.3. The drug loading efficiency was calculated using ultraviolet visible absorption method (λ=480 nm) followed by the equation:

LL% =

m1 − m0 m2 + m1 − m0

The m1, m2 and m0 represent the total mass of drug put in the reaction system, the


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total mass of TSMCN put in the reaction system and the mass of DOX not loaded in the supernatant, respectively. In order to make our hypothesis that DOX-loaded DOX/TSMCN could achieve controlled drug leakage efficiently more convincing, the drug release experiment was conducted as follows. Typically, samples of 1 mg DOX/MCN-COOH and DOX/TSMCN were equally dispersed in 3 mL various release media (pH 5.0 and pH 7.4 PBS)30 shaked at 120 rpm at 37 °C. At scheduled time, 1 mL volume of release medium was extracted and poured back to the release containers after determination at wavelength of 480 nm. Next, to further testify the DOX/TSMCN could control drug release in response to the temperature elevation efficiently, the NIR-induced drug release experiment was performed. The method was mostly similar to the above process. Differently, at specific time points (1 h and 6 h), the NIR laser was employed to illuminate the release media at 3.75 W cm-2 photo density for 5 min. 2.5 Photothermal properties of the TSMCN The ability of photothermal generators of TSMCN was explored by detecting the temperature changes under the NIR laser equipment with 808 nm wavelength (Changchun New Industries Optoelectronics Tech Co., Ltd., China) and a thermal infrared imager (LaserSight, Optris, Germany). In brief, the TSMCN suspensions containing different concentrations (5-100 µg mL−1) were illuminated by the NIR laser at a photo density of 2.5 W cm-2 for 3 min. To further study the effect of power density on photothermal heating, 0.1 mg mL−1 TSMCN suspensions were exposed to NIR laser by serial power densities (0.625-5 W cm-2) and the heating curves were



recorded by a thermal sensor. Pure water was set as a negative group with the same process. 2.6 Cellular photothermal effects induced by NIR laser The 4T1 cell line was cultured on 6-well cell culture plates with a concentration of 2.0 × 105 cells/well and incubated using RPMI-1640 culturing medium with 10% (v/v) FBS and 1% (v/v) streptomycin-penicillin for a day. Subsequently, in order to assess the cellular photothermal effects, the 4T1 cells were incubated with the TSMCN and the unmodified MCN-COOH at concentrations of 50 and 100 µg mL−1. After 2 h co-incubation, the cells were collected via removing the culturing medium and rinsed with cold PBS. Finally, the temperature of the cell suspensions was recorded during illuminated by laser at a photo density of 1.25 W cm-2 for 3 min. 2.7 Cellular uptake of the DOX/TSMCN The 4T1 cells were cultured in 24-well culture plates at a concentration of 5 × 104 cells/well and were incubated for 24 h. DOX solution and DOX/TSMCN (containing 5 µg mL−1 DOX) were simultaneously put into the wells and were further co-incubated for 2 h. For NIR group, fresh culture medium was explored to replace the growth medium and the wells were rinsed with PBS. Subsequently, the NIR with 808 nm was employed to illuminate the wells containing DOX/TSMCN sample and it takes 1 h to complete this process. The intracellular drug distributions of both free DOX and DOX/TSMCN groups were observed and imaged using CLSM (Leica Instruments Inc., Wetzlar, Germany). 2.8 Cytotoxicity study in vitro


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The MTT assay was conducted to estimate the in vitro nanoparticle cytotoxicity. To begin with, the 4T1 cells were cultured on 96-well plate at a concentration of 2.0 × 104 per well at 37oC environment containing 5% CO2 for a day. Subsequently, the cells were cultured with free DOX, TSMCN and DOX/TSMCN at a series of DOX concentrations (from 0.01 to 10 µg mL−1) for a day. For NIR irradiation group, after incubated for 20 h, fresh medium was used to replace the growth medium and then was illuminated by the 808 nm NIR laser at 2.5 W cm-2 power density for 3 min. Next, 50 µL sterile MTT solution (20 mg MTT powder dissolved in 10 mL PBS) was put into each well and continued to be co-incubated for another 4 h. Finally, the mixed solution was poured out and then the formed formazan crystals was dissolved by DMSO. Microplate reader was taken to determine the absorption of each well (SpectraMax M3, Molecular Devices, USA) (λ=570 nm). To further visualize the effect of photothermal therapy, 4T1 cells were cultured on 24-well cell culture dishes at a concentration of 5 × 104 / well. After incubation for 2 h, cells were illuminated using NIR laser with an 808 nm wavelength at 2.5 W cm-2 power density for different times. Besides, the different concentrations (25 and 50 µg/mL) of TSMCN were also added into the 24-well plates illuminated by NIR laser at 2.5 W cm-2 for 2 min. The 4T1 cells were rinsed several times after irradiation and co-stained using (propidium iodide) PI and calcein AM according to the manufacturer’s instructions and then imaged by CLSM. The intracellular ﬂuorescence was quantified using ImageJ software. 2.9 In vivo combination therapy



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The female Balb/c mice (18-22 g) was offered by the Animal Center of the Shenyang Pharmaceutical University. The mice were subcutaneously administrated with 1 × 106 4T1 cells that dispersed in serum-free RPMI-1640 culture medium in the right rear flank. When the tumor size reaching about 100 mm3, the mice bearing tumor were divided into several groups randomly: i) a control group (PBS injection); ii) DOX group (10 mg/kg); iii) DOX/TSMCN group (equal the DOX concentration of 10 mg/kg); iv) TSMCN+NIR group; and v) DOX/TSMCN +NIR group. The NIR laser equipment with emission at 808 nm was employed as a source of laser light. After two hours of administration, the mice from NIR treatment group was irradiated for 3 min using NIR laser at a power density of 1.25 W cm-2. The measurement of the body weight and tumor size were carried out every two days during the period of treatment. The calipers and the following equation were applied to measure and compute the size of the tumor respectively. The formula is that tumor volume = width2 × length /231. The major organs (heart, liver, spleen, lung and kidney) and tumor

were

collected

for

hematoxylin

and

eosin

(H&E)

staining

and

terminal-deoxynucleoitidyl transferase mediated dUTP nick end labeling (TUNEL) assay to determine the systemic toxicity of DOX and intratumoral late apoptosis respectively on the 15th day. 2.10 Biodistribution study and histological examination The tumor-bearing mice were divided into several groups randomly when the volume of tumor reached about 200 mm3 and administrated with 100 µL of a DOX solution and DOX/TSMCN suspensions (with irradiation or not) at an identical DOX


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concentration of 10 mg/kg through intravenously injected. At the predetermined time points (2, 6 and 12 h), the mice were euthanized and sacrificed, the tumor and major organs (heart, liver, spleen, lung and kidney) were collected. The distribution of DOX in all organs were monitored by using IVIS® system and the Bruker MI SE software at the optimal wavelength (λex = 470 nm and λem = 535 nm)32. Finally, The DOX amount in organs was determined by Bruker MI SE software. 3 Results and discussion 3.1 Synthesis and characterization of MCN, MCN-COOH and TSMCN The MCN were prepared via a called “silica-assisted” strategy28. In this work, TEOS served as an inorganic precursor, phenolic resols as a polymer precursor and CTAC as a template. The HF was used to remove the silica embed in the walls of the carbon and the MCN were obtained. To make it easily functional and turn to hydrophilic, the ammonium persulfate and concentrated sulphuric acid were applied to oxidize MCN. Next, the liposome was prepared for further modification. The MCN-COOH was coated by liposome through vortexing. Transmission electron microscope (TEM) images showed that MCN-COOH with a diameter of about 70 nm had a uniform spherical morphology (Fig. 1 A). Cryo-TEM was performed to testify the existence of lipid membrane. As exhibited in Fig. 1 B, a ring structure of about 3.8 nm thick appeared on the periphery of MCN-COOH after covering with liposome. Similarly to the structure of the graphitic carbon atoms33, the graphitic domains identified by the G band (1593 cm-1) and D band (1346 cm-1) were observed in MCN-COOH from the Raman spectrum (Fig. S1). Moreover, we could see that the



TSMCN had a smoother surface than that of the MCN-COOH from the images of AFM (Fig. 1 C-F) owing to the coating of the liposome. The N2 adsorption and desorption test was performed to investigated the mesoporous structure parameters of the MCN, MCN-COOH and TSMCN. As shown in Fig. 1 G, the MCN had a surface area as high as 913 m2/g. The total pore volume was calculated to be 1.28 cm2/g and the corresponding pore size was mainly distributed in 3.8 nm. After carboxylation, the BET surface area of MCN-COOH and their corresponding total pore volume were slightly decreased, while they were almost disappeared after liposome coating, indicating that liposome had coated on the surface of the MCN-COOH. The detailed parameters of these nanoparticles are given in Tab S1. As displayed in Fig. S2, the transformation of carboxyl group at 1720 cm-1 from appearance to disappearance in MCN-COOH and TSMCN verified that the MCN-COOH and TSMCN were successfully prepared. The ξ potential of MCN-COOH underwent great change after the coating of liposome, varying from -19.2 mV to -1.7 mV, which was similar to the potential of liposome (Fig. 1 H). The result was ascribed to that the neutrally charged PEG embed in liposome had a shielding effect on negatively charged MCN-COOH. As can be seen from the Fig. S7, the Tm of TSMCN was 40oC, which was slightly lower than that of the as-prepared liposome (40.7oC), indicating that the TSMCN were thermo-sensitive. X-ray photoelectron spectroscopy (XPS) results revealed the appearance of binding energy of P 2p in TSMCN, while there was not any in MCN-COOH, which proved the successful covering of liposome on the surface of MCN-COOH (Fig. S8). As is known, the good dispersion stability is necessary for


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intravenous administration of nanoparticles. From the Fig. S3, a better dispersion stability was observed for TSMCN compared to the naked MCN-COOH, since the TSMCN retained the characteristic (steric stabilization effect) of the liposome after liposome coverage. In detail, the liposome containing PEG chains could prevent the nanoparticles from direct contact, thus forming numerous particle-particle gaps (hot spots) and avoiding the aggregation of the nanoparticles into the lager aggregates34-36. Meanwhile, after covering the MCN-COOH with liposome, the hemolysis ratio of RBCs was negligible at tested concentrations, and the same at an extreme high concentration of 1000 µg/mL. By contrast, the hemolysis ratio of naked MCN-COOH increased in a concentration-dependent manner within the concentration range of the test (Fig. S4). The results demonstrated that TSMCN were biocompatibility and had a promising potential for intravenous drug delivery.

Fig. 1 (A) TEM image of MCN-COOH. (B) Cryo-TEM image of TSMCN. AFM images of (C-D) MCN-COOH and (E-F) TSMCN. (G)The N2 adsorption and



desorption curves of MCN, MCN-COOH and TSMCN (the inserted graphs were the pore diameter distribution curves). (H) Zeta potential of liposome, MCN-COOH and TSMCN. 3.2 Photothermal effect in vitro The heat production capacity of TSMCN was explored by monitoring the temperature changes under NIR irradiation. As displayed in Fig. 2 A, the temperature of TSMCN suspensions at a concentration of 100 µg/mL elevated rapidly and increased by 42.3oC under an 808 nm laser at the photo density of 2.5 W cm-2 for 3 min. In contrast, the temperature of the control group (water) had rarely changed at the same experimental conditions. Such excellent photothermal conversion effect of TSMCN was mainly ascribed to that the TSMCN had intensive absorption at the wavenumber of 808 nm as shown in Fig. S5. And the near infrared imaging photographs of TSMCN suspensions and water are shown in Fig. S6. Furthermore, the photothermal effect of TSMCN was concentration-dependent. The final temperature of TSMCN suspensions elevated with the concentration increase as can be seen from the Fig. 2 A. In addition, the TSMCN suspensions also exhibited an increase in temperature with the increase of power intensity, indicating a power intensity-dependent manner (Fig. 2 B). No apparent difference was observed in the process of temperature changes after continuous NIR irradiation for four cycles (Fig. 2 C), demonstrating a good thermal stability of TSMCN. To further confirm the potential of TSMCN to serve as a PTT agent, the comparison of thermal stability between TSMCN and indocyanine green (ICG) was conducted. ICG is a


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FDA-approved commercial PTT agent37. As displayed in Fig. 2 F and G, the full wavelength absorption spectra and the color of TSMCN suspensions did not change apparently after NIR irradiation, but the absorption peak of ICG almost disappeared and the color of ICG solution changed obviously under the same condition. Hence, TSMCN had a good photothermal effect and thermal stability, which was promising for the application of PTT. Next, the 4T1 cells were cultured with MCN-COOH and TSMCN suspensions to further testify the photothermal effect of the nanoparticles at cell level. As shown in Fig. 2 D and E, the cells without administered any preparations showed little temperature change under NIR irradiation at a photodensity of 2.5 W cm-2. However, the temperature of cell suspensions raised by 13oC and 23oC after pretreatment with 50 and 100 µg/mL MCN-COOH, respectively. The temperature of the other groups treated with TSMCN elevated much more than that treated with MCN-COOH. The results could be explained by the following points. As we all know that the cell membrane is negatively charged38. The ξ potential values of the MCN-COOH and TSMCN were -19.2 mV and -1.7 mV, respectively, as shown in Fig. 1 H. Only a little of MCN-COOH was taken up by cells owing to repulsive interaction between the negative charge of MCN-COOH and cell membrane. As for the elevated temperature for MCN-COOH group might be attributed to the high absorptivity of MCN-COOH. By contrast, the ξ potential of TSMCN was almost neutral, leading to a better integration with the cells. The above results indicated the good photothermal conversion capability and excellent biocompatibility of TSMCN, making TSMCN



have the potential for cancer therapy as photothermal agents.

Fig. 2 (A) Photothermal heating profiles of the TSMCN suspensions with series of concentrations varying from 5 to 100 µg/mL. (B) Photothermal heating profiles of the TSMCN suspensions under series of power intensities. (C) Heating of a suspension of TSMCN for four NIR irradiation on/off cycles exposed to laser at 2.5 W cm-2 power density. Photothermal profiles of 4T1 cells pretreatment with various concentrations of MCN-COOH (D) and TSMCN (E) at 1.25 W cm-2. UV-vis absorption spectra and photographs of (F) TSMCN suspensions and (G) ICG solutions irradiated by NIR laser at 2.5 W cm-2 photo density for 3 min. The DOX release curves of (H) DOX/TSMCN and DOX/MCN-COOH in pH 7.4 and 5.0 PBS and (I) DOX/TSMCN in presence /absence of NIR laser at 1 h and 6 h. 3.3 Drug loading efficiency and in vitro thermal-sensitive release


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In order to evaluate whether the TSMCN is suitable as drug delivery system, the DOX was loaded into the TSMCN. The drug loading capacity of TSMCN could reach up to about 43%. After coating with liposome, the drug loading capacity reduced to 32%. The reduced drug loading capacity was due to the weight loss of DOX absorbed on the surface of MCN-COOH and the increased weight of coated lipid bilayer. As shown in Fig. S5, the UV-vis spectra were applied to evaluate the success of drug loading. The free DOX had an absorption peak at a wavelength of 485 nm, whereas the DOX loaded DOX/TSMCN exhibited a slight bathochromic shift (from 485 to 505 nm) in the absorption spectrum. The result was similar to DOX loaded onto grapheme, indicating that the DOX was adsorbed into the TSMCN via π-π interactions39-40. Subsequently, the drug release property of the DOX/TSMCN was studied. By contrast, the drug release property of the DOX/MCN-COOH was estimated. The pH 7.4 PBS and 5.0 PBS were chosen to imitate the normal physiological environment and tumor microenvironment (TME), respectively. The release rate of drug from MCN-COOH could reach up to 25% and 46% in pH 7.4 and 5.0 PBS within 30 min (Fig. 2 H), exhibiting the rapid release properties. In comparison, the cumulative release rates of drug from the DOX/TSMCN were markedly decreased owing to that the lipid bilayer restricted the drug diffusion both in pH 7.4 and 5.0 PBS. Only about 22% of the loaded DOX released from TSMCN within 24 h in pH 7.4 PBS. The accumulated release percentage of drug from DOX/TSMCN increased to 48% within 24 h in weak acid release medium as expected due to the pH-dependent electrostatic



interactions between the MCN-COOH or TSMCN and DOX and the detachment of DOX from MCN-COOH or TSMCN under a low pH condition41. Furthermore, the bilayer modification of lipid can hinder the drug release from the TSMCN slightly thanks to the diffusion resistance. Especially in acidic condition, the bilayer modification of lipid cannot completely restraint drug release, because the release rate of DOX stimulated by the density gradient overperformed the restriction effect of the bilayer modification of lipid 24. By contrast, TSMCN had a strong retardation effect on drug release in pH 7.4 PBS environment. Considering thermo-sensitive of the liposome and the good photothermal conversion capacity of TSMCN, the NIR-induced drug release was evaluated. As seen from the curves in Fig. 2 I, the drug release curves presented ladder-shaped expedited release properties at the irradiated time points in both pH 7.4 and pH 5.0 PBS environment under illumination by NIR laser equipment at 3.75 W cm-2 photo density. The drug release rate was markedly enhanced when exposed to a momentary NIR irradiation (5 min). After NIR laser was removed, the DOX release rate again tended to be stable. This result might be attributed to that the local increased temperature produced by TSMCN exceeded the Tm (40.7oC) of the liposome. But the liquidity of the lipid bilayer could be increased under NIR and the molecular thermodynamic movement of DOX and promoting the drug release from the TSMCN. Besides, the increased temperature could reduce the electrostatic and the hydrophobic π-π interactions between the TSMCN and DOX. Consequently, DOX/TSMCN could efficiently release drug under NIR irradiation stimulus at the acidic conditions, which


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would minimize the side effect of anticancer drugs and enhance antitumor efficiency. 3.4 NIR-induced cellular uptake 4T1 cells was chosen as model cells to monitor cellular uptake of drug via CLSM. The images (Fig. 3 A) showed that the free DOX could enter the cell nuclei after incubation for 2 h. In comparison, only a small proportion of drug entered the cytoplasm in the DOX/TSMCN group. To further estimate the NIR irradiation effect on cellular uptake, fresh culture medium was explored to replace the growth medium and each well was illuminated by NIR laser. Interestingly, the DOX fluorescence in fresh medium was weaker than that in untreated group, which might result from that DOX concentration in the medium was lower than that in cell and proportional intracellular DOX escaped into the culturing medium. Excitingly, when the 24-wells plate exposed to a NIR laser, the DOX fluorescence was enhanced compared to without NIR irradiation group, suggesting that the DOX/TSMCN could be delivered to the cells and the NIR laser could enhance the drug release rate from the nanoparticles. The results might be explained by that the elevated temperature resulted from NIR irradiation could increase the molecular thermodynamic movement and enhance cell membrane penetrability and the sensitivity to DOX. The result proved cellular uptake of drug-loaded carrier and the potential application for the cancer treatment.



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Fig. 3 (A) Cellular uptake in the presence/absence of NIR laser (Scale bars = 10 µm). The cell viabilities of single chemotherapy (B) and combined therapy (C) (2.5 W cm-2, 3 min). Tab 1 IC50 values in different formulation groups for growth inhibition of 4T1 cells after incubated for 24h. (*p

Thermo-sensitive Lipid Bilayer Coated Mesoporous Carbon

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