Phosphorylcholine-Based Stealthy Nanocapsules Decorating TPGS

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Controlled Release and Delivery Systems

Phosphorylcholine-based stealthy nanocapsules decorating TPGS for combatting multidrug resistant cancer Liu Gan, Hsiang-I Tsai, Xiaowei Zeng, Wei Cheng, Lijuan Jiang, Hongbo Chen, Xudong Zhang, Jinxie Zhang, and Lin Mei ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b00152 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 25, 2018

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Phosphorylcholine-based stealthy nanocapsules decorating TPGS for combatting multidrug resistant cancer ∥ ⊥∥ ⊥ ⊥ Gan Liu,*,†, Hsiang-I Tsai,‡, , Xiaowei Zeng,† Wei Cheng,‡, Lijuan Jiang,‡, Hongbo Chen,† Xudong Zhang,△ Jinxie Zhang, ‡,⊥ Lin Mei*,†

†

School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, No. 132 Waihuan East Rd., Guangzhou University City, Panyu District, Guangzhou, 510275, P. R. China ‡

School of Life Sciences, Tsinghua University, Hai Dian, Beijing 100084, P. R. China

⊥

The Shenzhen Key Lab of Gene and Antibody Therapy and Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Nanshan District, Shenzhen, Guangdong Province, 518055, P. R. China △

Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, EB3911 Oval Drive, Campus Box 7115, Raleigh, NC 27695, USA

Corresponding Authors *Email: [email protected]; *Email: [email protected]. Author Contributions Gan Liu and Hsiang-I Tsai contributed equally to this work.

ABSTRACT: Improving the anticancer efficacy of chemotherapeutics not only demands for efficient delivery into tumor sites, but also always needs to combat the multidrug resistance of cancer. Here we attempted to conquer both these problems by decorating D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) onto a phosphorylcholine-based stealthy nanocapsule. This TPGS-decorated stealthy nanocapsule, referred as nBSA-TPGS-Dox, conjugated anticancer drug doxorubicin (Dox) through an acid-responsive benzoic-imine bond. nBSA-TPGS-Dox was demonstrated to be stable in PBS and exhibited acid-responsive Dox release behavior. In vitro results showed this nanocapsule could be efficiently uptaken by the Dox-resistant HepG2/ADR human liver cancer cells through clathrin-mediated endocytosis and greatly prevented the Dox efflux, causing much more cytotoxicity than free Dox and non TPGS-decorated nBSA-Dox. Furthermore, nBSA-TPGS-Dox exhibited much prolonged in vivo half-life compared to conventional PEGylated


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nanoparticles and achieved excellent tumor accumulation. Finally, this TPGS-decorated stealthy nanocapsule performed outstanding suppression of Dox-resistant tumor, much superior than non TPGS-decorated nBSA-Dox and free Dox. Thus, this TPGS-decorated stealthy nanocapsule provides a novel powerful nanomedicine platform for combatting multidrug resistant cancer. KEYWORDS: chemotherapy, multidrug resistance, stealthy nanocapsule, TPGS, intracellular delivery INTRODUCTION In recent years, nano-drug delivery systems have been widely proved to enhance cancer therapy, for that they would not only prolong the half-life of drugs in vivo, but also improve the accumulation in solid tumor through the enhanced permeability and retention (EPR) effect.1-8 Recently we developed a poly(2-methacryloyloxyethyl phosphoryl choline) (PMPC)-based anticancer drug doxorubicin (Dox)-loaded stealthy nanocapsule (nBSA-Dox) by in situ radical polymerization of MPC and benzaldehyde monomers around bovine serum albumin (BSA) following conjugating Dox.9, 10 nBSA-Dox exhibited much longer half-life than conventional PEGylated nanoparticles, accumulated efficiently in tumor, released conjugated Dox rapidly in tumor microenvironment-responsive manner and led to significant tumor suppression, demonstrating a potential Dox delivery nanoplatform for improved cancer therapy. In addition, since the stealthy nanocapsule was prepared by the radical polymerization of monomers around the protein, it can be tailored to be multifunctional by adding different monomers. For example, adding amine-containing monomer can tailor the surface of nanocapsule with amine groups for further modification of functional groups. In view of that cancer chemotherapy are facing several problems, such as short half-life, severe side effects and multidrug resistance, it is of value to prepare multifunctional stealthy nanocapsules through surface modification. Multidrug resistance is often caused by the overexpressed ATP-binding cassette (ABC) transporters on the cell membrane of drug-resistant cancer cells,11-15 such as P-glycoprotein, etc. D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), a safe pharmaceutic adjuvant approved by FDA, has been widely used as an excellent emulsifier and surface decoration of nanopharmaceutical materials.16-18 Feng group and many other groups reported that TPGS proved preferable merit of efficient intracellular uptake19,

20

and certain inhibition effect on P-glycoprotein for

overcoming multidrug resistance.21-25 TPGS inhibits the efflux of P-glycoprotein by affecting the conformation and activity of ATPase26. Therefore, we envision that



decorating TPGS onto the PMPC-based stealthy nanocapsule would not only achieve much prolonged circulation and excellent tumor accumulation, but also enhance the intracellular delivery and efflux inhibition of drug, combining efficient in vivo tumor delivery and overcoming multi-drug resistance. As far as is known, there is no such nanocapsule reported any before. Herein, a TPGS-decorated stealthy nanocapsule was designed to conjugate Dox for intracellular delivery and combatting multidrug resistance. The PMPC-based BSA nanocapsule (nBSA) was synthesized by in situ polymerization of the monomers, MPC and N-(3-Aminopropyl) methacrylamide hydrochloride (Apm), and degradable cross-linker glycerol dimethacrylate (GDA) around the protein BSA. The TPGS and benzaldehyde group (BzA) modified nBSA (nBSA-TPGS-BzA) was obtained after modification of the nBSA with TPGS-NHS and N-Succinimidyl 4-forMylbenzoate (SFB), followed by Dox conjugation through an acid-responsive benzoic-imine bond (Figure 1). Its size, morphology, structure composition, drug loading content, surface properties, in vitro stability including drug release behavious were tested. Its in vitro cellular uptake, cytotoxicity and mechanism of overcoming drug resistance were assessed in both HepG2 and Dox-resistant HepG2 human liver cancer cells. Pharmacokinetics in vivo was also studied. Its in vivo NIR fluorescence imaging and antitumor efficiency were further investigated on the HepG2/ADR xenograft tumor models.


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Figure 1. (A) The synthesis of TPGS-decorated and Dox-conjugated stealthy nanocapsule nBSA-TPGS-Dox. (B) Combatting multidrug resistant cancer by nBSA-TPGS-Dox, including keep stealthy during blood circulation, efficiently accumulate in tumor, enter the cancer cells and inhibit the P-glycoprotein efflux, release Dox under intracellular acidic environment followed by its diffusion into the nucleus and intercalation with the DNA.

RESULTS AND DISCUSSION Synthesis and characterization of nBSA-TPGS-Dox. nBSA-TPGS-BzA was prepared by modification of TPGS-NHS and BzA onto nBSA. Agarose electrophoresis showed that nBSA was positively charged (Figure S1) but BSA was negatively charged, indicating that BSA had been entirely encapsulated. In XPS spectra, the signals of N and P elements in nBSA-TPGS are much weaker than those in nBSA (Figure 2C), so a part of PMPC had been covered by TPGS modification. C1s peaks of nBSA-TPGS at 286.2 and 284.9 eV corresponding to C-O and C-C



respectively are significantly stronger than those of nBSA (Figure 2D), also directly verifying successful TPGS modification. Since the O1s peak of nBSA-TPGS-BzA at 530.6 eV representing carbonyl O is stronger than that of nBSA-TPGS (Figure 2E), it was proved that nBSA-TPGS had been successfully modified with BzA group to allow further Dox loading. After Dox loading, agarose electrophoresis results demonstrated that the signal of nBSA-TPGS-Dox in the light field coincided with the FITC fluorescence of FITC-labeled nBSA-TPGS-Dox, which proved that Dox had been conjugated into nBSA-TPGS-BzA, similar to the process into nBSA-Dox (Figure S1). As shown in Figure 2B, nBSA-TPGS-Dox and nBSA-Dox have consistent fluorescent emission spectra with that of Dox, though with much lower intensities, which should result from the fluorescence quenching induced by Dox aggregation in nanogels. Zetasizer detected that the particle sizes of nBSA-Dox and nBSA-TPGS-Dox were (19.5 ± 2.1) and (21.4 ± 2.3) nm (Figure 2A), and their Zeta potentials were (-1.9 ± 0.3) and (-2.1 ± 0.3) mV respectively. Therefore, the particle size of nBSA was only slightly increased after TPGS modification, and the potential was weakly negative, which better benefited long circulation in vivo.19 TEM image of nBSA-TPGS-Dox displays spherical nanoparticles sized approximately 25 nm, being in accordance with DLS results (Figure 2A). The Dox loading content of nBSA-TPGS-Dox (12.4%), which was calculated by UV-visible spectroscopy, was close to that of nBSA-Dox (14.2%). The above results verified that nBSA-TPGS-Dox and nBSA-Dox had been obtained indeed.


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Figure 2. (A) Size distributions of nBSA-Dox and nBSA-TPGS-Dox in PBS and TEM image of nBSA-TPGS-Dox . (B) Fluorescence spectra of Dox, nBSA-Dox and nBSA-TPGS-Dox with a Dox concentration of 25 µg/mL. (C) XPS spectra of nBSA and nBSA-TPGS. (D) XPS narrow scan for the C1s peaks of nBSA and nBSA-TPGS and (E) O1s peaks of nBSA, nBSA-TPGS and nBSA-TPGS-BzA.

In vitro stability and drug release behavious. The in vitro stability of nBSA-TPGS-Dox was determined. We have previously proven that nBSA-Dox remained fairly stable in PBS for two weeks. As expected, nBSA-TPGS-Dox had stable particle size in PBS at 37 °C over two weeks (Figure 3A). nBSA-Dox is stable because PMPC is a highly hydrophilic material and further surface modification with TPGS was proved to not affect the hydrophilicity of carrier owing to the hydrophilic PEG segment. Afterwards, in vitro release of Dox from nBSA-TPGS-Dox was detected. We have found that nBSA-Dox underwent apparent acid-responsive release, because the Schiff base formed between benzaldehyde and amino group remained stable at pH 7.4 but dissociated under acidic conditions27. Likewise, Dox release from nBSA-TPGS-Dox was detected at pH 7.4, 6.5 and 5.0 respectively. At pH 7.4, Dox release could hardly be observed within 12 h, suggesting that the Schiff base was indeed stable in this case (Figure 3B). In contrast, about 58.4% of Dox was released at pH 6.5 and 79.7% at pH



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5 after 6 h, indicating that nBSA-TPGS-Dox had obvious acid response because of Schiff base breakage under acidic conditions. Accordingly, similar to nBSA-Dox, nBSA-TPGS-Dox was extremely stable in blood and normal tissues, but it rapidly released Dox in tumor microenvironment and tumor cell endosomes.

Figure 3. (A) Stability study of nBSA-Dox and nBSA-TPGS-Dox in PBS at 37 °C over two weeks (n = 3). (B) In vitro Dox release behavious of nBSA-TPGS-Dox in culture media at different pH.

Cellular uptake study. To study whether nBSA-TPGS-Dox was subjected to endocytosis, CLSM was used to observe both HepG2 cells and Dox-resistant HepG2/ADR cells cultured respectively with Dox, nBSA-Dox and nBSA-TPGS-Dox at pH 7.4 for 3 h. With red fluorescence, Dox can be directly used for observation. As shown in Figure 4A, free Dox managed to enter the cytoplasm and cell nucleus quickly in 3 h, whereas nBSA-Dox basically failed to deliver Dox to cells, it's conform for our previous research. It’s totally different from nBSA-Dox that the cells of

nBSA-TPGS-Dox

group

exhibited

Dox

fluorescence

obviously.

Since

nBSA-TPGS-Dox was unable to release Dox at pH 7.4, this intracellular Dox fluorescence should be attributed to the cellular uptake of nBSA-TPGS-Dox. We also prepared nBSA-TPGS-Dox-FITC

through

covalently modifying FITC

onto

nBSA-TPGS-Dox and investigated its cellular uptake. The results showed the cells incubated

with

nBSA-TPGS-Dox-FITC

emitted

profound

Dox

and

FITC

fluorescences simultaneously and their distributions overlapped (Figure S2), indicating that nBSA-TPGS-Dox-FITC entered the cells before releasing Dox and achieved intracellular Dox delivery. These results confirmed that TPGS-decorated nBSA-Dox enabled intracellular delivery of Dox through cellular uptake of the nanocarrier. In Dox-resistant HepG2/ADR cells, free Dox was also observed to enter the cells, but much weaker than HepG2 cells due to the Dox resistance of HepG2/ADR cells. In the other hand, the cellular uptake efficiency of


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nBSA-TPGS-Dox by HepG2/ADR cell was almost the same as that of HepG2 cells, suggesting that nBSA-TPGS-Dox, quite different from free Dox, delivered Dox through nanoparticle-based cellular uptake pathway. Furthermore, the cellular uptake efficiency of Dox formulations was quantified. In Figure 4B, the uptake efficiency of free-dox by HepG2/ADR cells were only 50% of that of HepG2 cells. On the contrast, the uptake of nBSA-TPGS-Dox by HepG2/ADR cells was just slightly weaker than that of HepG2 cells, significantly higher than the uptake of free Dox by HepG2/ADR cells. Therefore, this directly confirmed that TPGS-decorated nBSA-Dox would maintain efficient uptake of Dox by Dox-resistant cells.

Figure 4. Cellular uptake of nBSA-TPGS-Dox by HepG2 and HepG2/ADR cells. (A) CLSM images and (B) Cellular uptake efficiency of HepG2 and HepG2/ADR cells after incubation with Free Dox, nBSA-Dox, nBSA-TPGS-Dox for 3 h (scale bar: 20 µm). Dox concentration was 20 µg/mL in all the samples.

In vitro cytotoxicity study. To verify the lethality of nBSA-TPGS-Dox against drug-resistant cancer cells, we conducted MTT assay for HepG2 and HepG2/ADR cells. In the meantime, free Dox and nBSA-Dox with identical Dox doses were utilized as controls. The cells were incubated with various concentrations of Dox formulations for 24, 48 and 72 h respectively. After incubation for 24 h, Dox markedly killed HepG2 cells, with a growth inhibition rate of 53% at 25 µg/mL (Figure 5A), because free Dox diffused into the cell cytoplasm rapidly and then entered the cell nucleus.28 Although nBSA-Dox also worked, its effects were far weaker than those of Dox, being in accordance with the above-mentioned low cellular uptake efficiency. On the contrary, nBSA-TPGS-Dox was more effective than free Dox, with an inhibition rate of 67% at 25 µg/mL after 24 h of culture. The IC50 values of Dox and nBSA were calculated as 22.6 and 61.4 µg/mL respectively, and yet that



of nBSA-TPGS-Dox was only 9.1 µg/mL (Table 1S). As the incubation time extended, the lethality of the three Dox formulations on cells were all apparently augmented, but nBSA-TPGS-Dox remained significantly more effective than the other two (Figure 5B and 5C). The above results proved that surface modification of nBSA-Dox with TPGS dramatically enhanced the cytotoxicity. On the other hand, the lethality of free Dox on HepG2/ADR cells was much weaker than that on HepG2 cells, i.e. 20% of the cells were killed at 25 µg/mL after 24 h. Accordingly, HepG2/ADR cells were highly resistant to Dox. Likewise, the cell killing ability of nBSA-Dox was weaker than that of free Dox. Contrarily, 24 h of culture with 25 µg/mL nBSA-TPGS-Dox killed 51% of cells. The inhibition rate reached 78% at 72 h, which was considerably higher than that of free Dox (Figure 5D and 5F, Table 2S) but slightly lower than that of HepG2 cells by nBSA-TPGS-Dox. Accordingly, nBSA-TPGS-Dox effectively overcame the resistance of cells to Dox.

Figure 5. Cytotoxicity of free Dox, nBSA-Dox and nBSA-TPGS-Dox at the same Dox dose to HepG2 cells after incubation for: (A) 24 h, (B) 48 h and (C) 72h and to HepG2/ADR cells after incubation for: (D) 24 h, (E) 48 h and (F) 72h.

Mechanism of overcoming drug resistance. After confirming that nBSA-TPGS-Dox overcame the cell resistance to Dox, we further clarified the mechanism. Drug resistance is generally related with the high expression of drug efflux pump of the cell membrane29. As the main efflux pump for drug-resistant HepG2/ADR cells, P-glycoprotein can reverse the cellular uptake of free Dox. However, nBSA-TPGS-Dox can escape P-glycoprotein by cellular uptake through endocytosis of nanoparticles. We thus used several endocytosis inhibitors to explore


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the uptake mechanism for nBSA-TPGS-Dox. Chlorpromazine (CMZ), an inhibitor of clathrin-mediated

endocytosis,

significantly

inhibited

the

endocytosis

of

nBSA-TPGS-Dox in a dose-dependent manner, whereas neither genistein (an inhibitor of caveolae-mediated endocytosis) nor cytochalasin D (CD, an inhibitor of macropinocytosis-dependent endocytosis) obviously inhibited the cellular uptake (Figure 6A). Therefore, nBSA-TPGS-Dox mainly underwent clathrin-mediated endocytosis which escaped efflux proteins to maintain efficient endocytosis of Dox by drug-resistant cells.

Figure 6. nBSA-TPGS-Dox overcomes drug resistance through clathrin-mediated endocytosis and inhibition of drug efflux. (A) Uptake efficiency of nBSA-TPGS-Dox on HepG2/ADR cells using typical endocytosis inhibitors: CD, CMZ and genistein. (B) Doxorubicin efflux from HepG2 and HepG2/ADR cells at 8 h after incubation nBSA-TPGS-Dox.

On the other hand, we quantified the drug efflux of HepG2/ADR cells. It is known that P-glycoproteins could pump out large amounts of antitumor agents with various functions and structures, thereby inducing drug resistance by reducing their concentrations in cancer cells.30 Herein, the efflux rate of HepG2/ADR cells for free Dox was twice that of HepG2 cells after 8 h, reaching 90% (Figure 6B and S3). For nBSA-TPGS-Dox, however, the efflux rate of HepG2/ADR cells was only 30% which was equivalent to that of HepG2 cells. Collectively, conjugating Dox with nBSA-TPGS significantly suppressed the efflux of P-glycoproteins, so the augmented cytotoxicity can mainly be ascribed to the cellular uptake of nanocarrier and the plummeting of Dox efflux after decorating TPGS (Figure 7).



Figure 7. Proposed mechanism of overcoming drug resistance by nBSA-TPGS-Dox in Dox-resistant HepG2/ADR cells.

In vivo pharmacokinetics and biodistribution study. We have previously reported that nBSA-Dox enhanced the enrichment of Dox in tumors by significantly extending its half-life in vivo9, so we evaluated the in vivo pharmacokinetics of nBSA-Dox modified with TPGS. nBSA-TPGS-Dox was injected into the tail vein of mice to detect the changes of plasma Dox concentration with prolonged time. Similar to our previous study, we measured the fluorescence intensity of Dox, quantified its concentration and thereafter performed fitting using a two-compartment model. After entering the blood, the concentration of free Dox dropped sharply to almost undetectable at 2 h (Figure 8A), whereas nBSA-Dox greatly prolonged the circulation time of Dox in plasma, like we found before. Also, nBSA-TPGS-Dox remarkably extended such time and raised the plasma concentration, rendering Dox still detectable at 48 h. The elimination half-life of nBSA-TPGS-Dox (27.6 h) was slightly shorter than that of nBSA-Dox (38.1 h) (Table 3S). Possibly, surface modification with TPGS partially attenuated the invisibility of PMPC, but the half-life remained much longer than that of free Dox or common nanocarrier (~10 h).31, 32 Afterwards, we analyzed the in vivo biodistribution and tumor enrichment capability of nBSA-TPGS-Dox with NIR fluorescence imaging. An NIR dye, Alexa 750, was first bound to nBSA-TPGS-Dox through stable covalent bonds. We have reported that nBSA-Dox could be efficiently enriched in tumors.9 Similar to the


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results after nBSA-Dox-Alexa 750 injection, major tissues (tumors included) in the mice injected with nBSA-TPGS-Dox-Alexa 750 emitted obvious fluorescence from the second hour (Figure 8B). Accordingly, nBSA-TPGS-Dox-Alexa 750 fast penetrated each tissue, followed by continuous enrichment in tumors in subsequent 24 h (Figure S4). Quantification suggested that the tumor enrichment efficiencies of nBSA-TPGS-Dox-Alexa 750 and nBSA-Dox-Alexa 750 were comparable. The biodistribution results showed that the fluorescence intensities of tumor tissues in the mice injected with nBSA-TPGS-Dox-Alexa 750 and nBSA-Dox-Alexa 750 significantly surpassed those of other non-tumor organs including the heart, liver, spleen, etc. (Figure 8C and S4). Hence, they were targeted to tumors more efficiently. The enrichment of a nanocarrier in tumors is predominantly controlled by its half-life and penetration ability.33,

34

Although nBSA-TPGS-Dox-Alexa 750 had a

shorter half-life than that of nBSA- Dox-Alexa 750, their tumor enrichment efficiencies were equivalent, which should probably be attributed to the former’s more potent penetration into tumors. We then studied the tumor penetration ability in vitro by using a 3D-cultured HepG2/ADR tumor spheroid model. Indeed, nBSA-TPGS-Dox had a higher penetration ability than that of nBSA-Dox (Figure 8D), probably owing to the longer retention time resulting from the stronger interaction of TPGS-modified nBSA-Dox with cells.

Figure 8. (A) Plasma Dox concentrations of nBSA-TPGS-Dox along with time postinjection. (B) Time-dependent NIR fluorescence images of HepG2/ADR cells xenograft-bearing SCID nude mice after treatment with nBSA-TPGS-Dox-Alexa 750 through tail vein injection. The tumors



were circled in the dotted lines. (C) Biodistribution of nBSA-TPGS-Dox in nude mice determined by quantitative analysis of the fluorescence intensity of the organs. (D) CLSM images showing in vitro penetration of nBSA-TPGS-Dox into 3D-cultured HepG2/ADR tumor spheroids.

In vivo antitumor efficacy. We finally assessed the in vivo antitumor effects of nBSA-TPGS-Dox. Every four days, the mice bearing HepG2/ADR cell xenograft were injected with normal saline, Dox, nBSA-Dox and nBSA-TPGS-Dox respectively once, three times in total. We have verified that the growth of HepG2 cell xenograft treated with nBSA-Dox was almost completely inhibited, with better antitumor effects than those of free Dox. As expected, free Dox or nBSA-Dox failed to significantly inhibit Dox-resistant HepG2/ADR cell xenograft after 14 days. Nevertheless, after treatment with nBSA-TPGS-Dox at the same dose, the tumor growth was entirely suppressed, and the volume plummeted from 101 mm3 to 59 mm3 (Figure 9A). Apparently, nBSA-TPGS-Dox allowed the suppression of drug-resistant tumor. Taking the comparable tumor enrichment abilities of nBSA-TPGS-Dox and nBSA-Dox into consideration, the former exerted better antitumor effects mainly by overcoming the drug resistance of tumor cells. Meanwhile, the body weight of mice treated with nBSA-TPGS-Dox hardly changed (Figure 9B), indicating mild tissue toxicity compared to that of free Dox.35 Moreover, Figure 9C visualizes the tumors of each group before and after treatment, further demonstrating the remarkable tumor suppression ability of nBSA-TPGS-Dox.

Figure 9. Antitumor efficacy of nBSA-TPGS-Dox upon nude mice bearing HepG2/ADR


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xenograft. (A) Tumor volume changes after treatment with saline, Dox, nBSA-Dox and nBSA-TPGS-Dox. (B) Body weight changes of tumor-bearing mice after treatment with saline, Dox, nBSA-Dox and nBSA-TPGS-Dox. (C) Tumor images of groups treated with saline, Dox, nBSA-Dox and nBSA-TPGS-Dox before and after treatment at day 14.

CONCLUSIONS A TPGS-decorated stealthy nanocapsule nBSA-TPGS-Dox was designed for intracellular delivery of Dox and overcoming multidrug resistance. nBSA-TPGS-Dox conjugated Dox through benzoic-imine bond and exhibited acid-responsive Dox release behavior.. It was demonstrated to efficiently deliver Dox into drug-resistant cancer cells through clathrin-mediated endocytosis and greatly inhibit the Dox efflux, leading to severe cytotoxicity against drug-resistant cancer cells. Furthermore, nBSA-TPGS-Dox also exhibited much prolonged in vivo half-life and excellent tumor accumulation. Thus, this nanocapsule performed outstanding suppression of drug-resistant tumor, providing a potential nanoplatform for clinical applications.

ACKNOWLEDGEMENTS The authors acknowledge the financial support from National Natural Science Foundation of China (51703258, 81771966, 31270019), Natural Science Foundation of Guangdong Province (2015A030313848), Guangdong Natural Science Funds for Distinguished Young Scholar (2014A030306036), Guangdong Special Support Program, Science and Technology Planning Project of Guangdong Province (2016A020217001). ASSOCIATED CONTENTSupporting Information Supplementary information related to this article can be found online at: Materials and experimental section (including synthesis and characterization of nanocapsule, size, morphology, structure composition, Drug loading content, X-ray photoelectron spectroscopy, Fluorescence spectroscopy, in vitro drug release behavious, Cellular uptake, in vitro cytotoxicity, biodistribution, in vivo anti-tumor efficacy, and pharmacokinetics study) Notes The authors declare no competing financial interest.



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For Table of Contents Use Only

Phosphorylcholine-based stealthy nanocapsules decorating TPGS for combatting multidrug resistant cancer ∥

Gan Liu,*,†, Hsiang-I Tsai,‡,

⊥, ∥

Hongbo Chen,† Xudong Zhang,

⊥

Xiaowei Zeng,† Wei Cheng,‡, Lijuan Jiang,‡, △

⊥

Jinxie Zhang, ‡, Lin Mei*,†


⊥

Phosphorylcholine-Based Stealthy Nanocapsules Decorating TPGS

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