A CD44-Targeting Programmable Drug Delivery System for

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Biological and Medical Applications of Materials and Interfaces

A CD44-targeting programmable drug delivery system for enhancing and sensitizing chemotherapy to drug resistant cancer Min Zhang, Yi Ma, Zhaohui Wang, Zhihao Han, Weidong Gao, Qiumei Zhou, and Yueqing Gu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19798 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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ACS Applied Materials & Interfaces

A CD44-Targeting Programmable Drug Delivery System for Enhancing and Sensitizing Chemotherapy to Drug Resistant Cancer Min Zhang†‡∥, Yi Ma†∥, Zhaohui Wang†, Zhihao Han†, Weidong Gao†, Qiumei Zhou†, Yueqing Gu†* †State

Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Screening,

Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University, Nanjing 210009 (China) ‡Institute

of Biomedical Materials and Engineering, College of Materials Sciences and Engineering, Qingdao University, Qingdao, 266071 (China)

KEYWORDS: Programmable sequential effect; CD44-targeting; Drug-resistant cancer; Sensitized therapy; Chondroitin sulfate

ABSTRACT Programmable drug delivery system holds great promise to enhance cancer treatment. Herein, a programmable drug delivery system using chondroitin sulfate based composite nanoparticle (CBN) was developed for enhancing and sensitizing chemotherapy to drug resistant cancer. The 1

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nanoparticle was composed of a crossed-linked chondroitin sulfate (CS) hydrogel shell and hydrophobic cores containing both free drugs and CS-linked prodrugs. Interestingly, the nanoparticle could mediate tumor-specific CD44 targeting. After specific cellular uptake, the payloads were suddenly released due to the decomposition of the CS shell, and the free drug molecules with synergistic effect induced tumor-specific cytotoxicity rapidly. Subsequently, the inner cores of the nanoparticles sustainedly release their cargos in drug resistant tumor cells to keep the effective drug concentration against the drug efflux mediated by p-glycoprotein. CS disassociated from the outer shell sensitized cancer cells to the antitumor drugs through downregulation of Bcl-XL, an anti-apoptosis protein. Such programmable drug delivery system with specific tumor targeting and sensitized therapy is promising for rational drug delivery and provide more versatility for controlled release in biomedical applications. 1. INTRODUCTION Despite tremendous efforts made in anticancer drug research over past decades, conventional drugs still face some serious drawbacks in tumor therapy1-3. According, the drug delivery systems have been explored due to the potential of a more effective treatment4-13. As widely known that ideal drug delivery systems should satisfy the conditions of no leakage in the delivery process and easy unloading the cargoes once arriving at the target sites.14 In order to meet these criteria, various stimuli-responsive drug delivery systems were proposed. Stimuliresponsive drug delivery systems are specialized nanosized drug delivery carriers equipped with environmental sensitive modalities within their structures15, 16. These drug delivery systems can release their encapsulated cargos in response to specific environmental stimuli, such as light17, temperature18, pH19, enzyme20, reactive oxygen species21, ultrasound22, 23, and redox agents24. However, most stimuli-responsive drug delivery systems usually release their guests simultaneously once attacked by stimulus25, lacking of sustained effective drug concentration against the drug efflux mediated by p-glycoprotein, which greatly impedes their practical application in drug resistant cancers with drug-efflux function. Therefore, a controlled drug 2


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delivery system with a programmable drug release function to quickly release a large amount of drug and sustain effective therapeutic concentration becomes an attractive alternative for drugresistant cancers. Antibodies, kinase inhibitors, endogenous molecules and lectin saccharides are widely used in target therapy. However, Some exogenous molecules might cause immune response, which lead to serious side effects. Endogenous molecules such as folic acid26, 27 and chondroitin sulfate are increasingly used as alternative tumor targeting ligands because of better biocompatibility. Chondroitin sulfate (CS), a natural polysaccharide28, has universally acknowledged good druggability, for it has been used as anti-inflammatory drug in Europe29. CS possesses a strong affinity for CD44 receptors30-32, which is highly expressed on various types of cancer cells, and determines the tumorigenic and metastatic capacities of cancer cells.

33, 34

More importantly, as

an anti-inflammatory drug, CS inhibits the synthesis of the pro-inflammatory enzyme COX-235, which directly regulates the phosphorylation of Akt and subsequently decreases levels of Bcl-XL, a Bcl-2 family member with anti-apoptotic functions.36 And the down-regulation of Bcl-2 increases sensitivity of tumor cells to chemotherapeutic drugs37, which could further reverse cancer drug resistant. Therefore, CS was not only an attractive candidate as targeting ligand for building structure of the drug delivery system, but also an amplifier of the therapeutic efficacy. Herein, we report a programmable sequential drug delivery system using CS based composite nanoparticles (CBN) to enhance and sensitize chemotherapy to overcome drug resistant cancer. As illustrated in Scheme 1a, anticancer drug Paclitaxel (PTX) and sunitinib (Su) with synergetic effect were used as therapeutic agents. PTX was connected with CS to form CS-connected prodrug (CP) and self-assembled with free drugs (PTX+Su) into smaller nanoparticles(CPN) 38-40. Then, the free drugs and drug-loaded nanoparticles CPN were encapsulated into a cross-linked 3



CS-based hydrogel shell to form this programmable composite nanoparticles, PTX/Su@CBN. The CS shells were cross-linked with GSH-sensitive disulfide bond, which enabled the shell to specifically degrade under high GSH concentration in tumor cells after CD44 mediated internalization. Then free PTX and Su located in the CS-shells rapidly diffused in tumor cells and exerted synergetic effect, while the drug-loaded cores CPN were also released out. CPN provided a second, sustained drug release, and maintained an effective concentration in drug resistant tumors against the drug efflux mediated by p-glycoprotein. Meanwhile, CS disassociated from the hydrogels, causing Bcl-XL down-regulation and increased sensitivity of tumor cells to chemotherapeutic drugs, which could further improve the therapeutic effect of drug resistant cancer (Scheme 1b). This study provided a strategy for prepare smart drug delivery systems, which can achieve programmable drug release and enhanced treatment, is of great potential to realize more rational drug administration for the therapy of drug resistant cancer. 2. RESULTS 2.1 Synthesis and characterization of CBN. The CS-based nanoparticles were composed of hydrogel shells and drug-loaded cores. The chemical structure of CS-connected prodrug CP was confirmed using FTIR and 1H-NMR (Figure S1). CBN were characterized by TEM (Figure 1a). The nanoparticles were dispersed and spherical, and had a negative surface zeta potential (Figure S2a). The ratio of PTX to Su in PTX/Su@CBN was 1:40 (w/w). Further quantification found that 20.7% PTX and 58.4% Su was distributed in CS shell and 79.3% PTX and 41.6% Su was in the inner core. After drug loading, PTX/Su@CBN showed a smaller size than CBN (Figure 1b), which was indicative that the addition of hydrophobic drugs to the inner particles improved their assembly. Moreover, the electric attraction between drug and nanoparticles may also contribute 4


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to a more compact structure. Both CBN and PTX/Su@CBN were stable through long-term storage (Figure S2b). As shown in Figure 1g, the particle size of CBN changed into two diameter distributions in 10 mM GSH, for the CS shells were degraded and the cores with smaller particle were released out. After 12 h incubation with 10 mM GSH, the nanoparticles were mostly degraded (Figure 1c) and the hydrophobic dye loaded in CBN was released (Figure 1d). TEM was employed to confirm the responsiveness of the CBN to GSH (Figure 1e). The nanoparticles remained spherical after an incubation with 10 µM and 2 mM GSH. By contrast, CBN disintegrated into irregularly shaped components in 10 mM GSH. The disassembly of CBN was a result of the cleavage of the disulfide linkages in high GSH concentrations, which led to the release of payloads from the interior of the nanoparticles in high concentration of GSH. The programmable drug release of CBN was designed as, first, rapid release after cell uptake and, second, sustained release in the tumor cells. The release behaviors of two kinds of drugs were investigated. During the initial 30 minutes, free drug molecules loaded in CS shells had a burst release behavior in the presence of 10 mM GSH (Figure 1f). Respectively, about 53.8% of PTX and 59.9% of Su are released. Then, a following sustained release was observed within 24 hours. The two-stage drug release profile could confirm the core-shell structure of CBN. Also, such a release behavior may help maintain an effective drug concentration in tumor cells against drug efflux, thus enhancing the therapeutic effect of drug resistant cancer. As GSH concentration in normal tissues is much lower than that in tumor environment41, only about 5.9% PTX and 8% Su were detected at 24 h under 10 uM GSH (plasma GSH concentration), while about 17.8% PTX and 20.7% Su were release in 2 mM GSH condition (normal tissue GSH concentration). The negligible drug leakage in low GSH concentration promised low systemic toxicity in vivo. 5



Therefore, the proposed drug delivery system was supposed to remain stable in the delivery process after intravenous administration and release anticancer agents specifically in tumor in a programmed manner. To further evaluate the stability of CBN in vivo, the concentration of free drugs in blood was studied. Samideh Khoei et al42, 43 investigated the in vivo behavior of drug-loaded nanocarriers through measuring the mean plasma concentration of free drug, which could clearly illuminate the prolonged elimination half-time. As shown in Figure S3, both of the free drugs displayed one-compartment model, whereas PTX/Su@CBN followed a muti-compartment model, showing a longer elimination time. More importantly, being constructed as GSH-sensitive nanoparticles, PTX/Su@CBN showed little drug release in blood, which further confirming the stability of nanoparticle in circulation. 2.2. In vitro targeting ability. Targeting specificity of CBN was evaluated on cancer cell lines MDA-MB-231, HepG2, U87MG, and MCF-7, the normal cell line L02 and MSCs (normal primary human adipose-derived mesenchymal stem cells). Fluorescein was loaded into CBN (Flu@CBN) for cell imaging and hyperspectral microscopy was used to image the intracellular CBN to exclude the possibility that differences in Flu@CBN uptake affected the fluorescence signal. As shown in Figure 2a, bright fluorescence was observed in MDA-MB-231 cells, where Z-axis images acquired at different depths (Figure 2b) indicate that CBN accumulated in the cytoplasm. A gradual decrease in fluorescent signal was observed across tumor cell lines, which is consistent with their CD44 expression levels (Figure 2c). Of note, there was nearly no signal detected in normal cells MSCs and L02, despite high expression of CD44 on MSCs. The low uptake of MSCs was possibly because the different isoforms of CD44 expressed on MSCs and tumor cells. Therefore, further experiments were carried out to confirmed the hypothesis. Anti6


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CD44 antibody was incubated with MDA-MB-231 and MSCs cell lines to visualize CD44 expression. As shown in Figure 3, both the cancerous and normal cells displayed bright fluorescence after antibody staining, confirming abundant CD44 expression on these cell lines. Next, the cellular uptake of CBN was evaluated. Obvious signal was observed in MDA-MB-231 cells, but not in MSCs after Flu@CBN incubation. Similar phenomenon was observed in both 3D spheres or 2D adherent cultures, indicating that CS specifically binds to CD44 on cancer cells, but not normal cells. These results demonstrated that the CS based nanoparticles were internalized through CD44 expressed on cancerous cells, but not noncancerous cells, which could avoid unnecessary toxicity towards normal cells. It was further speculated that the different targeting effects of CS was due to the variance of CD44 iosforms between normal and tumor cells. As shown in Figure S4, the fluorescent signal of the CD44 antibody was almost obliterated in normal cells after CD44s silencing, while the tumor cells retained bright fluorescence, implying that normal cells primarily express CD44s and CD44v may be the major isoform in tumor cells. To investigate binding of CS with CD44 receptors after gene silencing, CSfluorescein (CS-Flu) conjugate was synthesized. The intracellular accumulation of CS-Flu was unchanged in MDA-MB-231 cells following CD44s knockdown. By contrast, silencing of total CD44 blocked the uptake of CS-Flu into tumor cells. Under these conditions, uptake of CS-Flu was negligible as proved by minimal fluorescence. Therefore, CS binding was probably mediated by CD44v exclusively, which may explain the tumor-specific recognition by CS. 2.3. In vivo targeting ability. To evaluate the in vivo tumor targeting ability of CBN, a near infrared (NIR) fluorescent dye, Cypate, was encapsulated into CBN (Cy@CBN). As shown in Figure 4a, the fluorescence signal appeared in the tumors at 4 h, peaked at 12 h, and remained until 72 h in all tumor models. Meanwhile, there was no accumulation of the free NIR dye in the 7



tumors. Moreover, the fluorescence signal in the MDA-MB-231 tumor was stronger than in the HepG2, MCF7, and U87MG tumors. Consistent with the in vitro results, the fluorescence signals in the tumors was lower in relation to a decrease in CD44 expression (Figure 4d). Fluorescence imaging of different organs (Figure 4b) excised from tumor-bearing mice revealed that at 12 h post-injection, CBN had accumulated only in tumor tissue and had been nearly cleared from other organs. NIR dye served as a control. Confocal images of tissue sections confirm the accumulation of CBN in tumors (Figure 4c), supporting that this nanosystem has an enhanced ability to target tumors and only negligible accumulation in normal tissues. To confirm the specificity of CBN to tumor tissues, breast tumor and normal tissues were excised from the tumor-bearing mice and analyzed by laser confocal microscopy (Figure 5). Although both normal and tumor tissues showed certain extent of CD44 expression, obvious CBN signal was only observed in tumor tissues, indicating that CBN could specifically accumulate in CD44 overexpressed tumor tissues. Along with the cell imaging results, these data supported that the difference of CD44 isoforms on tumor and normal cells might induce the different binding ability. 2.4. In vitro antitumor efficacy and molecular mechanism. Cytotoxicity from PTX/Su@CBN and the free drugs combination (PTX+Su) was compared by performing cell viability analysis on MDA-MB-231, HepG2, and MCF7 cell lines. As shown in Figure S5, both of the two groups displayed a dose-dependent increase in antitumor efficacy. The IC50 value of each group was calculated based on MTT assay (Table S1). Notably, the IC50 value of PTX/Su@CBN was the lowest in all groups, which can be attributed to their superior tumor targeting ability, programmable drug release and drug sensitization. Apoptosis of PTX-resistant cancer cells (MDA-MB-231/Taxol) due to PTX, PTX+Su, and PTX/Su@CBN was evaluated by 8


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flow cytometry. As shown in Figure 6a-d, the PTX+Su-treated cells had a larger apoptotic population (51.1%) than those treated with PTX alone (14.5%), suggesting that combination therapy enhance the apoptotic potential. Notability, 72.8% of PTX/Su@CBN-treated cells were apoptotic, significantly enhancing the inhibition of drug-resistant tumor cells. MTT assays were further confirmed the efficacy of PTX/Su@CBN against MDA-MB-231/Taxol (Figure S6). More importantly, CS has been reported to interfere with nuclear factor-κB (NF-κB) and decrease COX-2 levels, which subsequently leads to down-regulation of Bcl-XL. The effect of CS on COX-2 and Bcl-XL down-regulation was confirmed by Western blot (Figure 6e and 6f). This increased the sensitivity of cancer cells to chemotherapeutic agents (Figure 6i) and caused enhanced anticancer toxicity by PTX/Su@CBN. Meanwhile, decreased expression of COX-2 and Bcl-XL was observed following incubation with PTX/Su@CBN, suggesting that the CS component of the nanosystem effectively neutralizes the drug-stimulated antiapoptotic effects mediated by Bcl-XL up-regulation. ELISA assays and immunofluorescence imaging were carried out to further elucidate the influence of CS on the signaling pathway (Figure 6g and 6h). Furthermore, we detected the other apoptotic related proteins, Bax and Caspase-3. After incubation with CS, neither of the proteins' expression changed (Figure S7). Consistent with the result of flow cytometry (Figure S8), we speculate that CS would not induce cell apoptosis under the working concentration, which implied that CS could reverse cancer drug resistant without any toxicity. All these results implied that CS not only served as a hydrophilic moiety for the drug delivery nanosystem, but also contributed to tumor selectivity and therapeutic efficacy enhancement. 2.5. The biodistribution and in vivo antitumor efficacy of PTX/Su@CBN. To measure the plasma concentrations of PTX and Su, the structure of PTX/Su@CBN was destructed and the 9



released drugs were collected and extracted. As shown in Figure S9a, PTX and Su entrapped in PTX/Su@CBN exhibited longer blood circulation even at 12 h post-injection. Meanwhile, free PTX and Su were cleared quickly from the blood stream. The AUC and T1/2 of PTX/Su@CBN and free drugs were calculated using Winolin. As shown in Table S2, PTX/Su@CBN exhibited a longer elimination half-life and a larger area under the curve than free drugs. Furthermore, the amounts of drugs in different tissues at 12 h post-injection were measured (Figure S9b), and the drug (PTX and Su) concentrations were nearly 5-fold higher in the tumor tissues of the PTX/Su@CBN-treated group than PTX+Su-treated group. Meanwhile, the normal organs retained little to none of the drugs following PTX/Su@CBN treatment with the exception of minimal drug accumulation in the liver. This is possibly due to the capture of nanoparticles by the reticuloendothelial system. These results demonstrated that PTX/Su@CBN greatly increased the tumor accumulation of anticancer drugs, and it was expected to achieve a more potent tumor inhibition effect in vivo. Next, MDA-MB-231/Taxol tumor-bearing mice were used to evaluate the antitumor efficacy of the PTX/Su@CBN with free drugs (PTX+Su) at the same dose as control. In addition, a double dose of PTX+Su was also assessed as a high-dose control. As displayed in Figure 7, CBN exhibited superior tumor-growth inhibition compared to that of the PTX due to the tumor targeting ability of CBN, resulting a higher accumulation of PTX. Notably, PTX/Su@CBN inhibited tumor growth more potently than other treatments at the same dose, which was due to specific tumor targeting, programmable drug release, and drug sensitivity enhancement of PTX/Su@CBN. Histologic assays also revealed observable tumor necrosis with decreased tumoral cellularity in PTX/Su@CBN-treated tumors. Meanwhile, PTX/Su@CBN had a similar antitiumor efficacy as the high dose of PTX+Su, indicating that PTX/Su@CBN can significantly 10


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inhibit tumors at a relatively lower dose. Moreover, the administration of PTX/Su@CBN caused notable down-regulation of CD31, a marker of blood vessel formation, indicating synergistic effects between these two drugs when incorporated into one system. Compared to the free solution of the two drugs, PTX/Su@CBN reduced the systemic toxicity of these chemotherapeutic reagents. As shown in Figure 7d, the free multidrugs caused evident damage to spleen and kidney tissues, which was notably decreased in PTX/Su@CBN-treated mice. Bodyweight measurements demonstrate an improved quality of life for the mice after treatment with PTX/Su@CBN, as there was less weight lost in this treatment group than in free drugtreated mice (Figure 7e). The enhanced antitumor properties and reduced toxicity of the PTX/Su@CBN compared to PTX+Su alone translated into significant improvements in the survival of mice with MDA-MB-231/Taxol-derived xenogenic breast tumors. The median survival time of each group (saline, CBN, PTX, PTX+Su, PTX+Su (double dose)) was shown in Table S3. PTX/Su@CBN treated group achieved a higher survival rate of 70% on day 28, which also demonstrated a better health condition during the therapy, confirming the enhanced therapeutic efficacy and reduced toxicity of PTX/Su@CBN. In summary, these results demonstrate the potential translational applications of PTX/Su@CBN for treatment with multidrugs using a delivery system that substantially reduces systemic toxicity and enhances antitumor activity in drug-resistant cancer. 3. CONCLUSION In this study, we successfully synthesized a CD44-targeting programmable drug delivery system for enhancing and sensitizing chemotherapy to drug resistant cancer. The proposed nanosystem was composed of crossed-linked chondroitin sulfate hydrogel shells and hydrophobic cores containing both free drug and CS-linked produgs. PTX and Su were used as 11



anticancer agents in this work, which also have synergistic efficacy. The nanosystem can keep its structural integrity without leakage of drugs in drug delivery process. After cellular uptake mediated by tumor specific CD44, the proposed nanosystem can successfully achieve programmable drug release. In presence of high GSH concentration, the outer shells of CBN degraded and the encapsulated free drugs were rapidly released as the first burst release stage. The disassembled CS increased the sensitivity of drug resistant tumor cells to anticancer agents and further enhanced the treatment efficiency. Subsequently, the drug loaded cores released to provide the second sustained release stage, which maintained the effective drug concentration in drug resistant tumor cells against the drug efflux. Such well designed nanosystem with programmable drug release, tumor specific targeting and sensitization of tumor cells to chemotherapeutics is promising to achieve a more effective treatment in drug resistant cancer. In addition, the nanosystem designed in this study provides more versatility for loading different drugs with synergistic efficacy, and also provides a versatile strategy for developing novel drug delivery systems for biomedical applications. 4. METHODS 4.1. Materials. All cell lines were purchased from American type culture collection (ATCC). Chondroitin Sulfate (CS) and hyaluronic acid (HA) are provided by Yuanmu Bioengineering Co., Ltd. Succinic anhydride, 1-(3-(Dimethylamino) propyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), cystamine dihydrochloride, ethanediamine was purchased from Sigma-Aldrich. PTX and Su were purchased from Meilun Biotechnology Co., Ltd. The other chemicals were purchased from Sinopharm Chemical Reagent Co.,Ltd. Cypate (MW: 689) was prepared by our own research group following a protocol described by Achilefu et. al.44, 45 12


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4.2. Preparation of CBN. For synthesis hydrogel shell, CS (240 mg)

was activated by

EDC/NHS catalyst systems, and then cystamine dihydrochloride (500 mg) was added and stirred for 12 h. The obtained mixture was dialyzed against deionized water then lyophilized. CS-connected prodrugs (CP) were prepared through connect CS and anticancer drug PTX through ethanediamine. PTX (100 mg) and succinic anhydride (23.4 mg) was dissolved in CH2Cl2, and 4-Dimethylaminopyridine was subsequently added as catalyzer. After stirring for 48 h at 50 °C, the end product was extracted using deionized water. CS was activated by EDC/NHS catalyst systems, and then ethanediamine was drop added and stirred for 12 h. The mixture of acetylated PTX was added into the reaction solution. After 12 h reaction, CP were dialyzed against deionized water then lyophilized. The chemical structure of CP was characterized by FTIR and 1H-NMR. CPNs were prepared through a sonication method. To prepare PTX and Su loaded CBN (PTX/Su@CBN), PTX and Su was dissolved in DMSO then added drop-wise into CP solution with magnetic stirring. The solution was treated with a probe-type sonicator (NingBo Scientz, work time 2 s, rest time 5 s, 200W) for 10 min and dialyzed against deionized water. Finally, both free drugs and CS hydrogel shell were dissolved in deionized water and added drop-wise into the obtained mixture solution with magnetic stirring. After 12 h stirring, the final solution was sonicated and dialyzed against deionized water. Morphology and hydrodynamic diameter of the nanoparticles was examined by transmission electron microscopy (TEM, Philips FEI Tecnai G2 20s-TWIN, Netherlands) and dynamic light scattering (DLS, LPSA, Malvern Instruments, UK). The amount of PTX and Su loaded in the nanoparticles was extracted and measured by HPLC.

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4.3. Disassembly of CBN NPs using GSH. CBN were placed in dialysis bags and immersed in release media in the presence of various concentrations of GSH (10 µM, 2 mM, 10 mM) at 37 °C with gentle shaking. The size distribution and morphology change were monitored at each time point. Cypate, a hydrophobic dye, was loaded into CBN to visualize the stimuli-responsive drug release profile. After immersed in media with different concentration of GSH at 37 °C for 12 h, the morphology change was observed. The drug release profile of PTX/Su@CBN was analyzed using a similar dialysis method, with the release media containing various concentrations of GSH. At every designated time interval, 3 mL of dialysate was withdrawn and replaced with the same volume of fresh release medium. The concentrations of PTX and Su in the release media were determined by HPLC analysis. 4.4. In vivo release of PTX and Su in blood. Free drugs and PTX/Su@CBN with same dosage were intravenously injected into mice (~220 g). Blood samples were collected in heparinized tubes at each time point, followed by centrifugation to obtain plasma. Then the free PTX and Su were extracted from plasma, and the concentration of PTX and Su were determined by HPLC analysis. 4.5. Targeting of CBN to CD44 expressing cells. Fluorescein (Flu) was encapsulated in CBN (Flu@CBN) to evaluate the targeting capacity of CBN. The human cell lines L02 (normal liver cells), U87MG (human malignant glioma), HepG2 (human liver cancer cells), MCF-7 (human breast cancer), MDA-MB-231(human breast cancer) and normal primary human adipose-derived mesenchymal stem cells (MSCs) were incubated with Flu@CBN for 4 h and measured using laser scanning confocal microscope (LSCM, FV1000, Olympus, Japan) and hyperspectral microscopy after washed with PBS for 3 times. The nucleus was labled by Hoechst. 14


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To further study the targeting ability of CBN, 3D cultured MDA-MB-231 and MSCs were used. Cells were cultured in 96-well plates coated with a layer of sterilized agarose-based DMEM (2% w/v) at a density of 1×105 cells/mL. After 10 days culture, the mammospheres were collected for further experimental use.46 The spheres were incubated with Flu@CBN for 8 h, washed with PBS, and fixed in 4 % paraformaldehyde. The sample was then incubated with 3% BSA to block potential nonspecific binding, followed by incubation with primary antibody of CD44 (Abcam, Cambridge, U.K.). After washed 3 times and incubated in dark for 1 h with secondary antibody (goat anti-rabbit IgG tagged with Alexa Fluor 568 from Abcam, Cambridge, U.K.). 2D cultured cells were incubated with CBN for 4 h and incubated with CD44 antibody as mentioned above. The samples were then washed and examined using LSCM. To investigate binding mechanism of CBN, CS was conjugated with Flu to form CS-Flu. The experimental steps were as follows: Flu was activated by EDC/NHS catalyst systems, and then drop added into CS, which was dissolved in ethylenimine. After 12 h reaction, CS-Flu was dialyzed against deionized water then lyophilized. To confirm the CD44 receptor specificity, RNA interference experiment of CD44 were also performed. The siRNAs against CD44 and CD44s were designed and synthesized by Biomics Biotechnology Co., Ltd (Nantong, China). The siRNAs were transfected into cells using Lipofectamine 2000. After incubating with CS-Flu and CD44 antibody, the fluorescence signal was detected using LSCM. 4.6. In vitro imaging. All the animal experiments were conducted under the Animal Management Rules of the Ministry of Health of the People’s Republic of China and the guidelines for the Care and Use of Laboratory Animals of China Pharmaceutical University. Tumor models were generated by injecting tumor cells into the left axillary fossa of nude mice. Cypate, an NIR fluorescent dye, was encapsulated into CBN for in vivo imaging (Cy@CBN). 15



After injection of Cy and Cy@CBN into the tail vein separately, the tumor-bearing mice were imaged using the NIR fluorescence imaging system at different time points. Each treatment group (n=10) was sacrificed up to 24 h after injection, and harvested tissues were cut into small pieces to examine the fluorescence intensity using LSCM. To confirm the specificity of CBN to tumor tissues, Flu@CBN were intravenously injected into the MDA-MB-231 bearing mice. The mice were sacrificed up to 24 h post injection, the organs and tumors were harvested and cut into small pieces. After incubating with CD44 antibody, LSCM was used to evaluate the fluorescence intensity. The nucleus was labeled by Hoechst. 4.7. In vitro antitumor efficacy and molecular mechanism. The cytotoxicity of PTX/Su@CBN, PTX+Su and CS was evaluated by MTT assay. MDA-MB-231, HepG2 and MCF7 cells were grown in 96-well plates with 5×103 cells per well and incubated for 24 h. Then, various concentration drugs were added into the media and incubated with cells for 48 h. Afterwards, MTT was added to each well and incubated for 4 h and dimethyl sulfoxide was used to dissolve the formazan crystals. The optical density was measured at 560 nm. According to the MTT results obtained, the IC50 values of different groups were calculated by SPSS. PTX resistant cell line MDA-MB-231/Taxol was used to study the antitumor efficacy of PTX/Su@CBN in drug resistant tumor cells. Annexin V and propidium iodide (PI) kit (Invitrogen, CA, USA) was used to determine the mode and extent of apoptosis according to the manufacturer's instructions. MDA-MB-231/Taxol cells were cultured in six well plate and treated with PBS, CS, PTX, PTX+Su and PTX/Su@CBN. Subsequently, cells were incubated for 30 min in dark after adding annexin V-FITC and propidium iodide and flow cytometry was used to differentiate the stained cells. The cytotoxicity of CS, PTX, PTX+Su, PTX/Su@CBN was evaluated using MDA-MB-231/Taxol cells. Cells were grown in 96-well plates with 5ˣ103 16


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cells per well and incubated for 24 h. Then, various concentration drugs were added into the media and incubated with cells for 48 h. Afterwards, MTT was added to each well and incubated for 4 h and dimethyl sulfoxide was used to dissolve the formazan crystals. The optical density was measured at 560 nm. Western Blot: MDA-MB-231/Taxol cells were grown in a 6 well plate and incubated with PTX/Su@CBN, PTX+Su and CS for 24 h, saline was used as control. Then, cells were collected and lysed to isolate the proteins. Proteins were separated using electrophoresis and then transferred to nitrocellulose membranes. After blocked, membranes were incubated with antibody. Reactive bands were observed with chemiluminescence. To assess the pathway of apoptosis, NF-κB, COX-2, Akt, Bcl-XL were measured using ELISA kits. MDA-MB-231/Taxol cells were collected and lysed after treated with different groups. The level of proteins was monitored at 450 nm. MDA-MB-231/Taxol cells were treated with different groups followed by incubation with COX-2 and Bcl-XL antibody as mentioned above. The nucleus was labled by Hoechst. The fluorescein signal was detected using LSCM. 4.8. Distribution and therapeutic efficacy of PTX/Su@CBN in tumor-bearing mice. PTX/Su@CBN was intravenously injected into tumor-bearing mice, 12 h later, the mice were sacrificed and the tissues were removed immediately after perfusion with saline. Plasma and tissue concentrations of drugs were determined by HPLC analysis. Free drugs and PTX/Su@CBN with same dosage were intravenously injected into mice (~220 g). Blood samples were collected in heparinized tubes at each time point, followed by centrifugation to obtain plasma. The mixture was treated through sonication with high power to break the structure of the nanoparticles, and result in the drug release. Then the free PTX and Su were extracted, and the 17



concentration of PTX and Su were

determined by HPLC analysis. The pharmacokinetics

parameters AUC and T1/2 were calculated through Winolin. MDA-MB-231/Taxol tumor-bearing mice were randomly assigned into five groups (n=10) and treated with different injections (saline, PTX, CBN, PTX+Su PTX/Su@CBN, PTX+Su(double dose)). The tumor volume and body weight of each mouse were monitored every other two days over a period of 28 days. The median survival time of different groups was calculated using SPSS. To further investigate the therapeutic effects of these treatments, the mice were sacrificed, and the tissues were excised for histological examination after treatment. The tissue sections were stained with hematoxylin and eosin (H&E) and observed by a BX41 bright field microscopy (Olympus, Japan). For immunohistochemistry assay, sample tumors were collected after treatment for subsequent CD31 immunohistochemistry, according to the manufacturer’s instructions (KeyGen Biotech, Nanjing, China). 4.9. Statistical analysis. Significant differences were determined using the Student’s t-test where differences were considered significant when p < 0.05. All data are expressed as mean± standard error of the mean.

ASSOCIATED CONTENT Supporting Information. FTIR and 1H-NMR of CP; zeta potential of CS, CP and CBN; diameter changes of PTX/Su@CBN under different temperatures; plama concentrations of free drugs after intravenous injection with PTX/Su@CBN or PTX+Su; fluorescent images of CS-Flu incubated 18


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MDA-MB-231 and MSCs; MTT assay of MDA-MB-231, HepG2, MCF7 and MDA-MB231/Taxol cell lines; western blot analysis and apoptosis of CS incubated cells; in vivo retention of PTX/Su@CBN.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ∥These authors contributed equally.

ACKNOWLEDGMENTS We acknowledge financial support from the National Natural Science Foundation of China (NSFC 81220108012, 61335007, 81371684, 81000666, 81171395 and 81328012); the 973 Key Project (2015CB755504); and the Priority Academic Program Development of Jiangsu Higher Education.

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Scheme 1. Schematic illustration of the CS-based nanoparticle (CBN) as a tumor-targeting programmable nanosystem for enhancing and sensitizing chemotherapy to drug resistant cancer. (a) Fabrication of the tumor-targeting and programmable drug delivery system. The system is composed of a hydrogel shell and drug-loaded cores. (b) The specific cellular uptake and intracellular fate of CBN. After specific accumulated in tumor cells through CD44 mediated internalization, the outer hydrogel can degrade and the free drugs with synergistic effect release out to induced cytotoxicity rapidly. CS disassociated from hydrogels, causing Bcl-XL downregulation and increased chemotherapeutic response of drug resistant tumor cells. Simultaneously, the drug-loaded cores can provide a second, sustained release and keep an effective concentration for a longer period. 28


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Figure 1. Characterization of the nanosystem (CBN). (a) Morphology of CBN by and transmission electron microscope (TEM); (b) Dynamic Light Scattering (DLS) results of CP, CBN and PTX/Su@CBN; (c) Photographic images of CBN and (d) NIR dye, Cypate, loaded CBN (Cy@CBN) treated with different concentrations of GSH; (e) TEM images of CBN treated with different concentrations of GSH; (f)Drug-release profiles of PTX/Su@CBN in the presence of different concentrations of GSH; (g) DLS results of CBN treated with 10mM GSH after different times.

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Figure 2. Uptake of fluorescein-loaded CBN (Flu@CBN) by cells expressing different levels of CD44. (a) Hyperspectral microscopy and fluorescent images of different cells after a 2 h incubation with Flu@CBN; (b) three-dimensional re-construction image of MDA-MB-231 cells treated with Flu@CBN; and (c) linear fitting between relative fluorescent intensity of Flu@CBN and CD44 expression. (Blue: Hoechst, nuclear dye; Green: fluorescein loaded in CBN).

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Figure 3. Uptake of Flu@CBN in CD44-overexpressing cells in 2D and 3D cultures. (a) Fluorescence images of cellular uptake using 2D cultured cells after incubation with Flu@CBN; (b) Fluorescence images of cellular uptake using 3D cultured spheres of cells after incubation with Flu@CBN. (Red: anti-CD44 antibody; Green: Flu@CBN)

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Figure 4. Tumor-targeting efficacy of Cy@CBN in vivo. (a) Live in vivo fluorescent images of tumor-bearing mice up to 72h post-treatment with Cy@CBN; (b) Fluorescence imaging of tumors and other organs harvested from MDA-MB-231 tumor-bearing mice treated with Cy@CBN and free Cy at 2, 6, and 12 h; (c) Fluorescence imaging of sectioned tissues 12 hours after Cy@CBN treatment; (d) Linear fitting between the tumor/normal tissue ratio of Cy@CBN at 12h and relative CD44 expression of the specific cancer cell type.

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Figure 5. Uptake of Flu@CBN and CD44 expression in tumors and normal tissue. (Blue: Hoechst, nuclear dye; Red: anti-CD44 antibody; Green: Flu@CBN)

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Figure 6. In vitro anticancer activity of PTX/Su@CBN and the mechanism of CS sensitization of drug resistant tumor cells to drugs. (a-d) Apoptosis was analyzed using dual staining with Annexin V and PI. Cells in early apoptosis, late apoptosis, and necrosis are located in the lower right, upper right, and upper left quadrants of the dot plot (Annexin V positive and PI negative, Annexin V positive and PI positive, and Annexin V negative and PI positive), respectively; (e, f) Western blot analysis of COX-2 and Bcl-XL expression after cells were incubated with CS, PTX+Su, and PTX/Su@CBN, and quantitative analysis of the greyscales of different groups; (g) Enzyme-linked immunosorbent assay for NF-κB, COX-2, Akt, and Bcl-XL in cells treated with CS, PTX+Su, and PTX/Su@CBN; (h) Immunofluorescence assay of COX-2 and Bcl-XL expression in CS-treated and control cells; (i) Schematic of intracellular fate of PTX/Su@CBN.

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Figure 7. In vivo antitumor efficacy of PTX/Su@CBN. (a) Images of tumors collected from mice in different cohorts at day 28 post-treatment; (b) Survival rates over time of MDA-MB-231/Taxol tumor-bearing mice after different treatments; (c) Tumor growth curves in response to different treatments; (d) Vascular marker CD31 expression in tumors and H&E stained sections of tumors, spleens, kidneys; (e) Changes in bodyweight of mice following different treatments.

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A CD44-Targeting Programmable Drug Delivery System for

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