Targeted Transmembrane Delivery of Ca2+ via FA-nanogel for

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Biological and Medical Applications of Materials and Interfaces

Targeted Transmembrane Delivery of Ca2+ via FAnanogel for Synergistically Enhanced Chemotherapy Shan-Wen Hu, Jin Wang, Tingting Zhang, Xiang-Ling Li, Hong-yuan Chen, and Jing-Juan Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b04967 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019

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ACS Applied Materials & Interfaces

Targeted Transmembrane Delivery of Ca2+ via FA-nanogel for Synergistically Enhanced Chemotherapy Shan-Wen Hu1,2, Jin Wang1, Ting-Ting Zhang1, Xiang-Ling Li1*, Hong-Yuan Chen1, Jing-Juan Xu1*

1State

Key Laboratory of Analytical Chemistry for Life Science and Collaborative

Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China

2Shandong

Provincial Key Laboratory of Detection Technology for Tumor Markers,

College of Chemistry and Chemical Engineering, Linyi University, Linyi 276005, China

*Corresponding

author:

Email:

[email protected];

+86-25-89687924.

1

ACS Paragon Plus Environment

[email protected].

Tel/fax:

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Metal ions synergistically enhanced chemotherapy is a promising strategy for cancer treatment. However, targeting delivery of ions towards cancer cells remains challenging for decades. Herein, we developed a novel folic acid-nanogel (termed as FA-nanogel) with alkane chains as diffusion barriers for targeted transmembrane delivery of calcium ions into cancer cells. With the aid of hydrophobic diffusion barriers, the FA-nanogel showed a reduced and sustained speed for release of calcium ions, significantly prolonging the ions effect. Moreover, a pH-sensitive injectable hydrogel loaded FA-nanogel and chemotherapeutic drug 5-fluorouracil (5-Fu) was synthesized for investigating the synergistic effect of nanogel on chemotherapy. Both in vitro and in vivo experiments confirmed that the intracellular calcium ions was continuously increased due to the targeted delivery ability and ion sustained release ability of the smart FA-nanogel, and the tumor growth was effectively inhibited by the ions synergistic chemotherapy. This study not only provides a powerful nanoplatform for sustained transmembrane delivery of ions into malignant cells but also creates better conditions for improving the therapeutic efficacy of chemotherapy.

KEYWORDS: calcium ions, diffusion barriers, sustained release, nanogel, enhanced chemotherapy.

2


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1. Introduction Undoubtedly, over the past decades, chemotherapy has achieved great success in clinical cancer treatments1. However, malignant tumors with the relentless growth and metastasis features increase the difficulty of cancer chemotherapy. On the other hand, the side effects and the drug resistance of anti-cancer drugs also limit the efficacy of chemotherapy2-4. Developing

innovative

strategies

to

improve

efficacy

of

chemotherapy is urgent and necessary. Nowadays, the important roles of intracellular calcium signaling have been demonstrated in cancer chemotherapy5. Some interconnections have been found between calcium signaling and multidrug resistance. Florea and coworkers proposed intracellular calcium signaling could modulate expression of multidrug resistant genes which are targets to overcome drug resistance of anticancer drugs6. Moreover, high intracellular calcium concentration also has an anti-metastatic effect of some anticancer drugs causing cell death via apoptosis or necrosis7-9. Continuous supplementation of intracellular calcium has become an effective way to enhance chemotherapy. Simply exposing cells to calcium-related reagents is difficult to modulate intracellular calcium concentration as cells have very complicated mechanism to keep intracellular calcium concentration in physiological ranges. Several methods have been reported to modulate intracellular calcium signal, such as gene editing and using calcium cells to increase calcium channels10-12. A genius work has been proposed to open thermo-sensitive TRPV1 channels and cause Ca2+ influx13. 3



With the development of nanotechnology, using nanocarriers to directly transmembrane deliver ions into cells might offer an attractive pathway to enhance chemotherapy. More recent works about delivery of iron have been reported14,15, but the directly delivery of calcium ions into cancer cells remains challenging. With advantages of high stability, large loading capacity, excellent biocompatibility and biodegradability16,17, nanogels, consisted with three-dimensional crosslinked hydrophilic networks, have been widely applied to deliver drugs, proteins, DNA, RNA, and imaging molecules18-20. However, it is still difficult to use nanogels for ions delivery since the loop size is larger than Ca2+ ions size resulting in fast diffusion of ions and diffusion loss during delivery. Modifying nanogels with hydrophobic interfaces is a key prerequisite to diffusion control21,22, which not only creates barriers to control diffusion, but also provides a possibility to transmembrane delivery of calcium ions by nanogels. Herein, we rationally fabricated a novel targeted transmembrane calcium ions delivery system FA-nanogel, which achieved ions sustained release with the aid of hydrophobic diffusion barriers on the surface of nanogel (Scheme 1). Taking advantage of the diffusion barriers and targeting molecule, the FA-nanogel was able to deliver Ca2+ into cancer cells with sustained outwards speed, which significantly prolonged the positive effect of intracellular calcium ions. On the other hand, the FA-nanogel with chemotherapeutic drug encapsulated in the pH-sensitive injectable hydrogel, exhibited greatly improved therapeutic efficiency of chemotherapy in both in vitro and in vivo experiments. Therefore, the present work demonstrates a 4


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promising concept of ions transmembrane delivery system, which not only successfully overcome burst release of ions but also dramatically enhanced conventional chemotherapeutic drug ability for killing tumor cells.

Scheme 1. Diagram of the targeted transmembrane delivery of Ca2+ via FA-nanogel, resulting in enhanced chemotherapy towards cancer cells.

2. Experimental section 2.1 Materials Acrylamide (AM), N,N’-Methylenebis(acrylamide) (MBA), Sodium dodecyl sulfate (SDS),

ammonium

persulfate

(APS),

N,N,N’,N’-tetramethylethylenediamine

(TEMED), 1-Iodohexadecane, n-docosanoic acid, carbodiimide hydrochloride (EDC), N-hydroxysuccinimide N,N-dimethyl-formamide Poly(ethylene

glycol)

(NHS),

aspartic

(DMF), (PEG,

N,N’-dicyclohexylcarbodiimide,

acid,

hydrazine Mw

2000

5-Fluorouracil

sulfolane,

hydrate, Da), (5-Fu)

5


phosphoric

tetrahydrofuran

p-formyl and

benzoic

acid, (THF), acid,

4-(dimethylamino)


pyridine were analytically pure from Sigma and used as received without further purification. Folic acid and amido modified poly(ethylene glycol) (Mw 2000 Da, FA-PEG-NH2) was purchased from Haoyang Biotech (Shanghai, China). Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were bought from Invitrogen. 2.2 Synthesis and modification of FA-nanogel The nanogel was fabricated under a modified emulsion polymerization reaction22,23. 0.305 g AM was dissolved in 25 mL water with 0.00629 g SDS and 60 μL MBA. The mixture was stirred for 45 min under N2 atmosphere at room temperature. Then 1 mL water with 20 μL APS and 2.5 μL TEMED was added dropwise and the reaction last for 2 h. The reaction mixture was centrifuged to remove the unreacted reagents and washed three times before the solvent was replaced by 3 M calcium chloride (CaCl2) in water. From this point until the nanogel was added to hydrogel, the water used all contained 3 M CaCl2. Then the precipitate was transferred to dichloromethane containing 1 g mL-1 1-Iodohexadecane for 2 h before centrifugation and wash. 100 μL, 0.1g mL-1 behenic acid in dichloromethane was added to the nanogel and stirred for another 3 h before centrifugation and wash. After that, the product was activated by 200 μL EDC/NHS (40 mg/mL, 20 mg/mL) for 3 min and reacted with FA-PEG-NH2 for 12 h at 4 °C, followed by centrifugation and wash, dispersed in 1 mL PBS. 2.3 Synthesis of pH-responsive hydrogel The hydrogel was prepared from the precursors dibenzaldehyde-terminated PEG 6


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(DF-PEG) and polyaspartylhydrazide (PAHy). The synthesis of DF-PEG and PAHy were illustrated as the following procedure. DF-PEG was synthesized according to a previously reported work24. Briefly, THF(200 mL), DMAP (0.1 g), p-formyl benzoic acid (1.96 g, 6.52 mM) and N,N’-dicyclohexylcarbodiimide (3.36 g, 8.15 mM) were added to PEG2000 (6.52 g, 1.63 mM). The reaction lasted 24 h at 25 °C with N2 atmosphere. The generated white solid was precipitated with diethyl ether, and then recrystallized by THF and diethyl ether for three times. Finally, the product was dried under room temperature and stored for further use. The other precursor PAHy was prepared from poly(succinimide) (PSI). PSI was synthesized following a previous reported method25: L-Aspartic acid (40 g, 0.30 mol) was dispersed in sulfolane (200 mL) together with phosphoric acid (15 mmol) and stirred at 170 °C under N2 atmosphere. After 10 h, the reaction mixture was precipitated in excess methanol and successively washed with water until the pH of the suspension became neutral. The precipitate was washed with methanol and then dried at 80 °C in vacuo. Then PAHy was obtained from PSI via the following procedure26. PSI (3 g) was dissolved in DMF (40 mL) together with hydrazine hydrate (4.8 mL, 98.8 mmol), the mixture was stirred under 25 °C for 4 h. The solid product was filtered and washed with acetone, followed by recrystallization before dried out. For the formation of pH-responsive hydrogel, PAHy solution (3%, w/w in PBS) and DF-PEG solution (20%, w/w in PBS) were mixed together at a volume ratio of 7



1:1. 2.4 Cell culture and apoptosis Assay Normal immortalized human mammary epithelial cells (MCF-10A cells) and human cervical cancer cells (HeLa cells) were cultured and propagated at 37 °C with 5% CO2 atmosphere in a standard cell incubator. The cell culture medium contained DMEM mixed with 10% fetal bovine serum, 100 mg/L penicillin and 100 mg/L streptomycin. To determine the cytotoxicity of nanogel, the cell viability assay was performed using cytotoxicity detection kit. HeLa cells were digested and seeded in 96 well plates (1×104 per well) for 24 h. Then cells were incubated with DMEM, nanogel and FA-nanogel for 24 h, respectively. After that, the culture medium was discarded and the cells were washed with PBS for three times. Subsequently, MTT regents and DMSO solution were added to each well according to the manufacturer’s instructions. Finally, the absorption intensity at 540 nm was measured by using a Thermo Scientific Varioskan Flash 2.5 The properties of FA-nanogel in vitro HeLa cells and MCF-10A cells at the exponential growth phase were transferred to the petri dish for confocal laser scanning microscopy (CLSM). The control group was incubated at normal cultured environment without extra treatment. The cells of test sets (HeLa cells and MCF-10A cells) were incubated with FA-nanogel-Ca2+. Firstly, the FA-nanogel-Ca2+ (100 μg/mL, 100μL) was centrifuged, washed three times and dispersed in PBS. Then all cells in the test groups were incubated with FA-nanogel-Ca2+ for 12 h, followed by furo-2 staining and confocal laser scanning. 8


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For investigating the enhanced efficacy of chemotherapy via FA-nanogel-Ca2+ in vitro, HeLa cells (1×105 per well) were seeded in 6-well plates and cultured in the cell medium without buffer system for 24 h. Then the cells used were divided into four groups for flow cytometric analysis: (a) cultured with cell culture medium (as control group); (b) cultured with hydrogel containing FA-nanogel-Ca2+ only; (c) cultured with hydrogel containing chemotherapeutic drug 5-Fu only; (d) cultured with hydrogel containing FA-nanogel-Ca2+ and drug 5-Fu. The cells incubated with hydrogel in the fourth group were performed as the following procedure. Reagent A refers to PAHy dissolved in the prepared PBS containing nanogel (3%, w/w) and reagent B refers to DF-PEG solution in PBS containing 5 μM 5-Fu. Then reagent A (80 μL) and reagent B (80 μL) for the formation of hydrogel were mixed and added to cells at the exponential growth phase in 2 mL cell culture medium for each well. Besides, the hydrogel complexes for the second or the third group were formed by the above procedure just utilizing the reagent B without drug 5-Fu or the reagent A without nanogel, respectively. After 24 h incubation, the supernatant medium was discarded, and the cells were washed by PBS for three times. Then the cells were harvested, redispersed in PBS, stained with annexin V-FITC and propidium iodide (5 μL/sample) for 10 min at 37 °C, then further loaded to the Beckman Coulter FC500 Instrument. 2.6 The therapeutic efficacy of FA-nanogel enhanced chemotherapy in vivo In vivo therapeutic effect was performed on NU/NU mice (16-18 g) obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. HeLa cells were injected into the flanks of mice subcutaneously at density of 5×106 cells per mouse. When the 9



volume of tumors reached 80 mm3, the mice were divided into three groups with various conditions: PBS injection as control group (i), injection of drug 5-Fu (chemotherapy alone group, ii), injection of hydrogel loaded FA-nanogel and drug 5-Fu (enhanced chemotherapy group, iii). The injection of hydrogel into the mice was performed as the following procedure. Reagent A without FA-nanogel-Ca2+ (for the second group) or with FA-nanogel-Ca2+ (for the third group) and reagent B contained chemotherapeutic drug 5-Fu were injected separately to the same point around tumor in mice immediately. In total 21 days treatment, the nanomaterials were injected subcutaneously around the tumor site in mice and executed every 72 h, meanwhile the tumor volume was measured every 48 h. After 21 days, the tissues (liver, heart, spleen, kidney and lung) obtained from the sacrificed mice in different experimental groups were used for hematoxylin and eosin (H&E) assay. All animal operations were in accord with institutional animal use and care regulations approved by the Model Animal Research Center of Nanjing University (MARC). 2.7 Statistical analysis The results were presented as mean ± standard deviation. The statistical calculations was implemented using Student's t test via the statistical software SPSS (version 24.0). *P-values < 0.05 were considered significant, values of ** P-values < 0.001 were considered highly significant.

3. Results and discussion 3.1 FA-nanogel for targeted calcium ions delivery 10


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The strategy for synthesis of nanogel with hydrophobic diffusion barriers was illustrated in Fig. 1a (step i and ii). Firstly, the unmodified nanogel nanoparticles were rationally designed and successfully fabricated by emulsion polymerization reaction of AM and MBA, with APS and TEMED as reaction trigger, simultaneously coupled with SDS as reaction assistor. After 2 h, the nanogel with excellent hydrophilicity was gained. While aiming at sustained delivering calcium ions to cells across the membrane, the nanogel as nanocarrier should be equipped with hydrophobic diffusion barriers. The n-alkylation reaction between AM and 1-Iodohexadecane was utilized for the modification of hydrophobic diffusion barriers on the surface of nanogel. Briefly, the unmodified nanogel, which was synthesized in aqueous phase, was dispersed in dichloromethane (oil phase). Due to the high water content in the nanogel, the dichloromethane was prevent from penetrating into nanogel, hence the n-alkylation reaction was confined at the surface of nanogel and turned the surface of nanogel into hydrophobicity. Next, the size distribution of the as-prepared nanogel was determined by dynamic light scattering (DLS) measurements. Results demonstrated that the size distribution of nanogel was mainly in the range of 30-70 nm, and the average size was approximately 50 nm (Fig. 1b). Subsequently, the capability of nanogel with diffusion barriers for the calcium ions diffusion was investigated. Firstly, for loading of calcium ions, the nanogel delivery systems (with hydrophobic surface) and the unmodified nanogel (without hydrophobic surface) were filled with calcium chloride (CaCl2) during synthesis process, got rid of the redundant ions by centrifugation, washed and transferred two 11



types of nanogel into water, respectively. Then, the amount of calcium ions released from the nanogel or the unmodified nanogel was collected by centrifugation at different sampling time. Hence the supernatant was determined by ICP-MS. It can be seen that both of the calcium delivery systems had an obvious calcium ions release process at the first sampling time. With time prolonged, the release speed of the nanogel was much slower than that of the unmodified nanogel, and reached a nearly constant speed after 3 h (light gray column, Fig. 1c). Meanwhile, the release time of the nanogel with diffusion barriers can last to 24 h, achieving a sustained calcium ions release process. In comparison, the amount of released calcium ions from the unmodified nanogel was ultralow and undetectable by ICP-MS after 3 h (black column). All results revealed that the nanogel with diffusion barriers showed a more lasting and sustained ions release ability than that of the unmodified nanogel, which successfully avoided the explosive release and prolonged the ions effect. The differences of the release speed and release time between the nanogel (with diffusion barriers) and unmodified nanogel (without diffusion barriers) may be attributed to the following reasons. First of all, ions are much smaller than the loop size of gel network in unmodified nanogel and will get out of the nanogel due to the concentration gradient towards the outside, which is in agreement with the common feature of hydrogels21. When it comes to the nanogel modified with hydrophobic surface, the super-hydrophobic barriers herein acts as a wall, resulting in the separation of the interior domain of nanogel and the outside water environment, which will cut out the molecular transport across the nanogel, and thus significantly 12


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reduce the molecular diffusion speed. To maximize the selectivity of nanogel on specific malignancies and minimize the side effect to normal tissue, further modifications of nanogel for improving the efficiency of targeting cancer cells is necessary. As illustrated in Fig. 1a (step iii and iv), the hydrophobic surface of nanogel was carboxylated by behenic acid and then covalently bonded with the target molecule-folic acid and amido modified poly(ethylene glycol) (NH2-PEG-FA) for obtaining the intact ions delivery system FA-nanogel. Our previous work have revealed that FA can act as the targeting moiety for cancer cells27, as the molucule FA was highly bound to the folate receptor (FR), which is overexpressed in many types of malignant tumor28,29. Then, the calcium ions sustained release property of the FA-nanogel was also investigated by ICP-MS. As shown in Fig. 1c, there was no significant change of the release rate of calcium ions from the FA-nanogel (dark gray column), verifying that the modification of target molecules had no adverse effect on the release manner of calcium ions from the nanogel. Meanwhile, the surface of nanogel was converted from hydrophobicity into hydrophilcity after the modification of targeted functional group, which significantly enhanced the stability and biocompatibility of nanogel in complex physiological environment

26,30.

Furthermore, the cell viability assay verified this hypothesis (Fig.

S1). Nearly 90% of the HeLa cells survived after incubation with nanogel or FA-nanogel for 24 h, showing that the nanogel exhibited excellent biocompatibility and without inherently cytotoxicity for the cells.

13



Fig. 1 FA-nanogels with diffusion barriers. a) Schematic illustration for the formation of nanogel (step i and ii) and FA-nanogel (step iii and iv); b)The DLS results of the modified nanogels; c) The release curves of Ca2+ from nanogel without diffusion barriers, nanogel with diffusion barriers and FA-nanogel.

To assess the target efficiency and specificity of the FA-nanogel, confocal laser scanning microscopy microscopy (CLSM) of normal cells (normal immortalized human mammary epithelial cells, MCF-10A cells) and cancer cells (human cervical cancer cells, HeLa cells) were performed. In the experiments, three groups of cells were used. The first group (HeLa cells) as the control set was cultured under regular conditions supplied with complete cell culture medium, the second group (HeLa cells) and the third group (MCF-10A cells) as the test sets incubated with FA-nanogel loaded with calcium ions (named as FA-nanogel-Ca2+) for 12 h, and then washed with fresh cell medium. After these processes, the intracellular calcium concentration of each group was determined by Fura-2, a commercial Ca2+ fluorescent indicator, whose fluorescence intensity is linear corresponding with calcium ions concentrations within certain range. In order to clearly clarify the Ca2+ delivery and tumor targeting 14


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property of FA-nanogel-Ca2+, the fluorescence signal of control group was also detected, which was used as comparison to show the inherent amount of intracellular calcium. As presented in Fig. 2, an obvious increment of fluorescence intensity from Ca2+-Fura-2 chelator was witnessed in the second group cell (HeLa cells), which was quantized to be 4 folds higher than the untreated cells (Fig. S2). This proved that vast FA-nanogel-Ca2+ was ingested by cancer cells and delivered calcium ions into cells. Nevertheless, the third group normal cells treated with the same process produced the final result differently. Even after incubation with FA-nanogel-Ca2+ for 12 h, extremely negligible red fluorescence signal was observed in the normal MCF-10A cells, demonstrating that the smart ions delivery system (FA-nanogel-Ca2+) can discriminate normal cells and cancer cells, and specifically target cancer cells with overexpression of folate receptor. Given these evidence, we claimed the FA-nanogel was capable of targeted transmembrane delivery of ions to cancer cells.

Fig. 2 Targeted transmembrane delivery of calcium ions to cancer cells. Confocal images of HeLa cells without treatment as control, HeLa cells and MCF-10A cells incubated with FA-nanogel-Ca2+. The images from left to right represent: bright, fluorescence and merged images, respectively.

15



3.2 pH-responsive hydrogel for nanogel and drug delivery Attractively, recent reports have revealed that there is a strong correlation between intracellular calcium concentrations and chemotherapy of malignant tumor and high intracellular calcium concentration can significantly alleviate multidrug resistance and enhance the therapeutic efficacy8. Considering the excellent performance of FA-nanogel for targeted delivery of Ca2+ into cancer cells, we supposed FA-nanogel could be used as an assistor of chemotherapy for beating cancer. In order to investigate the potential capability of the smart FA-nanogel in promoting therapeutic efficacy, we synthesized pH-responsive hydrogel as carriers for simultaneously loading of sustained calcium ions delivery system FA-nanogel and various chemotherapeutic drugs. The pH-responsive hydrogel was synthesized following a typical method31, and the products of each step were characterized by 1H NMR spectrum (Fig. S3 and Fig. S4). The crosslinks in the hydrogel formed by PEG dialdehyde and α,β-polyaspartylhydrazide were covalent acylhydrazone bonds, which can be easily hydrolyzed in an acidic environment and further result the disintegration of the hydrogel. It is well-known that low pH value is the unique features of tumor microenvironment32-34, therefore this hydrogel is expected to act as an acidic pH-activatable carriers for controllable nanogel and drug release in tumor site and prevent the damage to normal cells, which greatly mitigate the side effects. Subsequently, various pH values aqueous buffer solutions, which mimicked the microenvironments of tumor tissue (pH 5.5 and 6.5) and healthy tissue (pH 7.4), were utilized for investigating the release profile of pH-responsive hydrogel. Firstly, upon 16


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the scanning electron microscope (SEM) characteristic, we measured the release profiles of nanogel from the hydrogel under different pH values. As shown in Fig. 3a, vast nanogel released from the hydrogel in acidic solution. With higher pH values of buffer solutions, the release amounts of nanogel were significantly decreased and no leakage of nanogel from the hydrogel was observed in the neutral solution (Fig. 3b and c). Meanwhile, for observing the chemotherapeutic drug controllable release from hydrogel, the absorption intensity of the cumulative release amount was collected at different time intervals. As can be seen in Fig. 3d, in the acidic solution mimicking tumor microenvironment (pH 5.5), high absorbance intensity of 5-Fu representing a high concentration of released drug was detected in the initial 5 h (curve blue),35 which indirectly revealed that vast encapsulated drug 5-Fu was released from the hydrogel (curve blue). With time prolonging, the cumulative drug release amount continuously increased, revealing that more and more the hydrogel was disintegrated in the acidic environment. In other buffer solutions, the released trend of entrapped drug from hydrogel was similar to that of the nanogel. The amount of released drug from hydrogel was decreased in the mildly acidic condition and just about half-released amount of the previous condition (pH 6.5, curve red). In the neutral condition, the hydrogel exhibited excellent stability and just few drugs were leaked from the hydrogel even over a period of 10 h (curve black). These outcomes illustrated that the pH-activatable hydrogel owned the potential capacities of preventing encapsulated cargo randomly leaking and targeting release the cargo at the target area, which could significantly mitigate side effects of healthy tissue and 17



maximize the therapeutic efficacy of tumor tissue.

Fig. 3 pH triggered nanogel and drug released from the pH-responsive hydrogel. (a-c) nanogel collected from hydrogel at pH 5.5(a), 6.5(b) and 7.4(c); (d) The release curves of drug 5-Fu from the pH-responsive hydrogel at different pH.

3.3 FA-nanogel enhanced chemotherapy in vitro To evaluate the efficacy of FA-nanogel-Ca2+ enhanced chemotherapy, quantitative analysis of cell apoptosis was performed after treating the malignant tumor cells (HeLa cells) with different conditions: (a) cultured with cell culture medium (as control group); (b) cultured with hydrogel containing FA-nanogel-Ca2+ only; (c) cultured with hydrogel containing chemotherapeutic drug 5-Fu only; (d) cultured with hydrogel containing FA-nanogel-Ca2+ and drug 5-Fu. To intuitively display the therapeutic efficiency, a dual stain strategy (FITC-annexin V/propidium iodide for labelling the early stage and late stage of cell apoptosis) was utilized to discriminate different stages of cell apoptosis by flow cytometric analysis. Annexin V is able to 18


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bond with phosphatidylserine (PS), a biomarker locates in inner side of the cell membrane and turns to the outer side of cell membrane in the early stage of apoptosis. And propidium iodide (PI) can just specifically enter the nucleus of the late apoptotic cells. As shown in Fig. 4, cells treated with FA-nanogel-Ca2+ alone (group b) exhibited comparable high cell viability as the control group a, and the total percentage of apoptotic cells in group b was less than 5%. The results demonstrated that the smart FA-nanogel-Ca2+ owned excellent biocompatibility and with negligible injury to cultured cells. A slightly higher apoptosis percent almost up to 29.4% was observed in the free drug 5-Fu treatments group, meaning that 5-Fu as a widely used anticancer drug was valid to cancer cells. In comparison, the total apoptosis percent of cells in group d, simultaneously treated with drug and nanogel, was drastically up to 75%, which was 45.6% higher than that of free 5-Fu treated group and was nearly 70% higher than that of FA-nanogel-Ca2+ treated group. These inspiring results validated that the calcium ions released from FA-nanogel-Ca2+ enhanced the therapeutic efficiency of chemotherapy and achieved synergistic effect in vitro.

19



Fig. 4 Flow cytometric analysis results of HeLa cells. (a) the control group, (b) cells treated with hydrogel containing FA-nanogel-Ca2+ only; (c) cells treated with hydrogel containing drug 5-Fu only (d) cells treated with hydrogel containing drug 5-Fu and FA-nanogel-Ca2+. Region Q1 represents live cells (AnnexinV-FITC-/PI-), region Q2 represents early apoptotic cells (AnnexinV-FITC+/ PI-), region Q3 represents late apoptotic cells (AnnexinV-FITC+/PI+), while quadrant Q4 represents necrosis cells (AnnexinV-FITC-/PI+).

3.4 In vivo therapeutic regimen in tumor-bearing mice Before evaluating the overall anticancer performance of the nanosystem on live animal models, we firstly studied the formation performance of the pH-responsive hydrogel in vivo. As the precursor PAHy and DF-PEG for hydrogel were water soluble with low viscosity and the hydrogel was only emerged when the precursor met together, which was beneficial for biotic injection. Then the precursor polyaspartylhydrazide (PAHy) and dibenzaldehyde-terminated PEG (DF-PEG) for hydrogel were implanted subcutaneously in mice and no abnormal behaviour of mice was witnessed, showing the excellent biocompatibility of these materials. After 20


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subcutaneous injection, the resulting hydrogel was strip off from the sacrificed mice for scene imaging and the weight assay. As illustrated in Fig. S5, the morphology image verified the successful formation of the designed hydrogel in vivo, and the detailed weight assay also revealed that the yield rate was up to more than 85% in reference with the injection amount. To study the biodistribution of the Ca2+ delivery system FA-nanogel in vivo, the fluorophore R6G labeled nanogel (FA-nanogel-R6G) with similar properties to FA-nanogel-Ca2+ was encapsuled in the hydrogel. Afterwards, the hydrogel were subcutaneously injected to the tumor-bearing mouse outside the tumor tissue and the fluorescence signal of the system with time was measured by in vivo imaging system. As shown in Fig.5, an obviously higher fluorescence signal in tumor than that of injection position was observed after 2 h, revealing that the high enrichment and uptake efficiency of nanogel in tumor site. With incubation time prolonged, the signal of nanogel in the tumor site was gradually decreased and the residual fluorescence signal was found around the emunctory after 72 h later (Fig. S6). The results suggested the FA-nanogel can be metabolized in tumor-bearing mice in 72 h, which provides accurate guidance for administration frequency and dosage of each time.

Fig. 5 In vivo imaging of tumor-bearing mice injected with FA-nanogel-R6G at different sampling time: (a) before injection; (b) 2 h after injection; (c) 72 h after injection. 21



Based on these motivating results, we further investigated the therapeutic efficacy of FA-nanogel-Ca2+ enhanced chemotherapy on NU/NU mice bearing HeLa tumor. When the tumor volume reached approximately 80 mm3, all mice treated with different conditions were divided into three groups: PBS as control group (i); the hydrogel loaded with drug 5-Fu as chemotherapy alone group (ii); the hydrogel loaded with FA-nanogel-Ca2+ and drug 5-Fu as Ca2+ enhanced chemotherapy group (iii). To qualitatively evaluate the therapeutic efficacy of FA-nanogel-Ca2+ enhanced chemotherapy, the tumor sizes of the mice conducted with different treatments were continuously monitored by a caliper for 21 days. The volume of tumor (TV) was calculated by TV=1/2× (tumor width)2 × tumor length36. As illustrated in Fig. 6a, the tumor of saline injected mice in the control group grew rapidly and the relative tumor volume (V/V0) was high up to 4.9±0.2 (curve black) at the end of treatment. Moderate growth inhibition of tumor was observed in the free drug treated group, meaning that the chemotherapy utilized drug 5-Fu alone had modest effect in cancer therapy. Interestingly, the tumor growth in the FA-nanogel-Ca2+ enhanced chemotherapy group was effectively repressed. And remarkably tumor growth inhibition (TGI=1Vtest/Vcontrol)37 rates of the third group was high up to 80.0% and 67.7% compared to the PBS control group and chemotherapy only group. The results revealed that Ca2+ released from the ions transmembrane delivery system FA-nanogel-Ca2+ significantly improved antitumor efficacy of drug 5-Fu and achieved enhanced synergistic antitumor effect in vivo. Meanwhile, the representative photographs of excised tumor tissue from mice in different groups also verified that the best therapeutic outcome 22


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was achieved in the FA-nanogel-Ca2+ combined drug 5-Fu group, suggesting that the Ca2+ can significantly enhance the therapeutic efficacy of chemotherapy and attain synergistic effect in antitumor treatment (Fig. 6b).

Fig. 6 In vivo therapeutic regimen in tumor-bearing mice. (a) The relative tumor volume (V/V0) of mice in each group changing with time and (b) representative photos of tumor tissues obtained after the total treatments. The tumor-bearing mice were treated as: (i) injection with PBS (control group), (ii) injection with hydrogel containing drug 5-Fu, (iii) injection with hydrogel containing drug 5-Fu and FA-nanogel-Ca2+. *P < 0.05, significance; **P < 0.01, high significance.

Moreover, the bodyweight of the mice were continuously monitored during the whole treatments to evaluate the systemic toxicity of the material, which was critical factor in biological studies and therapeutic applications. No obvious differences in bodyweight of mice among each group were observed (Fig. S7), suggesting no acute side effects was caused by the FA-nanogel-Ca2+. Further hematoxylin and eosin (H&E) staining was performed for the typical organs of the mice at the end of the treatments. As shown in Fig. 7, no detectable tissue damage or adverse effect of the major organ was observed, revealing the relatively safety of the ions transmembrane delivery system.

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Fig. 7 H&E stain of major organs from control group (i) and FA-nanogel-Ca2+ combined chemotherapy group (ii).

Conclusions In summary, we have developed a diffusion barrier modified ions transmembrane delivery system FA-nanogel, which preferentially release calcium ions into cancer cells and simultaneously enhance chemotherapy in oncotherapy. With the diffusion barrier, the FA-nanogel released Ca2+ in a sustained manner, which successfully avoided the explosive release and prolonged the ions effect. Incorporating the newly formulated

FA-nanogel-Ca2+

with

chemotherapy

based

on

the

injectable

pH-responsive hydrogel, in vivo anticancer therapy was conducted, achieving remarkably improved synergetic treatment effect compared to chemotherapy alone and simultaneously presenting better tumor inhibition. Another outstanding feature of the FA-nanogel was that the nanogel can be thoroughly metabolized within 3 days, which significantly depressed the toxic side effects on normal issue and organs in oncotherapy. Taken together, these positive results suggest that the FA-nanogel could not only be applied to the development of sustained ions-delivery systems but also in the fields of ions enhanced synergistic anticancer research.

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Acknowledgment We gratefully acknowledge the National Natural Science Foundation (Grants 21327902, 21535003, 21605072) of China and the Natural Science Foundation of Shandong Province (ZR2017PB004). This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Supporting Information Available: The details of materials characterization, the fluorescent intensity of nanogel or FA-nanogel-R6G, the formation of hydrogel and the body weight tests in vivo could be found in supporting information, which is available free of charge via the Internet at http://pubs.acs.org. Conflict of interest: The authors declare no competing financial interest.

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For TOC only:

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Targeted Transmembrane Delivery of Ca2+ via FA-nanogel for

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