Prodrug-Based Cascade Self-Assembly Strategy for Precisely

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Biological and Medical Applications of Materials and Interfaces

Prodrug-based Cascade Self-assembly Strategy for Precisely Controlled Combination Drug Therapy Wei Ha, Xiao-bo Zhao, Xin-yue Chen, Kan Jiang, and Yan-Ping Shi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05170 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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ACS Applied Materials & Interfaces

Prodrug-based Cascade Self-assembly Strategy for Precisely Controlled Combination Drug Therapy Wei Ha‡, Xiao-bo Zhao‡, Xin-yue Chen, Kan Jiang and Yan-ping Shi* CAS Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Lanzhou 730000 (P. R. China). KEYWORDS: prodrug, progressive self-assembly, supramolecular hydrogel, cascade release, combination therapy

ABSTRACT: The development of co-delivery systems for combination therapy that can load different drugs in single carrier and precisely deliver payloads (ratio and administration time) via programmable administration has proven to be challenging. By taking advantage of the increased dimension or space from particles self-assembly approach, we have developed a prodrug-based cascade self-assembly strategy to construct supramolecular hydrogel that can load different drugs in stages, and yet temporally/spatially release drugs by cascade disassembly of supramolecular hydrogel under different microenviroment. The cascade self-assembly mechanism has been investigated in detail by morphology evolution of prodrug micelles. Using tumor cell uptake, cytotoxicity assay and a tumor-bearing animal model, the effectiveness of the prodrug micelle-

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based cascade self-assembly system was studied, such as loading, controlling the drug ratio, and the administration time for possible therapeutic applications. These studies fully demonstrate the proof of concept, and open up an attractive new way to construct multidrug loaded carriers for combination therapy.

INTRODUCTION It is undeniable that nanoparticles-based drug delivery systems have become the most attractive strategy in systemic cancer drug delivery due to their great potential in improving drug solubility, prolonging drug circulation half-life, especially, better accumulation at the tumors.1,2 Many nanoparticles which are focused on single drug encapsulation, such as micelles,3,4 polymersomes5,6 liposomes,7,8 and inorganic nanoparticles,9,10 etc., have been developed to actively deliver drug to special tissues or organs. However, cancer treatment depending on a single drug remains suboptimal. In clinic, two or more kinds of therapeutic agents with different mechanisms have been administered in combination to synergistically prohibit cancer development.11-14 Therefore, great effort has been devoted to develop nanoparticle-based drug carriers which can combine two or more therapeutic approaches for combination therapy.15-21 These carriers have shown potential in loading different therapeutic agents and controlling their ratio. However, to realize their optimal synergetic effect, it is expected the different therapeutic agents should be administered one after another and, ideally, be released in different position of tumor cells for combination therapy.22 Generally, two or more different therapeutic agents can only be loaded on the same location of nanoparticles-based drug carriers due to their limited structure space, which is hard to control the release sequence and position of therapeutic drugs. Therefore, constructing superstructures using


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nanoparticles as building blocks, in other word, increasing the dimension of nanoparticles-based carriers, will be particularly useful for regulating the loading region for multi drugs due to the additional space for carriers, and controlling the release sequence as well as release position via different loading mechanism (physically trapping, non-covalent adsorption, covalent linkage, etc.). In recent years, using nanoparticle-based self-assembly strategy to construct multidimensional superstructures has drawn much more attention as an active and fruitful scientific area.23,24 Particle self-assembly could control the arrangement of particles by diffusionlimited and/or reaction-limited aggregation mechanism and lead to superstructures with better functionalities over those of original particles.25-30 Self-assembled superstructures maintained the original advantages of nanoparticles, and will not only provide more different space for drug loading but also significantly increase the design diversity for temporally and spatially cascade release of drugs. However, although a large number of particle self-assembly systems have been developed, much less effort has been devoted to their application in drug delivery area, particularly for combination therapy. Prodrug micelles, compared with other nanoparticles, are much more capable of loading different drugs at different position in carriers via different loading mechanism. Amphiphilic polymer-drug conjugates based prodrug micelles could efficiently load hydrophobic drugs at first due to its inherent structural features. Subsequently, the functionalized polymer at the surface of micelle can also be able to further self-assembly into superstructure by modulating inter-particle interaction caused by the change of solubility, configuration and/or chemical reaction of polymers. During the entire self-assembly process, another drug could be easily loaded into progressive self-assembled superstructures due to the increased dimension and/or space. Such a strategy can lead to a dual drug loaded superstructures with functionalities entirely different from


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Figure 1. Design concept for controlled combination drug therapy based on progressive selfassembly strategy using prodrug micelle as building blocks. or superior to these prodrug micelles, which is particularly useful for controlling the release sequence of different drug (Figure 1). As a proof of concept, the disulfide bond connected PEG modified camptothecin (CPT) prodrug micelles was selected as model particles in this work. The glutathione (GSH)-responsive cleavage of disulfide linkage is an extremely valuable stimulus inside cells due to the fact that the GSH concentration in the intracellular space is 2 to 3 orders higher than that in the extracellular fluids.31 An injectable three-dimensional hydrogel was further obtained successfully via the progressive self-assembly process after introduction of αCD. In supramolecular chemistry, the inclusion complexes formed by PEG chain threading a series of α-CD has become a very attractive and valuable model to construct self-assembled hydrogels and has been extensively applied in the biomedical field.32 The self-assembled supramolecular hydrogel further provides a suitable environment for entrapping another anticancer drug 5-flrorouracil (5-FU) which possess excellent water solubility. The cascade release of two drugs at different times and positions could be precisely controlled by their different loading mechanism as well as the programmed dissociation of hydrogel.


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EXPERIMENTAL SECTION Chemical synthesis and characterization. Synthesis of PEG-COOH: The PEG-COOH was synthesized via our previously reported method and its structure confirmed by direct comparison with our previously reported spectroscopic data.33 Scheme 1 Synthetic route of the PEG-COOH

O

OH n

+

O

O

O

O

O n

PEG-2000

COOH O

PEG-COOH

Scheme 2. Synthetic route for the CPT-S-S-OH

Synthesis of CPT-S-S-OH: CPT (0.20 g) and DMAP (0.21 g) were suspended in 15 mL dry CH2Cl2 under a N2 atmosphere. Triphosgene (56 mg) was then added under ice bath and stirred for 2 h at 0 °C. After that, 2 mL of dry THF solution containing 0.13 g 2-Hydroxyethyl disulfide was added dropwise and further stirred for 16 h at 0 °C. After filtration and removal of all the solvents by rotary evaporation, the residue was diluted with AcOEt (15 mL). The mixture was then washed with water, 0.1 N HCl and brine, respectively. After the removal of solvent in organic layer, the crude product was separated by silica column chromatography to give a pale yellow powder (CPT-S-S-OH, yield: 61%).1H NMR (400 MHz, CDCl3):8.43 (s, 1H), 8.23 (d,


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J=8.4 Hz, 1H), 7.95 (d, J=8.4 Hz, 1H), 7.84-7.87 (m, 1H), 7.67-7.71 (m, 1H), 7.44 (s, 1H), 5.71 (d, J=17.2 Hz, 1H), 5.29-5.41(m, 3H), 4.35-4.40 (m, 2H), 3.88-3.93 (m, 2H), 2.86-3.00 (m,4H), 2.16-2.31 (m, 2H), 1.02 (t, J=7.2 Hz, 3H).HRMS calcd for C25H25N2O7S2: 529.1083 [M+H]+; found, 529.1098 [M+H]+. Scheme 3. Synthetic route for the PEG-S-S-CPT O N

O O

N

O

O

S

S

+

OH

O

O

n

O CPT-S-S-OH

COOH O

PEG-COOH O

N

O

DCC, DMAP DCM

O

O N

O

O

S

S

O

O

O PEG-S-S-CPT

O

O n

Synthetic of PEG-S-S-CPT: To a solution of CPT-S-S-OH (0.53 g) and PEG-COOH (3 g) in anhydrous CH2Cl2 (200 mL), N,N′-dicyclohexylcarbodiimide (DCC, 0.41 g) and 4Dimethylaminopyridine (DMAP, 0.18 g) was added under the ice bath. The reaction was then quenched with methanol after stirring overnight at room temperature. The reaction was filtered and the filtrate was concentrated in vacuum. The resulted residue was separated by silica column chromatography to give a yellow solid. (PEG-S-S-CPT, yield: 79%).1H NMR (400 MHz, CDCl3): δ 8.40 (s, 1H), 8.19 (d, J=8.4 Hz, 1H), 7.94 (d, J=8.4 Hz, 1H), 7.82-7.851 (m, 1H), 7.657.69 (m, 1H), 7.32 (s, 1H), 5.69 (d, J=17.2 Hz, 1H), 5.30-5.40 (m, 3H), 4.35-4.36 (m, 2H), 4.204.28 (m, 4H), 3.59-3.70 (m), 3.36 (s, 3H), 2.84-2.94 (m, 4H), 2.58-2.61 (m,4H), 2.12-2.28 (m, 2H), 0.97-1.01 (m, 3H). 13C NMR (100 MHz, CDCl3): δ 172.1, 171.9, 167.2, 157.2, 153.4, 152.3,


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148.9, 146.5, 145.6, 131.2, 130.7, 129.7, 128.5, 128.2, 128.2, 128.1, 120.2, 95.9, 71.9, 70.5, 69.0, 67.0, 66.5, 63.8, 62.4, 59.0, 50.0, 37.1, 36.6, 31.9, 7.6. Synthetic of CyNH-S-S-PEG: To a mixture of CyNH-S-S-OH (0.59 g, 1 mmol) and PEGCOOH (3 g, 1.5 mmol) in anhydrous CH2Cl2 (200 mL), DCC (0.41 g) and DMAP (0.18 g) was added under the ice bath. The reaction was then quenched with methanol after stirring overnight at room temperature. The reaction was filtered and the filtrate was concentrated in vacuum. The resulted residue was separated by silica column chromatography to give CyNH-S-S-PEG, yield: 51%. 1H NMR (400 MHz, CDCl3): δ8.63 (d, 1H, J=15.2), 7.35-7.50 (m, 5H), 6.94-7.01 (m, 4H), 4.75 (s, 2H), 4.29 (s, 2H), 3.44-3.82 (m), 3.37 (s, 3H), 3.05-3.12 (m, 8H), 2.88-2.94 (m, 4H), 2.81-2.83 (m, 2H), 2.73-2.74 (m, 2H), 1.94-2.01 (m, 4H), 1.81 (m, 9H);

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C NMR (100MHz,

CDCl3):δ 178.8, 172.1, 170.5, 159.5, 153.1, 152.5, 146.3, 142.2, 141.5, 130.5, 129.4, 128.1, 127.9, 122.4, 119.8, 118.9, 115.9, 113.6, 109.4, 107.3, 71.8, 70.6, 69.0, 64.1, 59.0, 51.0, 45.8, 45.8, 29.7, 29.3, 28.9, 28.3, 21.8, 11.6, 8.7. The degradation of PEG-S-S-CPT prodrug and analysis of CPT release behavior by HPLC. To the solution of PEG-S-S-CPT (2 mg/mL, 1 mL) in water, different amount of dithiothreitol (DTT) (0, 2, 5, 10 mM) were added and the mixture was incubated at 37 °C, respectively. 0.1 mL of solution was removed at predetermined time intervals and detected by HPLC to monitor the degradation of PEG-S-S-CPT prodrug and the release of CPT. The self-assembly behavior of prodrug micelle. The effect of α-CD: Various amount of αCD (40, 60, 80 and100 mg) was dissolved in 1 mL of prodrug solution (20 mg/mL), respectively. Subsequently, the mixture was ultrasonicated for 5 min followed by standing for 72 h at room


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temperature. The samples were lyophilized and the morphologies of self-assembled superstructures were observed by scanning electron microscopy (SEM). The effect of PEG-S-S-CPT: To the solution of PEG-S-S-CPT (5, 10, 15 and 20 mg/mL, 1 mL) in water, 100 mg α-CD was added, respectively. Subsequently, all the samples were treated according to the above-mentioned method and observed by SEM. Preparation of 5-FU loaded supramolecular hydrogel. In brief, 2 mg 5-FU was added into 1 mL of PEG-S-S-CPT PBS solution (20 mg/mL). The solution was then mixed thoroughly by sonication to dissolve 5-FU completely. Subsequently, 100 mg α-CD was added to the mixed solution and ultrasonicated for 5 min followed by standing for 72 h at room temperature before measurements. Preparation of FITC loaded CyNH-S-S-PEG/α-CD hydrogel. In brief, 0.5 mg FITC was added into 1 mL of CyNH-S-S-PEG PBS solution (20 mg/mL). The solution was then mixed thoroughly by sonication to dissolve FITC completely. Subsequently, 100 mg α-CD was added to the mixed solution and ultrasonicated for 5 min followed by standing for 72 h at room temperature before measurements. GSH and DTT triggered hydrogel gel-sol transition. The hydrogel (1 mL) with 20 mg/mL PEG-S-S-CPT and 100 mg/mL α-CD formed in the bottom vials were sealed by caps, and then 10 mM DTT and GSH were added into vials, respectively. The vials were incubated at 37 °C in a water bath and taken out 30 min later for observation. The gel-sol transition behavior can be monitored visually by inverting the vials for 1.0 min to observe whether the hydrogels flowed.


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In vitro drug release. In the first stage, the 5-FU loaded PEG-S-S-CPT/α-CD supramolecular hydrogel prepared in cuvette (1.5 mL) was placed inversely in a tube containing 30.0 mL of PBS (pH 7.4) or NaAc-HAc buffer solution (pH 6.0), respectively. The cuvettes were incubated at 37 °C and the release medium was changed at predetermined time intervals. In the second stage, to the released sample solutions from stage 1 at determined intervals of time (20 h), 10 mM GSH was added and the solution was incubated at 37 °C, respectively. Subsequently, 0.1 mL of the solution was removed at predetermined time intervals and detected by HPLC. The same drug release experiments were carried out to further investigate the effect of GSH on the release behavior of CPT and 5-FU from prodrug/α-CD supramolecular hydrogel, in which all the release medium contained 10 mM GSH in different buffer solution. For all drug release experiments, the cumulative release of PEG-S-S-CPT, 5-FU and CPT were determined by HPLC. Cell Incubation and fluorescence imaging. HeLa cells were cultured according to the reported method and further utilized to detect cellular uptake of two drug released from hydrogel.34 For fluorescence microscopy, the cells were seeded onto culture dishes with appropriate density and cultured in culture medium for 24 h. Then, FITC and CyNH-S-S-PEG released from hydrogel in PBS (pH 7.4) were added to the cells, mixed by gentle shaking. Fluorescence images were obtained by using CLSM (Olympus Fluoview 1000) after washing the cells twice. In vitro anticancer activities. HL-60 (human promyelocytic leukemia) and HCT-116 (human colon cancer) cells were used in vitro cytotoxicity assays. The cytotoxicity of compounds against HL-60 cells was evaluated using MTT assay 35, while the cytotoxicity against HCT 116 cells was evaluated by sulforhodamine B assay.36 For HL-60 cells, the concentrations of CPT and PEG-SS-CPT prodrug were ranged from 0.004 to 0.5 µM while 5-FU ranging from 0.16 to 10 µM. For


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HCT-116 cells, the concentrations of all samples were ranged from 0.16 to 10 µM. The in vitro cytotoxicity results are shown in Table S1. In vivo anticancer activities. Chinese Kun Ming mice (male, 18–20 g) were obtained from Lanzhou University and all the operation process was conformed to the principles of laboratory animal operation regulation and was approved by the Experimental Animal Use and Care Committee, Lanzhou University. The mice were randomly divided into three groups (4 mice each group) and maintained under a temperature-controlled and sterile conditions for 12 h light/dark cycle. H22 tumor cells were injected into the right hind of mice. 1 week after injection, the treatments were started when the tumor model were well established. 0.2 mL PEG-S-SCPT/α-CD hydrogel, 5-FU loaded PEG-S-S-CPT/α-CD hydrogel, or PBS (control group) was subcutaneously injected to peritumor of each mouse per day for four consecutive days, respectively. Then the tumor weight of each mice were measured at predetermined time intervals (24 h after the treatment). RESULTS AND DISCUSSION Characterization of the PEG-S-S-CPT prodrug micells. CPT was chemically conjugated to mPEG (Mn=1900) through a series of reactions (Scheme 1-3, Experimental section) to form redox-responsive amphiphilic polymeric prodrug (PEG-S-S-CPT, Figure 2c).37-39 The chemical structures of the intermediate products and PEG-S-S-CPT were confirmed by 1H NMR and

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C

NMR spectra (Figure S1-3, Supporting Information). The amphiphilic PEG-S-S-CPT prodrug can form typical nanosized micelles (50-100 nm) in water with well spherical shape due to the aggregation of the hydrophobic CPT molecules (Figure S4, Supporting Information). The hydrophobic CPT was loaded in the core of micelles via the disulfide linkage and PEG chain


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were distributed on the surface of micelles, which will facilitate to further self-assembly with αCD. It should be noted that the PEG-S-S-CPT micelles contained 13.9 wt% CPT, as calculated from its molecular structure. This constant loading efficiency of prodrug micelles is much more beneficial to precisely control the drug ratio for drug combination over other nanoparticles-based drug carriers, whose drug loading content may vary from batch to batch.16-19

Figure 2. (a) The HPLC curves of PEG-S-S-CPT prodrug micelles upon the addition of DTT. (b) The release kinetics of CPT from PEG-S-S-CPT prodrug micelles upon the addition of DTT. (c) The mechanisms for CPT release from PEG-S-S-CPT prodrug micelles triggered by DTT/GSH. The activation of CPT from PEG-S-S-CPT micelles upon the addition of dithiothreitol (DTT) was investigated by using HPLC. As shown in Figure 2a, the distinct peak of prodrug at 3.8 min, decreased significantly with increasing the DTT concentration from 0 to 10 mM. The amount of released CPT (2.1 min) was also determined using HPLC. As shown in Figure 2b, when the concentration of DTT was increased to 10 mM, the accumulated release of CPT reached


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approximately 95% in 170 min, demonstrating the successful disulfide cleavage of PEG-S-SCPT followed by an intramolecular nucleophilic substitution and further removes a fivemembered ring thiolactone (Figure 1c).37-41 Moreover, the structure of prodrug has no change during the entirely release process without DTT (Figure S5, Supporting Information), and the released model drug CPT retains its original molecular structure, which is important to keep the activity of therapeutic agents. Therefore, these results indicate that the prodrug micelles have excellent stability and can targeted release the loaded drug inside tumor cells. The progressive self-assembly behavior of PEG-S-S-CPT prodrug micelles. To satisfy the demand for further constructing regular superstructures using the aforementioned prodrug micelle as building blocks, the key issues needs to be addressed is the introduction of shell-shell attraction among prodrug micelles. This is significant because the shell-shell attractive interactions could efficiently drive the self-assembly of the prodrug micelles via changing their distribution, shape, size and contact area, which determines the stability and the interior morphologies of the resultant superstructures. It is well-known that the pseudopolyrotaxane (PPR), formed between PEG chain and α-CD, has become a very attractive and valuable element to design and construct self-assembled superstructures.32,42 Strong hydrogen bond interactions between the adjacent PPRs could provide an efficient driving force to promote the self-assembly process.43,44 Therefore, in this work, α-CD was introduced to the prodrug micelles system to form PPR complexes with PEG chain which distributed onto the shell of micelles and further provide the requisite shell-shell attractive interactions. Different amount of α-CD were added into PEG-S-S-CPT micelles solultion (20 mg/mL, 1 mL), and all samples were mixed thoroughly by sonication. It was found the solution became turbid with the α-CD concentration increased from 40 to 80 mg/mL (Figure 3a-c), finally, only


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Figure 3 SEM images and optical photos of the superstructures made of PEG-S-S-CPT prodrug micelles (20 mg/mL) with different amount of α-CD. (a) 40 mg/mL, (b) 60 mg/mL, (c) 80 mg/mL and (d) 100 mg/mL. precipitate and sol in fluidity form was formed, respectively (Figure 3b, c). When the α-CD concentration reached 100 mg/mL, the hydrogel was formed during ultrasound process (Figure 3d). To further evaluate the morphology of the complex after introducing α-CD, the freeze-dried samples were observed by SEM. As shown in Figure 3a-c, only chaotic sheet-like aggregates were found while a typical porous superstructure was observed in Figure 3d. In this system, as previously mentioned, the shell-shell attraction interaction was governed by the hydrogen bond interactions between the adjacent PPRs. The formation process of PPR supramolecular structure has been widely proven to be a dynamic equilibrium process.45 At low concentration of α-CD, little amount of PPRs could be formed due to the fact that the α-CD tends to dethread from the PEG chain to water, which results in a weak shell-shell interaction as well as irregular clusters


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with fractal structures (Figure 3a-c). When the α-CD concentration reached 100 mg/mL, a large number of PPRs was formed because α-CD tends to retain over the PEG chain. Therefore, the PEG-S-S-CPT micelles can self-assembly into a 3D hydrogel which induced by the strong hydrogen bonds between PPRs.

Figure 4. (a) Schematic representation of programmed cascade self-assembly process of prodrug micelles. SEM images and optical photos of the supramolecular hydrogels (G1-G4) formed by α-


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CD (100 mg/mL) with different concentration of prodrug micelles. (b) G1, 5 mg/mL, (c) G2, 10 mg/mL, (d) G3, 15 mg/mL and (e) G4, 20 mg/mL. To further investigate the self-assembly process and the forming mechanism of the resultant hydrogel, the effect of PEG-S-S-CPT concentration on the self-assembly process under constant α-CD concentration (100 mg/mL) were studied in detail. α-CD (100 mg) was added into PEG-SS-CPT micelle solutions (1 mL) with different concentration (5-20 mg/mL) and then all samples were mixed thoroughly by sonication. As shown in Figure 4b-e, the homogeneous hydrogels (G1-G4) were successfully obtained over a broad range of PEG-S-S-CPT concentrations. The rheological results further confirmed the formation of supramolecular hydrogel, in which the storage modulus (G’) is much larger than its loss modulus (G’’) (Figure S6a, Supporting Information). Furthermore, a characteristic shear-thinning property (Figure S6b) was observed in rheological measurements of hydrogel, which is indispensable for injectable hydrogel.46 At high concentration of α-CD (100 mg/mL), a large amount of PPR which distributed onto the shell of micelles could be formed efficiently (Figure 4a). The XRD studies provide the proofs for PPR formation (Figure S7, Supporting Information), the typical diffraction peak (2θ = 19.8) of the crystalline formed by α-CD-PEG inclusion complexes can be observed obviously in the pattern of G1 to G4, which is identical with the extended rod channel structure of α-CD.47,48 Furthermore, the characteristic peak of PEG-S-S-CPT (19.1 º ) were barely observed in the pattern of G1-G4, which implies that the most of PEG chains distributed onto the surface of prodrug micelles were covered with α-CD molecules. The strong hydrogen bond interactions between the adjacent PPR modified particles will induce the particle self-assembly process, while the repulsive interactions among these particles could stabilize the assembled complexes. Finally, the morphologies of the resultant superstructures will be controlled by the balance


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between the attractive and repulsive interactions. As shown in Figure 4b, at low concentration of PEG-S-S-CPT (5 mg/mL), the obtained hydrogel clearly demonstrates a regular interior structure composed of a large amount of porous microspheres. This result indicated that the PPR modified micelles could self-assembly into stable porous microspheres firstly due to the limited shell-shell aggregation (Figure 4a). Subsequently, the hydrogen bond interactions among these microspheres could further provide the indispensable cross-links and scaffolds for hydrogel formation. It is obvious that the amounts of self-assembled microspheres in the system play a crucial role in determining the gelation time and interior structures of hydrogel. With increasing the concentration of prodrug micelles, as shown in Figure 4b-e, the interactions among the microspheres were significantly enhanced and the contact distance can be minimized, which results in the shell-shell coupling of microspheres and ultimately leads to the formation of homogeneous hydrogel with typical porous interior structure. The cascade self-assembly process of prodrug micelles could be further proved by the introduction of DTT or GSH. As shown in Figure S8, the hydrogel showed a gel-sol transition behavior after adding DTT or GSH into the system. It could be ascribed to the fact that the reduction of disulfide bond in PEG-S-S-CPT conjugates destroying the micelle structure and the following particle self-assembly process, result in the dissociation of supramolecular hydrogel. At the same time, it was observed that the gelation time for different gels could be reduced from 3 h to 5 min with increasing the prodrug concentrations from 5 to 20 mg/mL. Considering the aforementioned results, we have a reason to believe that the resultant hydrogel were actually constructed by a programmed cascade selfassembly process and such a approach is benefit to encapsulate multi drugs. In vitro release kinetics and cellular uptake studies. The hydrogel G4 was selected as carrier to encapsulate different amount of another anti-cancer drug with excellent water-


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Figure 5. (a) Schematic representation of the cascade release process of two drugs. 5-FU and prodrug release kinetics from the hydrogles at pH 7.4 (b) and 6.0 (c). 5-FU and CPT release kinetics from the hydrogles with the additional of 10 mM GSH at pH 7.4 (d) and 6.0 (e). solubility due to its highly hydrated porous structure and short gelation time. 5-FU has an excellent water-solubility and a different anti-cancer mechanism with CPT drugs, which is always combined with CPT drugs in clinic.49 The progressive self-assembled hydrogels demonstrated a temporally and spatially controlled release behavior of two drugs (Figure 5a). At first, 5-FU was sustained released from the hole of porous hydrogel through diffusion and the partial destruction of self-assembled supramolecular hydrogel. Meanwhile, a small amount of prodrug was released and no CPT was observed at pH 7.4 (Figure 5b). This result indicated that the supramolecular hydrogel and prodrug is stable in physiological environment. Subsequently,


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in the presence of high concentration GSH, CPT could also be activated effectively by the cleavage of disulfide bond in PEG-S-S-CPT micelles released from the first stage (Figure S11). It should be noting that such a cascade release behavior could be accelerated at acid environment (Figure 5c). This is mainly ascribed to the fact that the acidic environment could impact the PPR formation and decrease the stability of PPR formed between PEG and α-CD.50 Furthermore, the CPT molecule is easy to be protonated at acidic environment and will affect the structure of amphiphilic prodrug micelles (Figure S4), resulting in the destruction of the hydrogel. This structure-dependent drug release behavior could also be proved by the interior morphology changes of hydrogel during the release process. As shown in Figure S12, the hydrogel maintained the porous structure at pH 7.4 while the significant destruction of the hydrogels was observed at pH 6.0 during the release process. Such pH-responsive dual drug release behavior of injectable hydrogel is appealing due to the mildly acidic pH encountered in tumor microenvironment. When the GSH was introduced into hydrogel system directly, the release of 5-FU and CPT could also be observed concurrently due to the dissociation of the PEG-S-S-CPT prodrug micelles which acted as the building block of hydrogel (Figure 5d, e). The dramatically accelerated release of 5-FU was ascribed to the gel-sol transition behavior of hydrogel with addition of GSH. Such a morphology transformation can also be observed in SEM investigation (Figure S12). To further investigate the cellular uptake and monitor the drug release process of two drugs in cancer cells, a water-soluble fluorescence probe fluorescein isothiocyanate (FITC) and a novel turn-on NIR fluorescence, CyNH-S-S-PEG (Figure 6a) were selected as alternatives of 5-FU and CPT to construct self-assembled hydrogel (Figure S8) and simulate the drug release process. The synthesis of CyN-S-S-PEG is depicted in Figure 6a, the starting material of CyNH- S-S-OH and


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Figure 6. (a) Synthetic route for the CyNH-S-S-PEG and the mechanisms of the near-infrared fluorescent turn-on probe. (b) Absorption spectra and (c) fluorescence emission spectra (λex=697 nm) of CyNH-S-S-PEG (20 µM) before and after reacting with GSH (10 equiv) in HEPES buffer solution (pH 7.4, containing 10% DMSO as a co-solvent). PEG-COOH were prepared following the reported procedures.33,51,52 Finally, CyN-S-S-PEG was obtained by coupling of CyNH-S-S-OH and PEG-COOH via an ester bond. The overall chemical structures of CyN-S-S-PEG were verified by 1H NMR,

13

C NMR (Supporting Information,

Figure S9 and S10). To confirm that GSH could effectively activate the NIR fluorophore, the absorption and fluorescence spectra of CyNH-S-S-PEG upon the addition of GSH were investigated. As seen in Figure 6c, CyNH-S-S-PEG showed a deep blue color with the highest absorption peak at 588 nm and two smaller peaks at 556 nm and 633 nm in the absence of GSH (Figure 6b). The fluorescent probe displayed extremely weak fluorescence with a maximum peak at 705 nm (Figure 6c). However, in the presence of GSH, the fluorescence at 718 nm


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dramatically increased (Figure 6c) and a new absorption peak at 682 nm appeared, while the peaks at 556 nm, 588 nm, and 633 nm decreased significantly (Figure 6b), leading to an obvious color change of the solution from deep blue to cyan, which can be easily distinguished with the naked eye. This result demonstrated that the disulfide bond in CyNH-S-S-PEG or prodrug can be efficiently cleaved by GSH in cancer cells, leading to the NIR fluorescence turn on or release of CPT. Using the confocal laser scanning microscopy (CLSM), the cellular uptake of FITC and CyNH-S-S-PEG micelle release from hydrogel were investigated. In the HeLa cells, almost constant green fluorescence (FITC released from hydrogel) were observed during 15 min to 1 h incubation process at 37 ºC (see Figure 7b, panel 488 nm and Figure 7c), this result indicated that the released water-soluble drug in the first stage could get into the tumor cells rapidly (Figure 7a). Meanwhile, an enhanced red fluorescence were observed gradually during the incubation process (Figure 7b, panel 633 nm and Figure 7c), which implied that the prodrug micelles were internalized into the cells slowly and CPT or probe could be effectively activated in cancer cells which contain a high concentration of GSH (Figure 7a). In the presence of GSH, the dissociation of prodrug micelles within the cells could be demonstrated in vitro. As shown in Figure S4, the disruption of the micelle structure belong to PEG-S-S-CPT prodrug could be observed obviously after introducing GSH. Therefore, the CPT in hydrogel could be released at different space and time compared with 5-FU in the second release stage. All of these results indicated that the programmed cascade self-assembly of prodrug could encapsulate different drugs in different space and loading mechanism, and efficiently control the release sequence from entering the cells.


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Figure 7. (a) Schematic representation of the cascade release process of two fluorescence probes from entering the cells. (b) Fluorescence images of HeLa cells incubated with FITC and CyNHS-S-PEG micelle released from hydrogel. (c) Quantitative analysis of fluorescence intensity in cells. Combination effect in vitro and in vivo. The combination effect of 5-FU and PEG-S-S-CPT prodrug was then investigated by measuring their cytotoxicity against HL60 and HCT-116 cells. As seen in Table S1 in the Supporting Information, the cytotoxicity of PEG-S-S-CPT to HL60


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and HCT-116 cells is comparative to that of the free CPT. This can be ascribed to the fact that the disulfide bond will be broken owing to the high concentration of GSH in tumor cells, thus releasing the CPT. Furthermore, the combination of 5-FU and PEG-S-S-CPT showed enhanced cytotoxicity for each drug compared with each single modality treatment (Table S1). The combination index (CI) of 5-FU and prodrug was calculated according to the median-effect principle (Table S2 and S3).53,54 As shown in Figure 8a and b, for HCT-116 cells, the CI is

Prodrug-Based Cascade Self-Assembly Strategy for Precisely

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