Theranostic Prodrug Vesicles for Imaging Guided Codelivery of

Subscriber access provided by NEW YORK UNIV

Article

Theranostic Prodrug Vesicles for Imaging Guided Co-Delivery of Camptothecin and siRNA in Synergetic Cancer Therapy Hongzhong Chen, Huan Jia, Huijun Phoebe Tham, Qiuyu Qu, Pengyao Xing, Jin Zhao, Soo Zeng Fiona Phua, Gang Chen, and Yanli Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06936 • Publication Date (Web): 28 Jun 2017 Downloaded from http://pubs.acs.org on June 29, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8

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

ACS Applied Materials & Interfaces

Theranostic Prodrug Vesicles for Imaging Guided Co-Delivery of Camptothecin and siRNA in Synergetic Cancer Therapy Hongzhong Chen,† Huan Jia,‡ Huijun Phoebe Tham,†,§ Qiuyu Qu,† Pengyao Xing,† Jin Zhao,¶ Soo Zeng Fiona Phua,† Gang Chen,*,† and Yanli Zhao*,†,§ †

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371 ‡ School of Biological Science, Nanyang Technological University, Singapore §

School of Materials Science and Engineering, Nanyang Technological University, Singapore

¶

College of Chemical Engineering and Materials Science, Tianjin University of Science & Technology, China

Supporting Information ABSTRACT: The construction of prodrugs has been a popular strategy to overcome the limitations of chemotherapeutic drugs. However, complicated synthesis procedures and laborious purification steps make the fabrication of amphiphilic prodrugs rather difficult. By harnessing the concept of host-guest interaction, we designed and prepared a supra-amphiphile consisting of a dendritic cyclodextrin host and an adamantane/naphthalimide modified camptothecin guest through glutathione-responsive disulfide linkage. This host-guest complex could self-assemble in aqueous solution to give nanosized vesicles. When the disulfide bond in adamantane/naphthalimide modified camptothecin was cleaved by glutathione, the fluorescence of the freed adamantane/naphthalimide unit showed a significant red shift with enhanced intensity. Such glutathione-responsive fluorescence change allows for intracellular imaging and simultaneous monitoring of drug release in real-time. On account of abundant positively charged amine groups on supramolecular vesicle surface, siRNA (siPlK1) could be efficiently loaded on the vesicle. The gel retardation and fluorescence experiments proved that the siPlK1 was successfully bonded to the supramolecular vesicle. The vesicle with dendritic cyclodextrin ring exhibited negligible cytotoxicity even at high concentrations, avoiding the shortcoming of cytotoxicity from commonly used gene vectors. In vitro studies demonstrated that the loaded siRNA was transported into cancer cells to improve cancer therapeutic efficacy. Thus, we developed a prodrug-based supramolecular amphiphile via the host-guest interaction with better therapeutic performance than free camptothecin. The assembled system was utilized as a drug/gene vector to achieve combinational gene- and chemo-therapy with a synergistic effect, providing an alternative strategy to deliver both prodrug and therapeutic gene. KEYWORDS: bioimaging, cancer therapy, host-guest interactions, prodrugs, vesicles

Chemotherapy as a leading therapeutic approach has been widely used in clinical cancer treatment.1,2 However, it still faces serious drawbacks, such as the lack of specificity and side effects. As such, the construction of prodrugs has become an increasingly popular strategy to overcome these drawbacks, thus attracting tremendous attention in recent years.3 Prodrugs are modified drugs that exhibit low toxicity and can be converted to effective forms after the uptake by tumor or activated by external stimuli.4,5 The fabrication of prodrugs could improve water solubility of active drugs and extend the circulation time.6,7 Most importantly, selective activation of prodrugs reduces side effects, which is often achieved by the introduction of different functional groups that are responsive to different triggers. Such method significantly enhances the selectivity of the chemotherapy. Several stimulus approaches have been harnessed, including exogenous stimuli such as light irradiation and environmental temperature change,8,9 as well as endogenous stimuli such as pH change,10,11 elevating reactive oxygen species (ROS) amount,12,13 and increasing glutathione (GSH) concentration. GSH, as an important antioxidant in human body, can alleviate the damage caused by ROS. Interestingly, the concentration of GSH was found to be abnormally high in many cancer cell lines as compared to normal cells, which allows the design and fabrication of GSH-

responsive prodrugs by the introduction of the disulfide linkage as a cleavable linker.14–16 To achieve non-invasive real-time monitoring of drug distribution for targeted and effective cancer therapy, theranostic prodrugs should be developed by the incorporation of fluorophores into prodrugs. Fluorescence changes, including fluorescence turn-on or bathochromic shift after being triggered by intracellular or external stimuli, can be utilized to precisely monitor the drug release process and its therapeutic status.17 These fluorescence changes may also be employed for non-invasive imaging of cancer cells and monitoring the distribution of prodrugs in real-time. Similar to those common prodrugs, the theranostic prodrugs are also constructed by using functional units with stimuli-responsive capability.12,18,19 Nanoparticles with the size distribution below 200 nm prefer to accumulate at the tumor tissues rather than normal tissues by enhanced permeation and retention (EPR) effect, due to the mutation in the architecture of the vessels in tumor tissues.20–22 In light of the EPR effect, the delivery of the prodrugs could be enhanced either by the encapsulation of these prodrugs within nanosized carriers or the direct selfassembly into nanoparticles. Prodrug-loaded nanoparticles face some common disadvantages of the drug delivery systems

1 ACS Paragon Plus Environment


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

including poor drug loading content, premature drug leakage, and insufficient drug release.23–25 On the other hand, the direct self-assembly of amphiphilic prodrugs into nanoparticles has drawn lots of attention on account of higher drug loading content.26 However, there are still many challenges in selfassembled prodrug nanoparticles such as poor water solubility and stability, complicated organic synthesis and timeconsuming purification steps. The construction of supraamphiphiles could possibly overcome these limitations.27 Such systems are achieved by facile synthesis of separate host and guest moieties, followed by the host-guest complexation through noncovalent interactions such as hydrogen bonding, van der Waals and π-π interactions.28 Macrocycles such as cyclodextrin,29,30 cucurbituril,31,32 sulfonatocalixarene33 and pillararene34,35 are some frequently employed hosts in the construction of drug delivery systems, because of their low cytotoxicity and ease in the fabrication of water-soluble supramolecular architectures. On the other hand, small interfering RNA (siRNA) based gene therapy is an importance alternative, because siRNA can efficiently inhibit diseases related to the gene expression and silence specific proteins. However, the main challenge faced in the siRNA therapy is how to develop benign and efficient delivery vectors.36,37 Although viral vectors exhibit high transfection efficacy, their potential immune responses, inflammatory and gene control effect lead to safety considerations.38–40 It is thus crucial to construct gene delivery vectors without the abovementioned drawbacks.41,42 One of the siRNA that targets polo-like kinases (Plk1) overly expressed in several cancer cell lines abbreviated as siPlk1 has shown to improve the therapeutic efficacy of camptothecin (CPT) in the treatment of cancer.43 On account of the working mechanism, siRNA should normally be released faster than chemo-drugs, which validates the construction of prodrugs as an ideal gene delivery vehicle on account of its delayed controlled drug release upon entering into cancer cells.43–45 Without a safe and efficient vector for the co-delivery of CPT and siRNA, these advantages could not be harnessed. With these considerations, cyclodextrins could serve as non-viral gene carriers due to excellent biocompatibility, easy modification and suitable encapsulation of small guest molecules.46–48 Benefiting from the concept of supra-amphiphiles and considering the reasons outlined above, we herein developed a supramolecular theranostic prodrug vesicle based on hostguest interactions, and employed this vesicle as a vector to deliver siRNA as shown in Scheme 1. The prodrug was first constructed by the conjugation of adamantane (Ada)-modified

Page 2 of 8

naphthalimide with CPT via a GSH-responsive disulfide linkage. This prodrug (G1), is regarded as the guest molecule and hydrophobic component in the formation of supraamphiphiles. An amino dendrimer was grafted onto βcyclodextrin via click reaction to give hydrophilic host molecule (H1). The self-assembly of the host and guest molecules occurred in two steps. They first formed an inclusion complex to give a supra-amphiphile. Then, spontaneous self-assembly of the supra-ampliphile led to the formation of supramolecular vesicles. The vesicles display remarkably high prodrug loading content of 25.7%. Following which, siPlK1 was loaded by mixing this siRNA with the vesicle solution, and the obtained nanoparticles were incubated with cancer cells for therapeutic evaluations. After the internalization by cancer cells, siRNA escapes from the vesicle in lysosome, and the prodrug is cleaved by GSH along with an intramolecular cyclization to afford free CPT. A similar intramolecular cyclization leads to the release of Adamodified naphthalimide fluorophore (compound 3) accompanied by a significant fluorescence red shift. This fluorescence change enables color-tunable intracellular imaging and in vitro monitoring of the drug release in realtime. Our study provides a new strategy for the co-delivery of anticancer drugs and siRNA. Results and Discussion Design and synthesis of supramolecular amphiphile prodrug. The synthetic procedures of the dendritic cyclodextrin (H1) and Ada/naphthalimide modified CPT prodrug are shown in Scheme 2. An amino dendrimer was linked on β-cyclodextrin via Cu(I) mediated azide-alkyne cycloaddition, giving hydrophilic dendritic cyclodextrin as the host molecule (Scheme 2a). Compounds 5 and 6 were synthesized by following procedures reported previously.49,50 For the synthesis of the compound 4, compound 5 was reacted with adamantane carboxylic acid chloride in the presence of triethanolamine (TEA) in dichloromethane. Compound 4 was then reduced by hydrazine hydrate in the presence of Pd@C in ethanol to give compound 3. Compound 3 was reacted with triphosgene, followed by reacting with 2,2′-dithiolethanol to give compound 2. At last, compound 2 was treated with triphosgene and CPT in the presence of 4dimethylaminopyridine (DMAP) to afford prodrug G1 as the guest molecule. The overall chemical structures of compounds H1, 4, 3, 2, and G1 were fully confirmed by NMR and high resolution mass spectrometry (HRMS) as shown in the Supporting Information.

Scheme 1: Schematic illustration of the self-assembly process of the vesicle, and its therapeutic agent release and fluorescence turn-on mechanism. When the disulfide bond in prodrug G1 is cleaved by GSH in vitro, the fluorescence of the freed Ada/naphthalimide unit shows a significant red shift, and siRNA and CPT are released for the therapy.


Page 3 of 8

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


H1

Scheme 2: Synthesis of (a) dendritic cyclodextrin (H1) and (b) adamantane/naphthalimide modified CPT (G1). (a) CuSO4•5H2O, Lascorbic acid sodium salt, H2O, N2, 50 ºC, 48 h. (b) i: ethanol, N-boc-ethylenediamine, reflux, 6 h. ii: TFA, dichloromethane (DCM), room temperature, 3 h. iii: adamantanecarboxylic acid chloride, TEA, DCM, 0 ºC to room temperature, overnight. iv: Pd@C, hydrazine hydrate, ethanol, reflux, overnight. v: N,N-diisopropylethylamine (DIPEA), triphosgene, 2,2’-dithiodiethanol, THF/DCM. vi: CPT, DMAP, triphosgene, room temperature, overnight.

Self-assembly of supramolecular amphiphile prodrug. On account of supramolecular amphiphilic property, the hostguest inclusion complex can form higher order aggregates in water. To prepare amphiphilic supramolecular assembly, the Ada-containing prodrug G1 dissolved in DMSO was slowly injected into the dendritic cyclodextrin solution with molar ratio of 1:1 under the sonication, followed by dialysis against water. Transmission electron microscopy (TEM) images of the obtained aggregates exhibit vesicle-like morphology with the diameter about 110 nm (Figure 1a,b). The wall thickness of the vesicles was about 8 nm, which was consistent with the simulation results (Figure S1). Dynamic light scattering (DLS) was also employed to determine the size of the aggregates formed from H1 and G1 in aqueous solution. As shown in Figure 1c, the aggregates show an average hydrodynamic diameter (DH) about 120 nm with a narrow distribution, which is in accordance with the TEM results. The critical aggregation concentration (CAC) of the host-guest inclusion complex was determined by monitoring the hydrodynamic diameter changes upon increasing the concentration of the complex from 0.1 µM to 40 µM. Negligible DH changes were observed for the complex solution at low concentrations. Upon increasing the concentration to 1 µM, an obvious size change was detected, indicating that higher order aggregates were formed by the complex in the aqueous solution (Figure S2). The solution of the supramolecular amphiphilic vesicles with the concentration above the CAC displayed an obvious Tyndall effect, suggesting that a plenty of nanosized aggregates were formed by the host-guest inclusion complex (Figure 1d). The supramolecular vesicles also exhibited an excellent stability in aqueous solution for several months (Figure S3). GSH-responsive behavior of vesicles. Absorbance and photoluminescence studies were carried out to monitor the spectroscopic changes of the supramolecular vesicles upon the addition of GSH to phosphate buffer solution (PBS, pH = 7.4) containing the vesicles. As shown in Figure 2a, a wide absorption band from 300 nm to 500 nm centered at 365 nm was recorded in the absence of GSH, while a new absorption peak centered at 430 nm was observed after the treatment of the vesicles with GSH at 37 °C for 2 h. For the fluorescence spectra, the emission change of the supramolecular amphiphilic vesicles was recorded upon time after the addition of GSH (5.0 mM). A rapid emission intensity enhancement at

542 nm was observed, accompanied by a gradually decreasing emission intensity at 486 nm over 120 min (Figure 2). This obvious fluorescence red-shift endowed the supramolecular amphiphilic vesicles the ability for real-time monitoring the internationalization, drug release process, and intracellular imaging of the vesicles in cancer cells.

Figure 1: (a) TEM image of the vesicles self-assembled from H1 and G1, scale bar = 100 nm. Insert: enlarged TEM image of a vesicle, scale bar = 50 nm. (b) Enlarged TEM image of the vesicles, the thickness of the double-layer structure of the vesicle is about 8 nm, scale bar = 50 nm. (c) DLS data of the vesicles. (d) Photography showing the Tyndall effect of the supramolecular amphiphilic vesicles. Left: in the dark, right: in the presence of light.

Upon the treatment with GSH in PBS buffer (pH = 7.4) at 37 °C for over 2 h, the vesicle solution was further characterized by TEM and DLS. As compared with the solution in the absence of GSH, a wide size distribution from 500 nm to 1000 nm was observed in GSH treated vesicle solution, indicating that the vesicles were successfully degraded by GSH to result in large aggregates (Figure S4). TEM was employed to characterize the morphology, and defective capsule-like structures were observed with the size of over 500 nm. After the disulfide bond in G1 is cleaved by



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

GSH, the CPT segment is released. A complex formation between Ada-linked naphthalimide and dendritic cyclodextrin is still feasible, since supramolecular amphiphilic property of the host-guest molecules remains. Thus, large capsule-like structures were observed after the GSH treatment.

Page 4 of 8

Vesicles

Without With GSH

With GSH Without

Figure 3: (a) CPT and vesicle retention time in reverse-phase HPLC detected by UV absorption using 250 nm as the interrogation wavelength. The retention time range was from 10 min to 30 min. (b) Time-dependent change in the peak intensity at 16.15 min for the vesicles without and with the GSH treatment.

Figure 2: (a) Absorption and (b) fluorescence spectra of the supramolecular amphiphilic vesicle (5.0 µM) recorded in the presence and absence of GSH (5.0 mM). (c) Time-dependent fluorescence spectral changes observed when the supramolecular amphiphilic vesicle (5.0 µM) was treated with GSH (5.0 mM) at 37 °C in PBS buffer (pH 7.4). λex = 430 nm. (d) Time-dependent changes in fluorescence intensity at 540 nm for the vesicles without and with the GSH treatment.

The GSH-triggered release mechanism was then proposed. The disulfide bond was initially cleaved to afford two unstable intermediates in the presence of GSH. The Ada-attached fluorophore intermediate then underwent an intramolecular cyclization to give the Ada/naphthalimide fluorophore (compound 3) with the emission color change from blue to green (Figure S5). The CPT intermediate also underwent an intramolecular cyclization to release free CPT drug (Figure S5). Electrospray ionization mass spectrometry (ESI-MS) and reverse-phase high-performance liquid chromatography (HPLC) were employed to confirm the release mechanism of GSH induced disulfide cleavage followed by intramolecular cyclization (Figure 3). From the HPLC results, free CPT molecule exhibited a sharp retention time peak at 16.15 min with the absorption wavelength at 270 nm, and the vesicles showed a major peak at 27.10 min. After the treatment with GSH in PBS buffer of pH 7.4, vesicle samples were analyzed at different time points by HPLC. As shown in Figure S6, after the treatment with GSH for 20 min, a new peak at 16.15 min was observed, which could be assigned to free CPT. After the treatment of GSH for over 200 min, the intensity of the major peak at 27.10 min decreased upon time, while the intensity of the peak at 16.15 min enhanced. The ratio of CPT released from G1 reached 96% (Figure 3b), indicating that almost all the prodrug was converted to free CPT within 200 min in the presence of GSH. After treating the vesicles with GSH, peaks at 440.43, 545.24 and 972.83 m/z from the ESI-MS (Figure S7) could be assigned to compounds 3, CPT, and G1, respectively. The compound 3 and free CPT were detected after the reduction of G1 in the presence of GSH, indicating that the mechanism proposed above was reasonable.

Cellular uptake, cytotoxicity and in vitro antitumor effect of vesicles. To confirm the successful cellular uptake of the vesicles, confocal laser scanning microscope (CLSM) was employed to detect the cellular fluorescence changes. HeLa cells were first incubated with vesicles at a concentration of 2 µM for 12 h, followed by fixing for the characterization. As shown in Figure S8, strong fluorescence was observed in both blue and green channels, indicating that the vesicles were successfully internalized by HeLa cells. Observing luminescence in green channel also proved that prodrug can be efficiently cleaved to release the Ada/naphthalimide fluorophore when internalized by the cancer cells. Furthermore, the fluorescence observed in both blue and green channels validated the theranostic ability to monitor the distribution, accumulation, and real-time drug release of the supra-amphiphilic vesicles. The cytotoxicity of the dendritic cyclodextrin (H1) was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. As seen from Figure S9, H1 exhibited negligible cytotoxicity even at very high concentration (280 µg/mL), indicating that H1 is suitable as a cargo for the drug/gene delivery. To verify the anticancer efficacy of the vesicles, their cytotoxicity was also investigated by using MTT assay. When HeLa cells were incubated with free CPT, prodrug G1, and amphiphilic vesicles at concentrations of 0, 0.08, 0.16, 0.32, 0.63, 1.25, 2.5 5, 10 and 40 µM for 72 h respectively, dose-dependent cytotoxicity was observed. As shown from the results of the MTT assay in Figure S9, free CPT displayed similar cytotoxicity to G1 at low concentrations, but higher cytotoxicity at high concentrations. This was reasonable because G1 is activated under GSH mediated intramolecular cyclization to release free CPT. The response time is needed for its reduction by GSH, which was especially noticeable at high concentrations. The most interesting observation was that the vesicles exhibited much higher cytotoxicity than free CPT and G1 at higher concentrations (above 5 µM), while having a similar cytotoxicity at low concentrations. Corresponding half maximal inhibitory concentration (IC50) of CPT, G1 and vesicles was also determined, and the vesicles showed the best IC50 value as compared with others (Figure S10). The significant cytotoxicity enhancement indicates that the vesicles were preferably internalized by HeLa cells through the endocytosis. Based on the MTT assay data, the supramolecular amphiphilic vesicles were concluded to exhibit higher cellular


Page 5 of 8

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


uptake efficacy and better therapeutic performance than free CPT and prodrug. siRNA complexation with vesicles. On account of the abundance of free amine groups from dendritic cyclodextrin H1 in the vesicles, the vesicles were inherently able to be utilized as gene carriers. Most importantly, H1 displayed negligible toxicity, presenting its great advantage as compared to other toxic gene carriers. Thus, the supramolecular amphiphiles vesicles were also used as an siRNA vector for achieving synergetic gene- and chemo-therapy. siPlK1 that targets the PlK1 in cancer cells was chosen in this study, since it has been proven to improve therapeutic efficacy of CPT when concurrently administrated into cancer cells.43 The N/P (terminal amine/phosphate) ratio when loading siPlK1 onto the vesicles was optimized by using electrophoretic mobility experiments in agarose gel. As shown in Figure S11, complete retardation of siPlK1 was achieved at the N/P ratio of 25/1. To test the siPlK1 stability after binding with the vesicles, the siPlK1/vesicle complex was incubated with RNase A for over 120 min, and samples collected at different time points were analyzed using electrophoresis with 2% agarose gel. The presence of spots assigned to siPlK1/vesicle after the incubation of the complex with RNAse A (Figure S11b) demonstrates that the supra-amphiphiles vesicles are suitable as a gene delivery vector to bring about the stability to siPlK1.

result of the curvature increment. In this system, the curvature increment could be attributed to the surface charge changes. The amine groups from the dendrimers endow the vesicles with positively charged surface at neutral pH. After the interaction with the negatively charged siPlK1, the positive charges of the vesicles were partially neutralized by siPlK1. The electrostatic repulsion between the supramolecular amphiphilic H1/G1 complexes was decreased with the addition of siPlK1, leading to more compact nanostructures with the curvature increment. The hypothesis was proven by zeta potential measurements, in which the vesicles showed zeta potential values of +23.6 mV and +18 mV in the absence and presence of the siRNA, respectively (Figure 4d). Fluorescence emission spectra were used to investigate the interactions between the supramolecular amphiphilic vesicles and the siRNA. The siRNA used was labeled with Cy3 dye at the 5’ site. The supramolecular amphiphilic vesicle solution was observed to be colorless under room light, while Cy3siRNA solution was observed to be pink. After successful formation of siRNA-loaded vesicles, the color of the solution turned to dark orange as shown in Figure 5a. Upon the irradiation with UV lamp at a wavelength of 360 nm, the vesicle solution exhibited blue color, and no emission from Cy3-siRNA was observed under the same conditions. The Cy3-siRNA-loaded vesicles displayed nearly white emission in the presence of 360 nm UV irradiation (Figure 5b), probably due to Förster resonance energy transfer (FRET) between the vesicles and Cy3-siRNA, indicating that Cy3siRNA was successfully bound with the vesicles. The UV light with a wavelength at 360 nm cannot excite Cy3-siRNA, since its main absorption band is centered at around 550 nm (Figure 5c). This main absorption band overlaps very well with the emission of G1 in the vesicles. After the excitation with 360 nm light, a new emission peak around 570 nm from Cy3siRNA-loaded vesicles was observed, which was assigned to the emission of the Cy3 dye, indicating efficient FRET.

Figure 4: (a) TEM image of the supramolecular amphiphilic vesicles after the binding of siPlK1, scale bar = 100 nm. (b) Enlarged TEM image of the supramolecular amphiphilic vesicles after the binding with siPlK1, scale bar = 20 nm. (c) DLS data of the supramolecular amphiphilic vesicles loaded with siPlK1. (d) Zeta potential of the supramolecular amphiphilic vesicles without and with siPlK1 binding.

As shown from TEM images (Figure 4a,b), the siPlK1/vesicle complex was observed to have spherical nanostructures with uniform size of about 67 nm. Upon the interaction with siPlK1, the supramolecular amphiphilic vesicles underwent a size shrinkage to form smaller vesiclelike structures. The size distribution was determined by DLS, and the DH was about 72 nm with relatively narrow distribution (Figure 4c), which was in accordance with the results obtained from TEM. The curvature of the vesicles was regulated by many factors, including the charge distribution of the vesicles, the rigidity of molecules as well as hydrophilic and hydrophobic volumes. The smaller size is indicative of the higher curvature of the vesicles, and the size shrinkage was the

Vesicle+Cy3-siRNA Cy3-siRNA Vesicle

Figure 5: (a) Photography of the vesicle solution (100 µM), Cy3siRNA solution (100 µM), and Cy3-siRNA-loaded vesicle complex solution (50 µM with the N/P ratio = 25). (b) Photography of these solutions under the irradiation of the 360 nm light. (c) UV absorption spectrum of Cy3-siRNA. (d) Emission spectra of Cy3-siRNA (red curve), supramolecular vesicles (blue curve), and Cy3-siRNA-loaded vesicle complex (black curve). The excitation wavelength is 360 nm.

Cellular uptake, cytotoxicity and in vitro antitumor effect of siRNA-loaded vesicles. The successful delivery of siRNA in cellular level was also confirmed by the CLSM.



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

HeLa cells were incubated with Cy3-siRNA-loaded vesicles (2 µM vesicles and 20 nM siRNA) for 12 h, followed by fixation using 4% formaldehyde. As shown in Figure 6, blue fluorescence emission assigned to G1 was observed (the excitation of 405 nm laser), while strong green fluorescence emission observed (the excitation of 458 nm laser) was due to compound 3 generated from the cleavage of G1 by intracellular GSH. After the excitation with 561 nm laser for Cy3-siRNA, obvious red false color was observed in vitro, indicating that the supramolecular amphiphilic vesicles could successfully deliver the siRNA into HeLa cells. The results of the CLSM proved that the supramolecular vesicles could be utilized as a gene vector to deliver nucleotides into cancer cells for gene therapy.

Page 6 of 8

displayed higher therapeutic performance than original CPT drug in vitro. Furthermore, abundant amine groups at the terminal sites of the dendrimer unit provide binding sites for siRNA, enabling the supramolecular amphiphilic vesicles to be an siRNA delivery vector for chemo/gene combinational therapy. Detailed studies have confirmed the loading of siRNA onto the supramolecular vesicles. Cellular uptake studies have proven that the loaded siRNA could be transported into cancer cells for improving the cancer therapeutic efficacy. Therefore, the present strategy of employing supramolecular concept to prepare amphiphilic prodrug vesicles capable of real-time intracellular imaging and drug/gene co-delivery could be a general approach to develop simple and efficient therapeutic systems in the cancer treatment.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Synthesis and characterization details, simulation studies, TEM images, HPLC, ESI-MS, confocal ﬂuorescence images, cell viability, gel retardation, NMR spectra, and HRMS.

AUTHOR INFORMATION Corresponding Author Figure 6: (a) Confocal fluorescence images of HeLa cells incubated with Cy3-siRNA-loaded vesicles (2 µM) for 12 h: (i) blue channel at 450 ± 35 nm, (ii) green channel at 515 ± 30 nm, (iii) Cy3 channel, and (iv) overlap image generated from (i-iii). Scale bar = 20 µm. (b) Enlarged confocal fluorescence images of corresponding HeLa cells, scale bar = 10 µm.

*Email: [email protected]; [email protected].

The cytotoxicity of the siRNA-loaded vesicles was also investigated by the MTT assay. The siPlk1 was chosen as therapeutic active agent, while siRNA with non-active sequences (siNC) was employed as the negative control. After the incubation of HeLa cells with the vesicles (10 µM), siPlK1-loaded vesicles (10 µM of vesicles with 100 nM of siPlK1), and siNC-loaded vesicles (10 µM of vesicles with 100 nM of siNC) for 72 h, the MTT assay was employed to measure the cell viability. As shown in Figure S12, the supramolecular amphiphilic vesicles suppressed the cell viability to about 22% at the concentration of 10 µM. The siPlK1-loaded vesicles exhibited obvious viability inhibition enhancement of up to 11%, indicating successfully simultaneous chemo/gene cancer therapy. As for the negative control, siNC-loaded vesicles also displayed slight improvement in the cell viability suppression, which may be attributed to the size and surface changes of the vesicles. Conclusions Using host-guest interaction concept as the basis for the construction of supramolecular amphiphiles, we have successfully designed and fabricated GSH-responsive supramolecular theranostic prodrug vesicles. By taking advantage of the supramolecular strategy, the construction of the prodrug assembly is much easier in comparison to conventional synthetic approaches. The fluorophore segment of the prodrug could undergo a significant red shift after the disulfide bond cleavage followed by an intramolecular cyclization upon interacting with intracellular GSH. At the same time, free CPT drug was released. Thus, this system could be used for real-time cellular imaging and cancer therapeutics. The supramolecular amphiphilic prodrug vesicles exhibited excellent water solubility and stability, and

This research was supported by the NTU-Northwestern Institute for Nanomedicine, the SingHealth-NTU Research Collaborative Grant (No. SHS-NTU/009/2016), and the Singapore Academic Research Fund (No. RG121/16 (S)).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

REFERENCES (1) (2)

(3)

(4) (5)

(6)

(7) (8)

(9)

(10)

Allen, T. M.; Cullis, P. R. Drug Delivery Systems: Entering the Mainstream. Science 2004, 303, 1818–1822. Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as An Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2, 751–760. Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Jarvinen, T.; Savolainen, J. Prodrugs: Design and Clinical Applications. Nat. Rev. Drug Discov. 2008, 7, 255–270. Sherwood, R. F. Advanced Drug Delivery Reviews: Enzyme Prodrug Therapy. Adv. Drug Deliv. Rev. 1996, 22, 269–288. Liang, D.; Miller, G. H.; Tranmer, G. K. Hypoxia Activated Prodrugs: Factors Influencing Design and Development. Curr. Med. Chem. 2015, 22, 4313–4325. Cai, K.; He, X.; Song, Z.; Yin, Q.; Zhang, Y.; Uckun, F. M.; Jiang, C.; Cheng, J. Dimeric Drug Polymeric Nanoparticles with Exceptionally High Drug Loading and Quantitative Loading Efficiency. J. Am. Chem. Soc. 2015, 137, 3458–3461. Tong, R.; Cheng, J. Anticancer Polymeric Nanomedicines. Polym. Rev. 2007, 47, 345–381. Thapa, P.; Li, M.; Bio, M.; Rajaputra, P.; Nkepang, G.; Sun, Y.; Woo, S.; You, Y. Far-Red Light-Activatable Prodrug of Paclitaxel for the Combined Effects of Photodynamic Therapy and Site-Specific Paclitaxel Chemotherapy. J. Med. Chem. 2016, 59, 3204–3214. Jana, A.; Nguyen, K. T.; Li, X.; Zhu, P.; Tan, N. S.; Agren, H.; Zhao, Y. Perylene-Derived Single-Component Organic Nanoparticles with Tunable Emission: Efficient Anticancer Drug Carriers with Real-Time Monitoring of Drug Release. ACS Nano 2014, 8, 5939–5952. Duan, Q.; Cao, Y.; Li, Y.; Hu, X.; Xiao, T.; Lin, C.; Pan, Y.; Wang, L. pH-Responsive Supramolecular Vesicles Based on


Page 7 of 8

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

(11)

(12)

(13) (14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23) (24) (25)

(26)

(27)

(28)

(29)

(30)

(31)

ACS Applied Materials & Interfaces Water-Soluble Pillar[6]arene and Ferrocene Derivative for Drug Delivery. J. Am. Chem. Soc. 2013, 135, 10542–10549. Li, S.; Liu, L.; Jia, H.; Qiu, W.; Rong, L.; Cheng, H.; Zhang, X. A pH-Responsive Prodrug for Real-Time Drug Release Monitoring and Targeted Cancer Therapy. Chem. Commun. 2014, 50, 11852–11855. Saravanakumar, G.; Kim, J.; Kim, W. J. Reactive-OxygenSpecies-Responsive Drug Delivery Systems: Promises and Challenges. Adv. Sci. 2017, 4, 1600124. Liang, J.; Liu, B. ROS-Responsive Drug Delivery Systems. Bioeng. Transl. Med. 2016, 1, 239–251. Kim, E.; Kim, D.; Jung, H.; Lee, J.; Paul, S.; Selvapalam, N.; Yang, Y.; Lim, N.; Park, C. G.; Kim, K. Facile, Template-Free Synthesis of Stimuli-Responsive Polymer Nanocapsules for Targeted Drug Delivery. Angew. Chem. Int. Ed. 2010, 2, 4405– 4408. Lee, M. H.; Yang, Z.; Lim, C. W.; Lee, Y. H.; Dongbang, S.; Kang, C.; Kim, J. S. Disulfide-Cleavage-Triggered Chemosensors and Their Biological Applications. Chem. Rev. 2013, 113, 5071–5109. Lee, M. H.; Sessler, J. L.; Kim, J. S. Disulfide-Based Multifunctional Conjugates for Targeted Theranostic Drug Delivery. Acc. Chem. Res. 2015, 48, 2935–2946. Chen, H.; Tham, H. P.; Ang, C. Y.; Qu, Q.; Zhao, L.; Xing, P.; Bai, L.; Tan, S. Y.; Zhao, Y. Responsive Prodrug SelfAssembled Vesicles for Targeted Chemotherapy in Combination with Intracellular Imaging. ACS Appl. Mater. Interfaces 2016, 8, 24319–24324. Kumar, R.; Kim, E.; Han, J.; Lee, H.; Sup, W.; Min, H.; Bhuniya, S.; Seung, J.; Soo, K. Hypoxia-Directed and Activated Theranostic Agent: Imaging and Treatment of Solid Tumor. Biomaterials 2016, 104, 119–128. Maiti, S.; Hong, K. S.; Kim, J. S. An Activatable Prodrug for the Treatment of Metastatic Tumors. J. Am. Chem. Soc 2014, 136, 13888–13894. Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor Vascular Permeability and the EPR Effect in Macromolecular Therapeutics: A Review. J. Control. Release 2000, 65, 271–284. Fang, J.; Nakamura, H.; Maeda, H. The EPR Effect: Unique Features of Tumor Blood Vessels for Drug Delivery, Factors Involved, and Limitations and Augmentation of the Effect. Adv. Drug Deliv. Rev. 2011, 63, 136–151. Fang, J.; Seki, T.; Maeda, H. Therapeutic Strategies by Modulating Oxygen Stress in Cancer and Inflammation. Adv. Drug Deliv. Rev. 2009, 61, 290–302. Vallet-regí, M.; Balas, F.; Arcos, D. Mesoporous Materials for Drug Delivery. Angew. Chem. Int. Ed. 2007, 46, 7548–7558. Hoare, T. R.; Kohane, D. S. Hydrogels in Drug Delivery: Progress and Challenges. Polymer 2008, 49, 1993–2007. Allen, T. M.; Cullis, P. R. Liposomal Drug Delivery Systems: From Concept to Clinical Applications. Adv. Drug Deliv. Rev. 2013, 65, 36–48. Linderoth, L.; Peters, G. H.; Madsen, R.; Andresen, T. L. Drug Delivery by an Enzyme-Mediated Cyclization of a Lipid Prodrug with Unique Bilayer-Formation Properties. Angew. Chem. Int. Ed. 2009, 48, 1823–1826. Kang, Y.; Tang, X.; Cai, Z.; Zhang, X. Supra-Amphiphiles for Functional Assemblies. Adv. Funct. Mater. 2016, 26, 8920– 8931. Li, Z.; Zhang, Y.-M.; Wang, H.-Y.; Li, H.; Liu, Y. Mechanical Behaviors of Highly Swollen Supramolecular Hydrogels Mediated by Pseudorotaxanes. Macromolecules 2017, 50, 1141– 1146. Davis, M. E.; Brewster, M. E. Cyclodextrin-Based Pharmaceutics: Past, Present and Future. Nat. Rev. Drug Discov. 2004, 3, 1023–1035. Zhang, J.; Ma, P. X. Cyclodextrin-Based Supramolecular Systems for Drug Delivery: Recent Progress and Future Perspective. Adv. Drug Deliv. Rev. 2013, 65, 1215–1233. Lee, J. A. E. W.; Samal, S.; Selvapalam, N.; Kim, H.; Kim, K. Cucurbituril Homologues and Derivatives: New Opportunities

(32)

(33)

(34)

(35)

(36) (37)

(38) (39) (40) (41)

(42)

(43)

(44)

(45)

(46)

(47) (48)

(49)

(50)

in Supramolecular Chemistry. Acc. Chem. Res. 2003, 36, 621– 630. Ma, D.; Hettiarachchi, G.; Nguyen, D.; Zhang, B.; Wittenberg, J. B.; Zavalij, P. Y.; Briken, V.; Isaacs, L. Acyclic Cucurbit[n]uril Molecular Containers Enhance the Solubility and Bioactivity of Poorly Soluble Pharmaceuticals. Nat. Chem. 2012, 4, 503–510. Guo, D. S.; Wang, K.; Wang, Y. X.; Liu, Y. CholinesteraseResponsive Supramolecular Vesicle. J. Am. Chem. Soc. 2012, 134, 10244–10250. Yao, Y.; Xue, M.; Chen, J.; Zhang, M.; Huang, F. An Amphiphilic Pillar[5]arene: Synthesis, Controllable SelfAssembly in Water, and Application in Calcein Release and TNT Adsorption. J. Am. Chem. Soc. 2012, 134, 15712–15715. Yu, G.; Tang, G.; Huang, F. Supramolecular Enhancement of Aggregation-Induced Emission and Its Application in Cancer Cell Imaging. J. Mater. Chem. C 2014, 2, 6609–6617. Wang, Y.; Huang, L. A Window onto siRNA Delivery. Nat. Biotechnol. 2013, 31, 611–612. Jin, H.; Kim, A.; Miyata, K.; Kataoka, K. Recent Progress in Development of siRNA Delivery Vehicles for Cancer Therapy. Adv. Drug Deliv. Rev. 2016, 104, 61–77. Merkel, O. M.; Kissel, T. Nonviral Pulmonary Delivery of siRNA. Acc. Chem. Res. 2012, 45, 961–970. Mintzer, M. A.; Simanek, E. E. Nonviral Vectors for Gene Delivery. Chem. Rev. 2009, 109, 259–302. Guo, X. I. A.; Huang, L. Recent Advances in Nonviral Vectors for Gene Delivery. Acc. Chem. Res. 2012, 45, 971–979. Xia, Y.; Peng, L. Photoactivatable Lipid Probes for Studying Biomembranes by Photoaffinity Labeling. Chem. Rev. 2012, 113, 7880–7929. Liu, X.; Zhou, J.; Yu, T.; Chen, C.; Cheng, Q.; Sengupta, K.; Huang, Y.; Li, H.; Liu, C.; Wang, Y.; Posocco, P.; Wang, M.; Cui, Q.; Giorgio, S.; Fermeglia, M.; Qu, F.; Pricl, S.; Shi, Y.; Liang, Z.; Rocchi, P.; Rossi, J. J.; Peng, L. Adaptive Amphiphilic Dendrimer-Based Nanoassemblies as Robust and Versatile siRNA Delivery Systems. Angew. Chem. Int. Ed. 2014, 53, 11822–11827. Li, Y.; Liu, R.; Yang, J.; Ma, G.; Zhang, Z.; Zhang, X. Dual Sensitive and Temporally Controlled Camptothecin Prodrug Liposomes Codelivery of siRNA for High Efficiency Tumor Therapy. Biomaterials 2014, 35, 9731–9745. Tan, X.; Lu, X.; Jia, F.; Liu, X.; Sun, Y.; Logan, J. K.; Zhang, K. Blurring the Role of Oligonucleotides: Spherical Nucleic Acids as a Drug Delivery Vehicle. J. Am. Chem. Soc. 2016, 138, 10834–10837. Tan, X.; Li, B. B.; Lu, X.; Jia, F.; Santori, C.; Menon, P.; Li, H.; Zhang, B.; Zhao, J. J.; Zhang, K. Light-Triggered, SelfImmolative Nucleic Acid-Drug Nanostructures. J. Am. Chem. Soc. 2015, 137, 6112–6115. Wen, Y.; Zhang, Z.; Li, J. Highly Efficient Multifunctional Supramolecular Gene Carrier System Self-Assembled from Redox-Sensitive and Zwitterionic Polymer Blocks. Adv. Funct. Mater. 2014, 24, 3874–3884. Mellet, C. O.; Fernandez, J.; Benito, J. Cyclodextrin-Based Gene Delivery Systems. Chem. Soc. Rev. 2011, 40, 1586–1608. Li, J.; Jun, X. Cyclodextrin-Based Supramolecular Architectures: Syntheses, Structures, and Applications for Drug and Gene Delivery. Adv. Drug Deliv. Rev. 2008, 60, 1000–1017. Yu, T.; Liu, X.; Bolcato-Bellemin, A.; Wang, Y.; Liu, C.; Erbacher, P.; Qu, F.; Rocchi, P.; Behr, J.; Peng, L. An Amphiphilic Dendrimer for Effective Delivery of Small Interfering RNA and Gene Silencing in Vitro and in Vivo. Angew. Chem., Int. Ed. 2012, 51, 8478-8484. Makki, M.; Staneva, D.; Sobahi, T.; Bosch, P.; Abdel-Rahman, R.; Grabchev, I. Design and Synthesis of a New Fluorescent Tripod for Chemosensor Applications. Tetrahedron 2014, 70, 9366-9372.



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

Page 8 of 8

Table of Content

ACS Paragon Plus Environment

8

Theranostic Prodrug Vesicles for Imaging Guided Codelivery of

Recommend Documents