AIEgens for Drug Delivery Applications - ACS Symposium Series

1227. ISBN13: 9780841231580eISBN: 9780841231573. Publication Date (Web): September 27, 2016 ... Drug delivery system (DDS) that enables controlled dru...
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Chapter 11

AIEgens for Drug Delivery Applications

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Jing Liang,1 Youyong Yuan,1 and Bin Liu*,1,2 1Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore 117585 2Institute of Materials Research and Engineering, 3 Research Link, 117602 Singapore *E-mail: [email protected]

Lack of therapeutic efficiency and prevalence of side effects are the major problems in therapy development, which are usually caused by low drug concentration, poor biodistribution and limited drug targeting ability. Drug delivery systems (DDS) that can selectively deliver therapeutic agents to target site of action is highly demanded. This necessitates a controlled drug release mechanism which can respond to characteristic stimuli in target cells or tissues and a signal reporter that can keep track of drug trafficking, release and activation behaviors. AIEgens have demonstrated excellent versatility, biocompatibility and sensitivity as biosensing and bioimaging probes, which are highly compatible with DDS for tracking of drug location, signaling of drug activation and monitoring of therapeutic effects. These DDSs can easily incorporate other functional elements to achieve multi-modal therapy and imaging.

© 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Introduction As one of the most important applications in the biomedical realm, drug delivery is receiving increasing research interest, not only driven by the thriving nanotechnology, but also the need for personalized medicine to achieve higher therapeutic efficiency. Efficient drug delivery can significantly improve clinical effectiveness and reduce the treatment cycle. Drug delivery system (DDS) that enables controlled drug release at targeted sites is crucial for achieving good therapeutic effect with minimal systemic toxicity. A typical DDS consists of a carrying vehicle loaded with therapeutical agents via encapsulation, covalent and noncovalent binding. The carriers are usually biocompatible hydrogels, dendrimers, liposomes, micelles and inorganic nanoparticles (carbon nanotubes, gold, silver and silica) (1), which can be further functionalized with elements to improve their performance in longevity, cell uptake, stimuli-responsiveness and visual tracking ability. Drug delivery can be achieved via simple desorption of the loaded drug. However, to achieve on-demand drug delivery with improved drug release efficiency, DDSs are usually designed to respond to specific stimuli which causes physiochemical changes of the carriers to release cargo (2). Flurescence tagging is one of the most powerful tools for drug tracking due to its high sensitivity, versatility and good compatibility with biosamples. Fluorescence reporters are useful in revealing the information of biodistribution and drug delivery kinetics of the administered drug, which is crucial for therapy development and evaluation. Conventional DDSs based on quantum dots and small molecule dyes have limited application due to their high cytotoxicity or aggregation-caused quenching (ACQ) nature (3, 4). The fluorescence tag may also be hydrolyzed during intracellular tracking or affect the uptake behaviors of the naked drug. AIEgens with unique aggregation-induced emission (AIE) characteristics have emerged as promising materials for drug delivery applications. A series of light-up probes and nanoparticle probes have been developed based on AIEgens for biosensing and bioimaging (5–8), which are promising to be transformed into theranostic platforms by introducing drug or prodrug elements. Both specificity and controlled drug release functions are essential for increasing local drug concentration in pathological sites with enhanced therapeutic efficiency and reduced side toxicity. The cell specificity can be achieved by functionalization of targeting ligands which may have specific interaction with cell surface markers or preferential accumulation in target organelles. For nanoparticle based DDS, the enhanced permeation and retention (EPR) effect may also contribute to the targeting effect to tumor sites (9). On the other hand, the controlled drug release or on-demand drug delivery can be achieved by employing a stimuli-responsive mechanism. In light of the fact that many disease conditions such as cancer are associated with abnormalities in the microenvironment including change in acidity, temperature, redox potential and enzyme activity (10), chemical moieties that are responsive to these stimuli can be integrated into the system to allow for stimuli-regulated drug release. Furthermore, DDS can be designed to be responsive to external stimuli such as light, ultrasound 272 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

and magnetic field to introduce a different modality therapy and imaging such as photothermal and photodynamic therapy and magnetic resonance imaging (MRI). This chapter reviews the examples of AIEgens in theranostic applications, with a focus in delivering of chemotherapeutic agents. The examples are organized according to working mechanisms, each having different approaches in achieving therapeutic effects.

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DDS Based on Light-Up Probes AIEgen based light-up probes here refer to molecular probes that are typically are nonemissive in aqueous media and become highly emissive upon interaction with target analytes (11). The fluorescence turn on based on solubility change is especially useful for sensing of bioanalytes such as enzymes and small molecule reductant that may lead to cleavage of the AIEgen conjugates. Inspired by these designs, a few theranostic probes have been developed based on a Pt prodrug. Cisplatin is one of the most effective drugs used by clinic to treat a wide spectrum of malignancies as they can bind to DNA and cause cell apoptosis (12). However, the use of cisplatin is limited due to its severe side effects (13). As an alternative, the non-toxic Pt(IV) has been found to be an effective prodrug which can be activated by reducing agents such as ascorbic acid to resume the latent cytotoxicity (14). Taking advantage of this mechanism and AIE effect, light-up theranostic probes have been constructed. The first example of such probes was demonstrated for in situ monitoring of therapeutic response in 2014 (15). As shown in Figure 1A, the theranostic system is comprised of four major elements: (i) the chemotherapeutic prodrug platinum(IV), which can be reduced to active toxic Pt(II) form intracellularly; (ii) a tetraphenylsilole (TPS) unit with AIE property; (iii) an Asp-Glu-Val-Asp (DEVD) peptide sequence which can be cleaved by caspase specifically upon apoptosis activation; and (iv) a cRGD tripeptide as a targeting ligand that can bind αvβ3 integrin surface biomarker. This probe is essentially nonemissive due to good water solubility and it can be specifically taken up by cancer cells that overexpress αvβ3 integrin (Figure 1B). Upon entering the cells, the prodrug is reduced to its Pt(II) counterpart which triggers the cell apoptosis and activates caspase-3 to cleave the DEVD peptide and turn on fluorescence. The light-up response is thus able to real-time monitor the therapeutic effect of anticancer drugs in the early stage. Knowing how and when drugs are activated in cells are also important in therapy development and pharmacokinetic studies. Liu’s group has developed two theranostic AIE probes for in situ monitoring of drug activation. One example is based on a tetraphenylethene pyridinium (PyTPE) AIEgen conjugated to both Pt prodrug and cRGD targeting ligand, which is able to light-up upon drug activation and release of the highly fluorescent residue (16). The other example demonstrated a TPE-based probe that can simultaneously track two drugs, namely Pt prodrug and doxorubicin (DOX), which can produce synergistic anticancer effect (17). It takes advantage of the efficient energy transfer between TPE and DOX to report the drug location and activation through fluorescence changes. 273 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 1. Chemical structure of targeted theranostic platinum (IV) prodrug with built in apoptosis sensor (A) and schematic illustration of the probe for in situ evaluation of therapeutic response (B). Adapted with permission from ref. (15). Copyright 2014 American Chemical Society. Combinational therapy using multiple therapeutic modality can not only enhance therapeutic efficiency, but also offers additional advantages in overcoming drug resistance and inducing anticancer immunity (18). Recently, Liu’s group reported the combinatorial photodynamic and chemotherapy based on a light-up probe incorporating both a Pt prodrug and an AIE active photosensitizer (PS) (19). Upon entering the targeted cell, the probe is reduced by intracellular glutathione to generate cytotoxic cisplatin for chemotherapy, yielding a highly fluorescent PS residue that can not only report drug activation, but also generation reactive oxygen species (ROS) for photodynamic therapy (PDT) to kill cisplatin resistant cells. Another strategy to achieve targeted drug delivery and reduced systemic toxicity is by targeting altered redox status associated with certain organelles, in particular, mitochondria in cancer cells. Mitochondria play vital functions in eukaryotic cells and they are featured by negative membrane potential (20). As mitochondria in cancer cells usually have more negative membrane potential as compared to those in normal cells, targeting mitochondria offers a promising approach for cancer therapy with improved efficiency. As an example, Liu’s group demonstrated the selective cancer killing with a mitochondria targeting probe with both AIE and excited-state intramolecular proton transfer (ESIPT) characteristics (21). As shown in Figure 2, the probe AIE-mito-TPP consists of an AIE+ESIPT active fluorogen and two units of triphenylphosphonium (TPP) with lipophilicity and delocalized positive charge which can preferentially accumulate in mitochondria. It was found that HeLa cells incubated with the probe showed higher cytotoxicity as compared to fibroblast NIH-3T3 cells and cell viability further reduces with increasing probe concentration. Results further revealed that 274 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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the probe accumulated in mitochondria can decrease their membrane potential, causing generation of ROS and inhibition of ATP to achieve cancer cell killing. A similar strategy was used for combination chemotherapy and PDT using an AIE active PS (22). Among the various probes prepared, the probe with one TPP group (TPECM-1TPP) demonstrated only significant cytotoxicity under light irradiation while the one with two TPP groups (TPECM-2TPP) demonstrated high cytotoxicity both in dark and under light irradiation which are due to depolarization of mitochondria membrane potential and ROS generation. The findings elucidate the importance of molecular design in realizing mitochondria targeted therapy.

Figure 2. Molecular structures of mitochondria targeting AIE probes. A number of AIE probes have been reported to have specificity for mitochondria (23–27). While most of them are applied for mitochondria imaging and tracking due to their good biocompatibility, those who have high cytotoxicity have the potential to be used for the other purpose—killing the cells for chemotherapy applications. For example, Tang’s group has designed a mitochondria targeting probe TPE-In (23), which leads to significant reduction of cell viability upon staining HeLa cells with the probe. The anti-cancer effect was allegedly attributed to interaction of the probe with DNA due to the planarity of the probe. Organic Nanoparticle Based DDS Nanoparticle based DDSs typically consist of a drug molecule that is either covalently linked to an AIEgen or encapsulated within an AIEgen or AIEgen-polymer complex shell. Such DDSs usually are designed to have pH-responsiveness which enable drug release upon change in pH in the targeted organelles. 275 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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For example, Liang’s group has reported a self-indicating nanoparticle DDS based on an AIEgen for visualization of spatiotemporal drug release (28). A negatively charged TPE and the positively charged DOX were self-assembled into TPE-DOX nanoparticles (TD NPs) in aqueous medium via electrostatic interaction (Figure 3). These TD NPs were found to be pH responsive: at neutral pH, the TD NPs display weak fluorescence due to FRET from TPE to DOX; at lower pH, the charge reversion of TPE leads to dissociation of the NPs and subsequent recovery of blue fluorescence from TPE and red fluorescence from DOX. Confocal fluorescence images of the cells stained by TP NPs show that blue emission from TPE is observed in cytoplasm region and red emission from DOX is mainly localized in nucleus region, with some signal overlap with TPE in the lysosome. After 2 h incubation, nearly all DOX are translocated into nucleus. It was thus concluded that when taken up by cancer cells, TD NPs are transported into lysosomes where drug release occurs due to the low pH value of 5.5. The dissociated DOX will then enter nucleus indicated by red emission while the TPE remains in cytoplasm with blue emission. Therefore, by tracking the spatiotemporal transition the colors, the subcellular location of TD NPs, sites of drug release and action can be clearly visualized. The TD NPs were found to be more effective in inhibiting cancer cell growth as compared to free DOX at the same concentration. The DDS designed here is advantageous over conventional dyes and quantum dots based systems as it is free of ACQ effect, cytocompatible and does not affect drug functions. Based on a similar principle, another nanoparticle-based DDS was also reported by the same group using drug loaded self-assembly micelles (29). The nanocarrier is an amphiphilic polymer comprised of TPE and polyethylene glycol (PEG) conjugate. The nanomicelles were formed through hydrophobic interaction between TPE and DOX to leave the hydrophilic PEG arms as water soluble shell. Drug release is triggered when the micelles are transported into lysosomes, in which the protonation of DOX leads to electrostatic repulsion between drug molecules and reduced hydrophobic interaction between DOX and TPE that facilitates drug dissociation. It was found that the DOX-loaded micelles show higher anticancer efficiency than that of free DOX. The above two examples are nanoparticles with drug and AIEgen associated based on electrostatic or hydrophobic interactions and the drug release is triggered by pH-induced disruption of these interactions. Another approach to fabricate nanoparticles based DDSs is through covalent conjugation between the drug and AIEgen, and the pH responsiveness can be achieved using a pH sensitive linker. As an example, Liang’s group designed a probe with a TPE and DOX linked by a pH responsive hydrazone linker for dual-color fluorogenic drug tracking (30). The hydrazine bond is known to be cleavable under low pH environment and it does not produce pendent residues that may affect the drug properties (31, 32). The TPE-DOX conjugates can self-assemble into nanoparticles and they display weak fluorescence due to FRET between the two. Once they are in lysosomes, the hydrazine bond will be disrupted, releasing both free DOX and TPE to evoke a dual-color fluorescence response. By observing the dual color recovery, the kinetic drug release in live cells can be captured. 276 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 3. Schematic illustration of TD NP formation and mechanism of drug delivery. Adapted with permission from ref. (28). Copyright 2015 John Wiley and Sons. Based on similar principles, Liu and coworkers also reported the AIE DDSs using the pH responsive hydrazine linker (33, 34). Instead of linking the AIEgen and drug, the linker is designed to form the bridge between AIEgens and a hydrophilic polymer. As shown in Figure 4, the TPE can be either conjugated with a PEG chain or multiple TPE molecules can be conjugated with a dextran polysaccharide to form a copolymer. These amphiphilic conjugates are then used to encapsulate DOX to serve as a drug delivery carrier. The encapsulating polymer of the DDSs are biocompatible and they help to prevent premature drug release during blood circulation and minimize side effects. Upon cell uptake through endocytosis, they are both transported via endo/lysosomes in which pH-regulated drug delivery take place. The DDSs show dose-dependent cytotoxicity in HeLa cells and they are useful for both drug tracking and controlled drug delivery. The pH responsive DDSs illustrated above all target the lysosomal low pH environment within the cells. Liu and Tang recently has reported a nanoparticle probe Net-TPS-PEI-DMA for targeting tumor extracellular acidic microenvironment and its therapeutic applications (35). The tumor extracellular region (pH 6.5–7.2) is known to be more acidic than the blood and normal tissues (pH ~7.4), thus providing a hallmark for targeting tumors for drug release. In this work, the nanoparticle probe consists of a TPS AIEgen and a pH responsive charge-reversible polymer polyethyleneimine (PEI) modified with 2,3-dimethylmaleic anhydride as shown in Figure 5. The probe is negatively charged and almost nonemissive at physiological pH (7.4). Once it is in the acidic tumor environment, its charge is reversed which enables its electrostatic interaction with the positively charged cell membranes and components to turn on fluorescence. Unusually, this is the first example of nanoparticle based AIE probe that shows light-up response. The nanoparticle probe was also found to cause 277 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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cytotoxicity to cancer cell through suppression of Akt pathway and activation of the apoptotic pathway. The selective inhibition of cancer cells over normal cells renders them an effective tool for image-guided tumor therapy.

Figure 4. Schematic illustration of drug-loaded micelles based on TPE-polymer conjugates for cell imaging and pH-controlled drug delivery. Reproduced with permission from ref. (33, 34). Copyright 2015 the Royal Society of Chemistry.

Figure 5. Chemical structure of the probe Net-TPS-PEI-DMA and schematic illustration of the light-up nanoparticle probe for cancer cell imaging. Reproduced with permission from ref. (35). Copyright 2015 the Royal Society of Chemistry. 278 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Inorganic Nanoparticle Based DDS In addition to the stimuli-responsive DDSs, there are also delivery systems based on simple physical desorption of drugs. In such systems, an AIEgen and drug molecules are usually coloaded in a mesoporous structure and the drug molecules are slowly released in the desired location via physical desorption while the AIEgen can be used for imaging and drug tracking. For example, in Yu’s work in 2011, a mesoporous silica structure SBA-15 grafted with BTPE showed strong blue emission and was further loaded with a model drug ibuprofen (Figure 6) (36). The blue emission is enhanced after drug loading due to additional inhibition of intramolecular rotations. The drug delivery of the complex nanostructure was tested in simulated body fluid and decreased fluorescence was observed with increasing drug release due to lessened AIE effect. Thus the change of fluorescence intensity can be used to monitor drug release process. A similar strategy was reported based on an AIEgen bridged hollow hydroxyapatite nanocapsules for delivery of ibuprofen drugs in cancer cells (37). The nanostructure showed good biocompatibility and drug dosage dependent fluorescence changes.

Figure 6. Synthetic route for preparing mesoporous SBA-15 loaded with AIEgen and drug molecules. Reproduced with permission from ref. (36). Copyright 2011 the Royal Society of Chemistry. Similar work was reported by Tao and Wei using drug and AIEgen co-loaded mesoporous silica nanoparticles for cancer imaging and therapy (38). The AIEgen 9,10-distyrylanthracene derivative and cancer killer agent cetyltrimethyl ammonium bromide (CTAB) form amphiphilic complex first and they serve as structure-directed template for forming the mesoporous nanocomposite. Results show that A549 cells incubated with the nanocomposites yielded bright fluorescence and the cell viability dropped as a result of drug release and the cytotoxicity increases with increasing nanocomposite concentration.

Conclusions The unique physiological properties of AIEgens allow them to play different roles in DDS. The switching on and off of AIE light-up probes are dynamically linked to its solubility state regulated by drug release or enzyme activation. Capitalizing on fluorescence energy transfer between AIEgens and fluorescent drugs, multicolor response can be used for dynamic monitoring of fluorogen-drug separation. A number of Pt prodrug based theranostic probes have been designed and demonstrated for tracking of single and multiple drug activation, image-guided therapy in targeted cells and evaluation of therapeutic response. 279 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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AIEgens can not only serve as image contrast agent, but also as therapeutic agent if they exhibit cytotoxicity or photoactivity. Examples have been demonstrated for AIE photosensitizers for combination of chemotherapy and photodynamic therapy through targeting the mitochondria of cancer cells. When used in solid form, AIEgens display exceptionally high brightness with good photostability, allowing them to be used for self-indicating DDS. A simple strategy to track drug delivery was introduced, which involves encapsulation of both AIEgens and drug molecules with silica or other inorganic structures. The drug release can be monitored by measuring the change of fluorescence associated with desorption of drugs. A group of work have been demonstrated for spatiotemporal drug release using AIE-drug or AIE/polymer-drug nanoparticles through pH responsive mechanism. The nanoparticle based DDS are known to be better uptaken by cells via endocytosis and show homing effect to tumor tissues due to EPR effect. The examples covered in this review are bound to inspire the rational design of more advanced DDS based on AIEgens such as combitorial therapeutics. Through variation of therapeutic agents, recruitment of targeting elements and adoption of different internal and external stimuli, the applications of AIE DDS will be greatly expanded.

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