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Recent advances in biocompatible supramolecular assemblies for biomolecular detection and delivery
Chinese Chemical Letters 24 (2013) 351–358
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Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet
Review
Recent advances in biocompatible supramolecular assemblies for biomolecular detection and delivery Lei Wang, Li-Li Li, Horse L. Ma, Hao Wang * CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 January 2013 Received in revised form 4 March 2013 Accepted 5 March 2013 Available online 12 April 2013
Inspired by sophisticated biological structures and their physiological processes, supramolecular chemistry has been developed for understanding and mimicking the behaviors of natural species. Through spontaneous self-assembly of functional building blocks, we are able to control the structures and regulate the functions of resulting supramolecular assemblies. Up to now, numerous functional supramolecular assemblies have been constructed and successfully employed as molecular devices, machines and biological diagnostic platforms. This review will focus on molecular structures of functional molecular building blocks and their assembled superstructures for biological detection and delivery. ß 2013 Hao Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Supramolecular Assembly Biological detection Drug delivery
1. Introduction
2. Detection
Originated from the investigation and understanding of protein structures and some biological processes, supramolecular chemistry inherits the spontaneous self-assembly of building blocks from its natural counterparts, which is driven by a versatility of noncovalent interactions including hydrophobic effects, electrostatic interactions, p–p stacking, hydrogen bonding etc. Since the Nobel Prize was awarded to Lehn, Cram and Pedersen for their pioneering works on guest–host and supramolecular chemistry in 1987, supramolecular has undergone significant expansion. In order to investigate superstructures, the interactions among supramolecular building blocks during self-assembly and their robust functionalities with potential applications, synthetic chemists have developed different supramolecular systems ranging from liposomes to micelles. With the flourishing growth of interdisciplinary nanotechnology, supramolecular scientists could prepare desired nanostructures by using more sophisticated building blocks such as nanoparticles, polymers and carbon materials. One of the major challenges remaining for supramolecular chemists is the exploration of well-controlled and structuredefined superstructures for biological and biomedical applications. This review will focus on molecular structures of functional molecular building blocks and their assembled superstructures for biological detection and delivery.
2.1. Protein
* Corresponding author. E-mail address: [email protected] (H. Wang).
Tumor-specific or tissue-selective protein biomarkers overexpressed on cell surfaces are important targets for basic biological research and therapeutic applications. The identification of these proteins offers direct applications in therapeutics, forensic analysis and environmental monitoring. Based on the self-assembly and subsequent disassembly of fluorophores and ligand monomers with accompanied fluorescence signal changes (Fig. 1), I. Hamachi developed a strategy to identify these proteins controlled by the binding of ligands with target proteins (1-1–1-4) [1]. Later, the same unique supramolecular strategy for specific protein detection was carried out by using self-assembled fluorescent nanoprobes consisting of a hydrophilic protein ligand and a hydrophobic BODIPY fluorophore (1-5–1-11) [2]. In a similar supramolecular fashion, the proteins were detected by 19F-based MRI in an off/on mode. The supramolecular organic nanoparticles showed no 19Fbased MRI signals when aggregated, but produced a sharp signal in the presence of a target protein when disassembled. This ‘‘turn-on’’ response allows us to visualize the protein within live cells by 19FMRI and construct an in-cell inhibitor assay (1-12–1-14) [3]. In another case, the detection mechanism of self-assembled 19F NMR/ MRI nanoprobes was systematically investigated with improved sensitivity by rational design (1-12–1-22) [4]. Other PL-based sensing systems relying upon modulating the behaviors of supramolecular aggregation/disaggregation have also been developed to probe target proteins [5]. For example, a water
1001-8417/$ – see front matter ß 2013 Hao Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. http://dx.doi.org/10.1016/j.cclet.2013.03.018
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Fig. 1. The protein detection developed by I. Hamachi, the fluorescence probe is quenched when self-assembled in aqueous solution. The fluorescence is on after disassembled due to the protein recognition.
soluble perylene tetracarboxylic acid diimide derivative (2-1), bearing two positively charged groups, has an equilibrium between aggregated and monomeric forms. Therefore, strong emission originated from free monomers is observed in aqueous solution [6]. When the designed polyanionic aptamers were added to an aqueous solution of 2-1, strong electrostatic attractive interactions between the dye monomers/aggregates and the aptamers would occur, leading to rapid binding of the dye to the DNA and enhanced aggregation of 2-1. This behavior could result in a significant decrease in the intensity of the original emission originating from perylene monomers due to the ACQ effect. Upon the addition of lysozyme to the solution of 2-1 and aptamers, specific binding of lysozyme to aptamers would weaken the binding between the aptamers and the dye monomers/
aggregates. As a result, many more dye-monomer molecules were released; thus, an observable emission signal was turned on and the recovered luminescence intensity was directly proportional to the concentration of the added proteins. Based on some kind of special metal–metal and electrostatic interactions, the detection and quantification of lysozyme and thrombin have been achieved by means of the aggregation of platinum(II) complexes-induced spectral changes [7]. The addition of the corresponding aptamers (lysozyme and thrombin) could induce the spectral changes of 22, which shows a broad absorption shoulder with no clear band maximum emerged at ca. 538 nm and 512 nm, respectively, and the emission intensity at longer wavelength has also been observed to be significantly enhanced. These induced spectral signals were suggested to be related with the Pt(II)–Pt(II)
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Fig. 3. Schematic illustration of the structure and the color change of surface functionalized Au NPs in presence of target protein binders.
Fig. 2. The protein induced aggregation or disaggregation, leading to the fluorescence ON/OFF. The molecular structures from 2-1 to 2-4 are the fluorescence molecules used for protein detection.
interactions. It was also observed that adding various concentrations of target proteins, lysozyme or thrombin could cause a gradual decrease in the intensity of absorption and that of emission, owing to the high binding affinity of aptamers toward lysozyme or thrombin. This system can provide a new PL-based sensing strategy for label- and immobilization-free probing lysozyme or thrombin with good selectivity and specificity. Aggregation-induced emission (AIE) is attributed to restricted intramolecular rotations when the molecule aggregates through supramolecular assembly, which blocks the nonradioactive channel via the rotational energy relaxation processes and effectively populates the radioactive decay of the excitations. Such an abnormal phenomenon could be used for protein detection (Fig. 2). For example, H2A2HPS2+ (2-3) is an excellent ‘‘light-up’’ biosensor for the protein detection. Upon addition of bovine serum albumin (BSA), the dye solution becomes emissive. The PL intensity rises with an increase in BSA concentration and a 52-fold emission enhancement is achieved at a concentration of 500 mg/mL [8]. Also, human serum albumin (HSA) can be detected and quantitated by a readily accessible fluorescent bioprobe based on AIE tetraphenylethylene (2-4). The 2-4 enjoys a broad working range (0–100 nmol/L), a low detection limit (down to 1 nmol/L), and a superior selectivity to albumins [9]. Computational modeling suggests that the AIE luminogens dock in the hydrophobic cleft between subdomains IIA and IIIA of HSA with the aid of hydrophobic effects, charge neutralization, and hydrogen bonding interactions, offering mechanistic insight into the microenvironment inside the hydrophobic cavity. Another AIE co-self-assembled aggregation complex for 2-4 and myristoylcholine, once encountering AChE, could disassemble again, thus achieving an excellent detection concentration of AChE as low as 0.5 U/mL [10]. Supramolecular Au NPs have been successfully applied to colorimetric detection of proteins [11–13]. A diverse range of carbohydrate functionalized Au NPs have been prepared for the detection of carbohydrate binding proteins (Fig. 3). For example, the aggregation of b-D-lactopyranoside (3-1)-functionalized Au NPs has been utilized by Kataoka et al. for the detection of Recinus communis agglutinin (RCA120). The degree of colloidal aggregation was proportional to the protein concentration, thus allowing this method to be used in quantitative detection of lectin. Significantly,
a high sensitivity of lectin detection (lectin concentration of 1 ppm) has been achieved with this system [14]. Later, the density of Lac moiety on the particle surface has been modulated for controlling the concentration range of lectin detection [15]. The protein-directed assembly of gold glyconanoparticles has also been developed for facile and sensitive identification of protein– protein interactions. In an interesting study, binding partners of concanavalin A have been identified by utilizing the assemblies of Con A and mannose-modified (3-2) Au NPs, since the protein– protein interactions disrupt the initial nanoparticle–protein assemblies [16]. Another strategy based on Au NP harnesses the aggregation of nanoparticles, which was induced by the intact peptides in the absence of target proteases and could be dissembled elegantly by the protease-cleaving peptides. Later, Stevens et al. further simplified this approach by employing Au NPs decorated with Fmoc-protected peptides with a cysteine anchor inserted [17]. Presence of thermolysin in the system cleaved the peptide ligands, leading to Au NP’s dispersion in the solution along with a blue-tored color change with enhanced sensitivity (Fig. 4). On the basis of the enzymatic cleavage of DNA molecules, Mirkin et al. have developed a real-time colorimetric screening method for endonuclease activity by using DNA-mediated Au NP assemblies [18]. Simultaneous determination of the efficiencies of endonuclease inhibitors has been achieved utilizing the colorimetric endonuclease-inhibition assay. Similarly, detection of kinase, phosphatases, b-lactamase, and aminopeptidase, along with the screening of their activity has been achieved utilizing the enzyme-triggered Au NP assembly/disassembly approach [11]. Very interestingly, the ‘‘chemical nose approach’’ was introduced for detection of proteins by functionalized gold nanoparticles by Rotello et al. [19]. This approach relies on array-based sensing using selective recognition elements (Fig. 5). They fabricated a sensor array by using cationic Au NPs with various head groups and anionic poly(pphenyleneethynylene) (PPE) fluorescent polymer that serves as the fluorescence transduction element. In this sensor design, the cationic nanoparticles significantly quench the intrinsic fluorescence of the PPE polymer. Competitive binding of analyte proteins releases the PPE polymer, resulting in a fluorescence recovery. Linear discrimination analysis (LDA) was then used to identify unknowns from the training set. 2.2. Nucleic acids Detection of genetic mutations provides a crucial target for diagnostics, leading to a growing interest in nucleic acid-based
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Fig. 4. Schematic illustration of structure and thermolysin triggered dispersion of Au NP assemblies.
detection tests for the early diagnosis of many diseases. For a nucleic probe, a pyrene derivative (6-1, Fig. 6) bearing only one positively charged group has also been synthesized. Electrostatic attractive interaction between polyanionic nucleic acid and pyrene derivative induced the aggregation of pyrene, leading to the formation of an excimer [20]. Tang and his co-workers have reported the H2A2HPS2+ (2-3), which is a probe for protein detection. Also, this AIE molecule turns out to be a DNA fluorescence probe. The emission is turned on when herring sperm (hs) DNA is added to its buffer solution. The intensity is monotonically increased with an increase in the DNA concentration [8]. The linear range of I/I0 1 versus [hs DNA] plot is as wide as 0–100 mg/mL, with a correlation coefficient as high as 0.997. Similarly, HO-EPPS (6-2) can be used as a probe for RNAs by virtue of its AIE nature [8]. Progressive addition of RNA R6625 into alkalified HO-EPPS in the methanol/water mixtures leads to monotonically increased PL intensity with increasing DNA concentration. Fabrication of Au NPs functionalized with a DNA strand allowed researchers to tailor the properties of the nanoparticle probes according to the assay method (Fig. 7a). This discovery has stimulated extensive use of oligonucleotide-directed Au NPs aggregation for colorimetric detection of oligonucleotides and fabrication of structured assemblies [21,22]. In this approach, two ssDNA-modified Au NP probes were used for colorimetric detection of target oligonucleotides. The base sequences in the Au NP probes are designed so they are complementary to both ends of the target oligonucleotides. Au NP aggregation with
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concomitant color change is triggered by the presence of target oligonucleotides as a result of hybridization of the DNA strands. Highly specific base-pairing of DNA strands coupled with the intense absorptivity of Au NPs enables the subpicomolar quantitative colorimetric detection of oligonucleotides. This color change can be detected not only by naked eye and UV spectrometer, but also by surface plasmon resonance. In general, the surface plasmon resonance (SPR) of Au NPs is responsible for their intense colors. In solution, monodisperse 13-nm diameter Au NPs appear red and exhibit a relatively narrow surface plasmon absorption band centered at 520 nm in their UV–vis spectra. In contrast, a solution containing aggregated Au-NPs appears purple in color, corresponding to a characteristic red shift in the surface plasmon resonance of the particles from 520 nm to 574 nm. Other nanoparticles such as quantum dots could also be used as DNA/RNA detection (Fig. 7b) [23]. 3. Delivery 3.1. Drug delivery One of the greatest challenges facing chemotherapy today is developing drug delivery systems (DDSs) that are efficacious and therapeutic-selective. Both passive and active targeting approaches have been utilized with nanocarriers, most of which are made from amphiphilic supramolecular building blocks to form liposomes, micelles, hydrogels, and nanofibers. These DDSs have been proven to enhance targeted delivery of the therapeutics for cancer treatment. Recently, amphiphilic hyperbranched polymers have demonstrated unique structural characteristics and advantages in supramolecular self-assembly and drug delivery, such as globular
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Fig. 5. Illustration of ‘‘chemical nose’’ mechanism, the competitive binding between protein and polymer-NP complexes leads to the fluorescence recovery.
Fig. 6. Illustration of chemical structure of fluorescence molecules for nucleic acid detection.
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Fig. 7. Illustration of nucleic acids detection based on Au NP (a) and quantum dot (b).
architectures, chain end functionalities, improved micelle stability and drug loading ability. Yan and his co-workers [24–29] designed and synthesized some of amphiphilic hyperbranched polyphosphates for controlled drug delivery and release (Fig. 8). Generally, the phosphate amphiphilic polymers have a polar hyperbranched polyphosphate headgroup and many hydrophobic aliphatic tails, which can self-assemble into nanomicelles in water due to the amphiphilic structure. The self-assembled nanomicelles can contain small hydrophobic drug molecules for drug delivery [24,25]. Furthermore, the guest–host interactions have been involved in amphiphilic polymers. As the supramolecular hosts, such as cyclodextrin, cucurbituril and calixarene can possess a remarkable ability to include a wide range of guest molecules, via non-covalent interactions, into their hydrophobic cavities. The resultant drug delivery systems driven by the guest–host interactions can serve as nanocontainers for many guest drugs
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to increase the delivery selectivity and efficiency [26,27]. More sophisticated systems including pH-responsive (oxime) linkage (81) and reduction-responsive (disulfide) linkage (8-2) were designed to realize the pH-triggered and reduction-triggered intracellular delivery of hydrophobic anticancer drugs under physiological conditions [28,29]. Zhang et al. developed a new type of enzyme-responsive superamphiphile polymeric (8-3) systems utilizing the electrostatic interactions between a double-hydrophilic block copolymer and a natural enzyme-responsive molecule. The self-assembled spherical aggregates exhibit good responsive and releasing properties to phosphatase, which could be used to fabricate enzyme-responsive polymeric superamphiphiles for controlled self-assembly and disassembly with great potential in drug-delivery applications [30]. Another bola-form superamphiphile (8-4) platform based on a dynamic covalent bond that can self-assemble into micellar aggregates in aqueous solution was carried out, leading to the controlled release of guest molecules loaded in the micelles [31]. Supramolecular hydrogels are a useful class of materials to complement the well-established drug release systems based on biodegradable polymers. Stupp and co-workers [32,33] have developed and studied biocompatible peptide amphiphiles (PAs) programmed to self-assemble into high aspect ratio nanofibers. These nanofibers could be used as drug release system when the PAs conjugated covalently through a labile hydrazone bond to the target drug. Drug tethering was found not to alter the assembly of the PAs into filamentous aggregates. The small molecule drug could then be slowly released from the PAs gel into aqueous solution of hydrazide upon cleavage [32]. Later investigation was carried out with PAs (8-5) and the steroidal anti-inflammatory drug dexamethasone [33]. This material, along with similar drug conjugated peptide amphiphiles, could be useful as gel networks to provide prolonged and localized drug delivery at the site of injection. A supramolecular hydrogel based on D-amino acids reported by B. Xu and his co-workers, which resists hydrolysis catalyzed by proteinase K and offers long-term biostability, exhibits controlled release in vivo [34]. Their findings demonstrated that a 1 wt% supramolecular hydrogelator could significantly alter the drug release profile. By enhancing the
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Fig. 8. Amphiphilic supramolecular assembly to form micelle, nanofiber and hydrogel for drug delivery.
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Fig. 9. Self-assembled amphiphilic vesicle based on guest–host interactions for drug delivery.
interactions between the hydrogelator and the drug molecules, the supramolecular hydrogel could be used for other applications requiring long-term biostability or to prolong the duration of controlled drug release. A more sophisticated molecule (8-6), upon the action with enzyme, transforms into a hydrogelator, which self-assembles to form nanofibers and affords a supramolecular hydrogel of taxol with well-dispersed nanofiber networks. The taxol could be slowly released with cytotoxicity from the drug related hydrogel by drug self-delivery. This enzyme instructed selfassembly and hydrogelation of complex demonstrates a new, facile way to formulate highly hydrophobic drugs, such as taxol, into an aqueous form (e.g., hydrogel) without comprising their activity [35]. Liu and coworkers have developed the classic supramolecular host–guest assembly system [36,37] for drug delivery due to their unique structure characteristics with the cavities (Fig. 9). For example, amphiphilic self-assembled binary vesicle using p-sulfonatocalix[4]-arene (9-1) as the macrocyclic host and natural enzyme-cleavable myristoylcholine as the guest molecule can be dissipated by cholinesterase with high specificity and efficiency for controlled drug release. In another case, the nanosupramolecular binary vesicles based on host–guest complex formation between 9-1 and asymmetric viologen (9-2) can be disrupted to release hydrophilic doxorubicin from the interior of the vesicle by multiple stimuli. Furthermore, the guest–host interaction based supramolecular strategy was utilized together with the silica nanoparticles as nanocarriers for drug delivery [38–44]. 3.2. Gene delivery The development of safe and effective polycationic gene vectors is critical to gene therapy. To date, the overwhelming majority of
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polycationic vectors are covalent polymers including linear polycations, dendritic polycations, and peptides. It is well-known that the gene transfection efficiency is greatly related to structural parameters of cationic polymers such as amino types, charge density and charge distribution. However, optimization of these structural parameters generally requires a large number of covalent polymerizations or modifications, which makes the preparation of optimal polycations tedious and raises the cost of preparation. Due to the dynamic-tunable property and the potential use in biomedical applications, supramolecular polymers based on noncovalent interactions have attracted tremendous interests recently, which provide a new platform for designing supramolecular polycations for gene delivery. Tseng, Wang and coworkers have successfully developed a convenient, flexible, and modular synthetic approach for the preparation of size-controllable supramolecular NPs combining digital microfluidic system and supramolecular chemistry for targeted gene delivery (Fig. 10a) [45]. First, they developed a supramolecular approach for preparation of size-controlled nanoparticles, which showed excellent biocompatibility and delivery properties. Later, the small library of DNA encapsulated supramolecular nanoparticles with different sizes and RGD target ligand coverage was prepared for targeted gene delivery [46]. The results revealed that the size and target ligand coverage of DNARGD-SNP played a critical role in the target-specific gene delivery. Finally, using the digital microreactor, broad structural/ functional diversity can be programmed into a library of DNAencapsulated supramolecular nanoparticles (DNASNPs) by systematically altering the mixing ratios of molecular building blocks and a DNA plasmid. In vitro transfection studies with DNA SNPs library identified the DNASNPs with the highest gene transfection efficiency [47]. Zhu et al. [48–50] developed hyperbranched poly(amido amine)s containing different amounts of b-cyclodextrin (HPAACDs). In comparison to pure HPAA, the fluorescence intensity of HPAA-CDs was enhanced significantly while the cytotoxicity became lower. Ascribed to plenty of amino groups and strong photoluminescence, HPAA-CDs could be used as nonviral gene delivery vectors [48]. The transfection efficiencies of HPAA-CDs were similar to that of pure HPAA (Fig. 10a). Later, a charge tunable dendritic polycation was synthesized by a combination of the multifunctionality of dendritic polymers with the dynamictunable ability of supramolecular polymers [49]. The amino types and charge density of supramolecular dendritic polymers can be
Fig. 10. (a) Dendrimer based supramolecular nanoparticles for targeted gene delivery and (b) cyclen salt-containing cationic lipids formed cationic liposomes for gene delivery.
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Fig. 11. Self-assembled water channel systems from dendritic dipeptides 11-1, lipophilic ureidoimidazole 11-2 and hydrazide-functionalized pillar[5]arene 11-3.
easily controlled by only adjusting the ratios of various cationic bCD derivatives [50]. The materials can be used as efficient gene vectors with high transfection efficiency via noncovalent interactions. This novel class of supramolecular polycations expands the scope of nonviral gene vectors and provides a new strategy for designing and developing multifunctional materials via noncovalent interactions. Polymeric 1,4,7,10-tetraazacyclododecanes (cyclen) showed good DNA-binding ability and high transfection efficiency [51]. The protonated cyclen and imidazolium saltcontaining cationic lipids could form cationic liposomes (Fig. 10b), which could be used as non-viral gene delivery agents [52]. The gene transfection efficiencies were dramatically increased in the presence of calcium ion (Ca2+). 3.3. Small molecules delivery Water [53–55], ions [56], and other small molecules (such as amino acids [57] and sugars [58,59]) play a variety of functions in life. Most of the physiological processes depend on the selective exchange of ions or molecules between a cell and its environment, with water playing a crucial role in their translocation events. To understand the biological process mechanisms and the ‘‘beauty’’ of natural life, many artificial transportation channels have been extensively studied with the hope of facilitating the conduction of small molecules. Most transportation systems are based on biomimic supramolecular channel or pore structures. Percec et al. [53], for the first time, reported the diffusion of water and facilitated transport of protons with exclusion of the transport of other cations and anions through bilayer membranes (11-1, 11-3, in Fig. 11). Self-assembly through enhanced peripheral p–p stacking forms stable helical pores in bilayers. These pores, envisioned as ‘‘primitive aquaporins’’ transport water but do not exclude protons. The ion-exclusion phenomena are based on hydrophobic effects which is very important. Later, Barboiu and coworkers [54] prepared ureido imidazole monomers (11-2) and placed the urea and imidazole groups in a spatially separated configuration. The monomers self-assembled into I-quartet tubular architectures including water–wire arrays in the solid state and show water-channel conductance states in bilayer membranes. Hou and co-workers [53,57] proposed a very elegant artificial system that functions exclusively as single-molecular water
channels. Polydrazide-substituted pillar[5]arenes (11-3) were used to form tubular hydrogen-bonded superstructures which are robust when embedded in bilayer membranes [55]. The watertransport mechanism is strongly dependent on the length of the tubular assemblies. The longest hydrazide-substituted pillar[5]arene with a length of 3.5 nm perfectly fits the thickness of the bilayer and shows excellent activity for the transport of water and OH ions by a single-molecular transport mechanism. In the same group, they prepared the peptide-appended pillar[n]arene (n = 5, 6) derivatives and revealed that the molecules adopt a tubular conformation in solution and lipid bilayer membranes [57]. These molecules can efficiently mediate the transport of amino acids across lipid membranes at a very low channel-to-lipid ratio (EC50 = 0.002 mol%). In several cases, chiral selectivity for amino acid enantiomers was achieved, which is one of the key functions of natural amino acid channels.
4. Conclusion In conclusion, it has been more than 40 years since the emergence of supramolecular chemistry. Researchers have created different molecular building blocks and designed various weak driving-force systems for constructing supramolecular assemblies. With systematic investigation of the relationship between functional building blocks and the corresponding superstructures, people can rationally design the molecular building blocks and predict the functionality of the resulting superstructures. We believe that the next-generation of supramolecular assemblies will aim at the development of the functional devices and/or materials for healthcare services. We envision that the bio-supramolecular assemblies will open new avenues for disease diagnostics and therapeutics in the future. Acknowledgments This work was supported by National Basic Research Program of China (973 Program, No. 2013CB932701), the 100-Talent program of the Chinese Academy of Sciences, Beijing Natural Science Foundation (No. 2132053) and Young Scientists Fund of National Natural Science Foundation (No. 51102014).
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Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet
Review
Recent advances in biocompatible supramolecular assemblies for biomolecular detection and delivery Lei Wang, Li-Li Li, Horse L. Ma, Hao Wang * CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 January 2013 Received in revised form 4 March 2013 Accepted 5 March 2013 Available online 12 April 2013
Inspired by sophisticated biological structures and their physiological processes, supramolecular chemistry has been developed for understanding and mimicking the behaviors of natural species. Through spontaneous self-assembly of functional building blocks, we are able to control the structures and regulate the functions of resulting supramolecular assemblies. Up to now, numerous functional supramolecular assemblies have been constructed and successfully employed as molecular devices, machines and biological diagnostic platforms. This review will focus on molecular structures of functional molecular building blocks and their assembled superstructures for biological detection and delivery. ß 2013 Hao Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Supramolecular Assembly Biological detection Drug delivery
1. Introduction
2. Detection
Originated from the investigation and understanding of protein structures and some biological processes, supramolecular chemistry inherits the spontaneous self-assembly of building blocks from its natural counterparts, which is driven by a versatility of noncovalent interactions including hydrophobic effects, electrostatic interactions, p–p stacking, hydrogen bonding etc. Since the Nobel Prize was awarded to Lehn, Cram and Pedersen for their pioneering works on guest–host and supramolecular chemistry in 1987, supramolecular has undergone significant expansion. In order to investigate superstructures, the interactions among supramolecular building blocks during self-assembly and their robust functionalities with potential applications, synthetic chemists have developed different supramolecular systems ranging from liposomes to micelles. With the flourishing growth of interdisciplinary nanotechnology, supramolecular scientists could prepare desired nanostructures by using more sophisticated building blocks such as nanoparticles, polymers and carbon materials. One of the major challenges remaining for supramolecular chemists is the exploration of well-controlled and structuredefined superstructures for biological and biomedical applications. This review will focus on molecular structures of functional molecular building blocks and their assembled superstructures for biological detection and delivery.
2.1. Protein
* Corresponding author. E-mail address: [email protected] (H. Wang).
Tumor-specific or tissue-selective protein biomarkers overexpressed on cell surfaces are important targets for basic biological research and therapeutic applications. The identification of these proteins offers direct applications in therapeutics, forensic analysis and environmental monitoring. Based on the self-assembly and subsequent disassembly of fluorophores and ligand monomers with accompanied fluorescence signal changes (Fig. 1), I. Hamachi developed a strategy to identify these proteins controlled by the binding of ligands with target proteins (1-1–1-4) [1]. Later, the same unique supramolecular strategy for specific protein detection was carried out by using self-assembled fluorescent nanoprobes consisting of a hydrophilic protein ligand and a hydrophobic BODIPY fluorophore (1-5–1-11) [2]. In a similar supramolecular fashion, the proteins were detected by 19F-based MRI in an off/on mode. The supramolecular organic nanoparticles showed no 19Fbased MRI signals when aggregated, but produced a sharp signal in the presence of a target protein when disassembled. This ‘‘turn-on’’ response allows us to visualize the protein within live cells by 19FMRI and construct an in-cell inhibitor assay (1-12–1-14) [3]. In another case, the detection mechanism of self-assembled 19F NMR/ MRI nanoprobes was systematically investigated with improved sensitivity by rational design (1-12–1-22) [4]. Other PL-based sensing systems relying upon modulating the behaviors of supramolecular aggregation/disaggregation have also been developed to probe target proteins [5]. For example, a water
1001-8417/$ – see front matter ß 2013 Hao Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. http://dx.doi.org/10.1016/j.cclet.2013.03.018
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Fig. 1. The protein detection developed by I. Hamachi, the fluorescence probe is quenched when self-assembled in aqueous solution. The fluorescence is on after disassembled due to the protein recognition.
soluble perylene tetracarboxylic acid diimide derivative (2-1), bearing two positively charged groups, has an equilibrium between aggregated and monomeric forms. Therefore, strong emission originated from free monomers is observed in aqueous solution [6]. When the designed polyanionic aptamers were added to an aqueous solution of 2-1, strong electrostatic attractive interactions between the dye monomers/aggregates and the aptamers would occur, leading to rapid binding of the dye to the DNA and enhanced aggregation of 2-1. This behavior could result in a significant decrease in the intensity of the original emission originating from perylene monomers due to the ACQ effect. Upon the addition of lysozyme to the solution of 2-1 and aptamers, specific binding of lysozyme to aptamers would weaken the binding between the aptamers and the dye monomers/
aggregates. As a result, many more dye-monomer molecules were released; thus, an observable emission signal was turned on and the recovered luminescence intensity was directly proportional to the concentration of the added proteins. Based on some kind of special metal–metal and electrostatic interactions, the detection and quantification of lysozyme and thrombin have been achieved by means of the aggregation of platinum(II) complexes-induced spectral changes [7]. The addition of the corresponding aptamers (lysozyme and thrombin) could induce the spectral changes of 22, which shows a broad absorption shoulder with no clear band maximum emerged at ca. 538 nm and 512 nm, respectively, and the emission intensity at longer wavelength has also been observed to be significantly enhanced. These induced spectral signals were suggested to be related with the Pt(II)–Pt(II)
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Fig. 3. Schematic illustration of the structure and the color change of surface functionalized Au NPs in presence of target protein binders.
Fig. 2. The protein induced aggregation or disaggregation, leading to the fluorescence ON/OFF. The molecular structures from 2-1 to 2-4 are the fluorescence molecules used for protein detection.
interactions. It was also observed that adding various concentrations of target proteins, lysozyme or thrombin could cause a gradual decrease in the intensity of absorption and that of emission, owing to the high binding affinity of aptamers toward lysozyme or thrombin. This system can provide a new PL-based sensing strategy for label- and immobilization-free probing lysozyme or thrombin with good selectivity and specificity. Aggregation-induced emission (AIE) is attributed to restricted intramolecular rotations when the molecule aggregates through supramolecular assembly, which blocks the nonradioactive channel via the rotational energy relaxation processes and effectively populates the radioactive decay of the excitations. Such an abnormal phenomenon could be used for protein detection (Fig. 2). For example, H2A2HPS2+ (2-3) is an excellent ‘‘light-up’’ biosensor for the protein detection. Upon addition of bovine serum albumin (BSA), the dye solution becomes emissive. The PL intensity rises with an increase in BSA concentration and a 52-fold emission enhancement is achieved at a concentration of 500 mg/mL [8]. Also, human serum albumin (HSA) can be detected and quantitated by a readily accessible fluorescent bioprobe based on AIE tetraphenylethylene (2-4). The 2-4 enjoys a broad working range (0–100 nmol/L), a low detection limit (down to 1 nmol/L), and a superior selectivity to albumins [9]. Computational modeling suggests that the AIE luminogens dock in the hydrophobic cleft between subdomains IIA and IIIA of HSA with the aid of hydrophobic effects, charge neutralization, and hydrogen bonding interactions, offering mechanistic insight into the microenvironment inside the hydrophobic cavity. Another AIE co-self-assembled aggregation complex for 2-4 and myristoylcholine, once encountering AChE, could disassemble again, thus achieving an excellent detection concentration of AChE as low as 0.5 U/mL [10]. Supramolecular Au NPs have been successfully applied to colorimetric detection of proteins [11–13]. A diverse range of carbohydrate functionalized Au NPs have been prepared for the detection of carbohydrate binding proteins (Fig. 3). For example, the aggregation of b-D-lactopyranoside (3-1)-functionalized Au NPs has been utilized by Kataoka et al. for the detection of Recinus communis agglutinin (RCA120). The degree of colloidal aggregation was proportional to the protein concentration, thus allowing this method to be used in quantitative detection of lectin. Significantly,
a high sensitivity of lectin detection (lectin concentration of 1 ppm) has been achieved with this system [14]. Later, the density of Lac moiety on the particle surface has been modulated for controlling the concentration range of lectin detection [15]. The protein-directed assembly of gold glyconanoparticles has also been developed for facile and sensitive identification of protein– protein interactions. In an interesting study, binding partners of concanavalin A have been identified by utilizing the assemblies of Con A and mannose-modified (3-2) Au NPs, since the protein– protein interactions disrupt the initial nanoparticle–protein assemblies [16]. Another strategy based on Au NP harnesses the aggregation of nanoparticles, which was induced by the intact peptides in the absence of target proteases and could be dissembled elegantly by the protease-cleaving peptides. Later, Stevens et al. further simplified this approach by employing Au NPs decorated with Fmoc-protected peptides with a cysteine anchor inserted [17]. Presence of thermolysin in the system cleaved the peptide ligands, leading to Au NP’s dispersion in the solution along with a blue-tored color change with enhanced sensitivity (Fig. 4). On the basis of the enzymatic cleavage of DNA molecules, Mirkin et al. have developed a real-time colorimetric screening method for endonuclease activity by using DNA-mediated Au NP assemblies [18]. Simultaneous determination of the efficiencies of endonuclease inhibitors has been achieved utilizing the colorimetric endonuclease-inhibition assay. Similarly, detection of kinase, phosphatases, b-lactamase, and aminopeptidase, along with the screening of their activity has been achieved utilizing the enzyme-triggered Au NP assembly/disassembly approach [11]. Very interestingly, the ‘‘chemical nose approach’’ was introduced for detection of proteins by functionalized gold nanoparticles by Rotello et al. [19]. This approach relies on array-based sensing using selective recognition elements (Fig. 5). They fabricated a sensor array by using cationic Au NPs with various head groups and anionic poly(pphenyleneethynylene) (PPE) fluorescent polymer that serves as the fluorescence transduction element. In this sensor design, the cationic nanoparticles significantly quench the intrinsic fluorescence of the PPE polymer. Competitive binding of analyte proteins releases the PPE polymer, resulting in a fluorescence recovery. Linear discrimination analysis (LDA) was then used to identify unknowns from the training set. 2.2. Nucleic acids Detection of genetic mutations provides a crucial target for diagnostics, leading to a growing interest in nucleic acid-based
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Fig. 4. Schematic illustration of structure and thermolysin triggered dispersion of Au NP assemblies.
detection tests for the early diagnosis of many diseases. For a nucleic probe, a pyrene derivative (6-1, Fig. 6) bearing only one positively charged group has also been synthesized. Electrostatic attractive interaction between polyanionic nucleic acid and pyrene derivative induced the aggregation of pyrene, leading to the formation of an excimer [20]. Tang and his co-workers have reported the H2A2HPS2+ (2-3), which is a probe for protein detection. Also, this AIE molecule turns out to be a DNA fluorescence probe. The emission is turned on when herring sperm (hs) DNA is added to its buffer solution. The intensity is monotonically increased with an increase in the DNA concentration [8]. The linear range of I/I0 1 versus [hs DNA] plot is as wide as 0–100 mg/mL, with a correlation coefficient as high as 0.997. Similarly, HO-EPPS (6-2) can be used as a probe for RNAs by virtue of its AIE nature [8]. Progressive addition of RNA R6625 into alkalified HO-EPPS in the methanol/water mixtures leads to monotonically increased PL intensity with increasing DNA concentration. Fabrication of Au NPs functionalized with a DNA strand allowed researchers to tailor the properties of the nanoparticle probes according to the assay method (Fig. 7a). This discovery has stimulated extensive use of oligonucleotide-directed Au NPs aggregation for colorimetric detection of oligonucleotides and fabrication of structured assemblies [21,22]. In this approach, two ssDNA-modified Au NP probes were used for colorimetric detection of target oligonucleotides. The base sequences in the Au NP probes are designed so they are complementary to both ends of the target oligonucleotides. Au NP aggregation with
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concomitant color change is triggered by the presence of target oligonucleotides as a result of hybridization of the DNA strands. Highly specific base-pairing of DNA strands coupled with the intense absorptivity of Au NPs enables the subpicomolar quantitative colorimetric detection of oligonucleotides. This color change can be detected not only by naked eye and UV spectrometer, but also by surface plasmon resonance. In general, the surface plasmon resonance (SPR) of Au NPs is responsible for their intense colors. In solution, monodisperse 13-nm diameter Au NPs appear red and exhibit a relatively narrow surface plasmon absorption band centered at 520 nm in their UV–vis spectra. In contrast, a solution containing aggregated Au-NPs appears purple in color, corresponding to a characteristic red shift in the surface plasmon resonance of the particles from 520 nm to 574 nm. Other nanoparticles such as quantum dots could also be used as DNA/RNA detection (Fig. 7b) [23]. 3. Delivery 3.1. Drug delivery One of the greatest challenges facing chemotherapy today is developing drug delivery systems (DDSs) that are efficacious and therapeutic-selective. Both passive and active targeting approaches have been utilized with nanocarriers, most of which are made from amphiphilic supramolecular building blocks to form liposomes, micelles, hydrogels, and nanofibers. These DDSs have been proven to enhance targeted delivery of the therapeutics for cancer treatment. Recently, amphiphilic hyperbranched polymers have demonstrated unique structural characteristics and advantages in supramolecular self-assembly and drug delivery, such as globular
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Fig. 5. Illustration of ‘‘chemical nose’’ mechanism, the competitive binding between protein and polymer-NP complexes leads to the fluorescence recovery.
Fig. 6. Illustration of chemical structure of fluorescence molecules for nucleic acid detection.
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Fig. 7. Illustration of nucleic acids detection based on Au NP (a) and quantum dot (b).
architectures, chain end functionalities, improved micelle stability and drug loading ability. Yan and his co-workers [24–29] designed and synthesized some of amphiphilic hyperbranched polyphosphates for controlled drug delivery and release (Fig. 8). Generally, the phosphate amphiphilic polymers have a polar hyperbranched polyphosphate headgroup and many hydrophobic aliphatic tails, which can self-assemble into nanomicelles in water due to the amphiphilic structure. The self-assembled nanomicelles can contain small hydrophobic drug molecules for drug delivery [24,25]. Furthermore, the guest–host interactions have been involved in amphiphilic polymers. As the supramolecular hosts, such as cyclodextrin, cucurbituril and calixarene can possess a remarkable ability to include a wide range of guest molecules, via non-covalent interactions, into their hydrophobic cavities. The resultant drug delivery systems driven by the guest–host interactions can serve as nanocontainers for many guest drugs
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to increase the delivery selectivity and efficiency [26,27]. More sophisticated systems including pH-responsive (oxime) linkage (81) and reduction-responsive (disulfide) linkage (8-2) were designed to realize the pH-triggered and reduction-triggered intracellular delivery of hydrophobic anticancer drugs under physiological conditions [28,29]. Zhang et al. developed a new type of enzyme-responsive superamphiphile polymeric (8-3) systems utilizing the electrostatic interactions between a double-hydrophilic block copolymer and a natural enzyme-responsive molecule. The self-assembled spherical aggregates exhibit good responsive and releasing properties to phosphatase, which could be used to fabricate enzyme-responsive polymeric superamphiphiles for controlled self-assembly and disassembly with great potential in drug-delivery applications [30]. Another bola-form superamphiphile (8-4) platform based on a dynamic covalent bond that can self-assemble into micellar aggregates in aqueous solution was carried out, leading to the controlled release of guest molecules loaded in the micelles [31]. Supramolecular hydrogels are a useful class of materials to complement the well-established drug release systems based on biodegradable polymers. Stupp and co-workers [32,33] have developed and studied biocompatible peptide amphiphiles (PAs) programmed to self-assemble into high aspect ratio nanofibers. These nanofibers could be used as drug release system when the PAs conjugated covalently through a labile hydrazone bond to the target drug. Drug tethering was found not to alter the assembly of the PAs into filamentous aggregates. The small molecule drug could then be slowly released from the PAs gel into aqueous solution of hydrazide upon cleavage [32]. Later investigation was carried out with PAs (8-5) and the steroidal anti-inflammatory drug dexamethasone [33]. This material, along with similar drug conjugated peptide amphiphiles, could be useful as gel networks to provide prolonged and localized drug delivery at the site of injection. A supramolecular hydrogel based on D-amino acids reported by B. Xu and his co-workers, which resists hydrolysis catalyzed by proteinase K and offers long-term biostability, exhibits controlled release in vivo [34]. Their findings demonstrated that a 1 wt% supramolecular hydrogelator could significantly alter the drug release profile. By enhancing the
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Fig. 8. Amphiphilic supramolecular assembly to form micelle, nanofiber and hydrogel for drug delivery.
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Fig. 9. Self-assembled amphiphilic vesicle based on guest–host interactions for drug delivery.
interactions between the hydrogelator and the drug molecules, the supramolecular hydrogel could be used for other applications requiring long-term biostability or to prolong the duration of controlled drug release. A more sophisticated molecule (8-6), upon the action with enzyme, transforms into a hydrogelator, which self-assembles to form nanofibers and affords a supramolecular hydrogel of taxol with well-dispersed nanofiber networks. The taxol could be slowly released with cytotoxicity from the drug related hydrogel by drug self-delivery. This enzyme instructed selfassembly and hydrogelation of complex demonstrates a new, facile way to formulate highly hydrophobic drugs, such as taxol, into an aqueous form (e.g., hydrogel) without comprising their activity [35]. Liu and coworkers have developed the classic supramolecular host–guest assembly system [36,37] for drug delivery due to their unique structure characteristics with the cavities (Fig. 9). For example, amphiphilic self-assembled binary vesicle using p-sulfonatocalix[4]-arene (9-1) as the macrocyclic host and natural enzyme-cleavable myristoylcholine as the guest molecule can be dissipated by cholinesterase with high specificity and efficiency for controlled drug release. In another case, the nanosupramolecular binary vesicles based on host–guest complex formation between 9-1 and asymmetric viologen (9-2) can be disrupted to release hydrophilic doxorubicin from the interior of the vesicle by multiple stimuli. Furthermore, the guest–host interaction based supramolecular strategy was utilized together with the silica nanoparticles as nanocarriers for drug delivery [38–44]. 3.2. Gene delivery The development of safe and effective polycationic gene vectors is critical to gene therapy. To date, the overwhelming majority of
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polycationic vectors are covalent polymers including linear polycations, dendritic polycations, and peptides. It is well-known that the gene transfection efficiency is greatly related to structural parameters of cationic polymers such as amino types, charge density and charge distribution. However, optimization of these structural parameters generally requires a large number of covalent polymerizations or modifications, which makes the preparation of optimal polycations tedious and raises the cost of preparation. Due to the dynamic-tunable property and the potential use in biomedical applications, supramolecular polymers based on noncovalent interactions have attracted tremendous interests recently, which provide a new platform for designing supramolecular polycations for gene delivery. Tseng, Wang and coworkers have successfully developed a convenient, flexible, and modular synthetic approach for the preparation of size-controllable supramolecular NPs combining digital microfluidic system and supramolecular chemistry for targeted gene delivery (Fig. 10a) [45]. First, they developed a supramolecular approach for preparation of size-controlled nanoparticles, which showed excellent biocompatibility and delivery properties. Later, the small library of DNA encapsulated supramolecular nanoparticles with different sizes and RGD target ligand coverage was prepared for targeted gene delivery [46]. The results revealed that the size and target ligand coverage of DNARGD-SNP played a critical role in the target-specific gene delivery. Finally, using the digital microreactor, broad structural/ functional diversity can be programmed into a library of DNAencapsulated supramolecular nanoparticles (DNASNPs) by systematically altering the mixing ratios of molecular building blocks and a DNA plasmid. In vitro transfection studies with DNA SNPs library identified the DNASNPs with the highest gene transfection efficiency [47]. Zhu et al. [48–50] developed hyperbranched poly(amido amine)s containing different amounts of b-cyclodextrin (HPAACDs). In comparison to pure HPAA, the fluorescence intensity of HPAA-CDs was enhanced significantly while the cytotoxicity became lower. Ascribed to plenty of amino groups and strong photoluminescence, HPAA-CDs could be used as nonviral gene delivery vectors [48]. The transfection efficiencies of HPAA-CDs were similar to that of pure HPAA (Fig. 10a). Later, a charge tunable dendritic polycation was synthesized by a combination of the multifunctionality of dendritic polymers with the dynamictunable ability of supramolecular polymers [49]. The amino types and charge density of supramolecular dendritic polymers can be
Fig. 10. (a) Dendrimer based supramolecular nanoparticles for targeted gene delivery and (b) cyclen salt-containing cationic lipids formed cationic liposomes for gene delivery.
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Fig. 11. Self-assembled water channel systems from dendritic dipeptides 11-1, lipophilic ureidoimidazole 11-2 and hydrazide-functionalized pillar[5]arene 11-3.
easily controlled by only adjusting the ratios of various cationic bCD derivatives [50]. The materials can be used as efficient gene vectors with high transfection efficiency via noncovalent interactions. This novel class of supramolecular polycations expands the scope of nonviral gene vectors and provides a new strategy for designing and developing multifunctional materials via noncovalent interactions. Polymeric 1,4,7,10-tetraazacyclododecanes (cyclen) showed good DNA-binding ability and high transfection efficiency [51]. The protonated cyclen and imidazolium saltcontaining cationic lipids could form cationic liposomes (Fig. 10b), which could be used as non-viral gene delivery agents [52]. The gene transfection efficiencies were dramatically increased in the presence of calcium ion (Ca2+). 3.3. Small molecules delivery Water [53–55], ions [56], and other small molecules (such as amino acids [57] and sugars [58,59]) play a variety of functions in life. Most of the physiological processes depend on the selective exchange of ions or molecules between a cell and its environment, with water playing a crucial role in their translocation events. To understand the biological process mechanisms and the ‘‘beauty’’ of natural life, many artificial transportation channels have been extensively studied with the hope of facilitating the conduction of small molecules. Most transportation systems are based on biomimic supramolecular channel or pore structures. Percec et al. [53], for the first time, reported the diffusion of water and facilitated transport of protons with exclusion of the transport of other cations and anions through bilayer membranes (11-1, 11-3, in Fig. 11). Self-assembly through enhanced peripheral p–p stacking forms stable helical pores in bilayers. These pores, envisioned as ‘‘primitive aquaporins’’ transport water but do not exclude protons. The ion-exclusion phenomena are based on hydrophobic effects which is very important. Later, Barboiu and coworkers [54] prepared ureido imidazole monomers (11-2) and placed the urea and imidazole groups in a spatially separated configuration. The monomers self-assembled into I-quartet tubular architectures including water–wire arrays in the solid state and show water-channel conductance states in bilayer membranes. Hou and co-workers [53,57] proposed a very elegant artificial system that functions exclusively as single-molecular water
channels. Polydrazide-substituted pillar[5]arenes (11-3) were used to form tubular hydrogen-bonded superstructures which are robust when embedded in bilayer membranes [55]. The watertransport mechanism is strongly dependent on the length of the tubular assemblies. The longest hydrazide-substituted pillar[5]arene with a length of 3.5 nm perfectly fits the thickness of the bilayer and shows excellent activity for the transport of water and OH ions by a single-molecular transport mechanism. In the same group, they prepared the peptide-appended pillar[n]arene (n = 5, 6) derivatives and revealed that the molecules adopt a tubular conformation in solution and lipid bilayer membranes [57]. These molecules can efficiently mediate the transport of amino acids across lipid membranes at a very low channel-to-lipid ratio (EC50 = 0.002 mol%). In several cases, chiral selectivity for amino acid enantiomers was achieved, which is one of the key functions of natural amino acid channels.
4. Conclusion In conclusion, it has been more than 40 years since the emergence of supramolecular chemistry. Researchers have created different molecular building blocks and designed various weak driving-force systems for constructing supramolecular assemblies. With systematic investigation of the relationship between functional building blocks and the corresponding superstructures, people can rationally design the molecular building blocks and predict the functionality of the resulting superstructures. We believe that the next-generation of supramolecular assemblies will aim at the development of the functional devices and/or materials for healthcare services. We envision that the bio-supramolecular assemblies will open new avenues for disease diagnostics and therapeutics in the future. Acknowledgments This work was supported by National Basic Research Program of China (973 Program, No. 2013CB932701), the 100-Talent program of the Chinese Academy of Sciences, Beijing Natural Science Foundation (No. 2132053) and Young Scientists Fund of National Natural Science Foundation (No. 51102014).
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