Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and

Review pubs.acs.org/CR

Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy Chunlei Zhu, Libing Liu, Qiong Yang, Fengting Lv, and Shu Wang* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China of acceptors. The use of CPs as a new and highly sensitive biosensor element represents a novel and very promising direction in cross-disciplinary areas of chemistry, material sciences, and biological sciences. Many research groups have well demonstrated that CP-based biosensors can sensitively detect specific chemical and biological targets by employing CPs’ unique opto-electronic properties. In this field, many papers have given detailed reviews in the last ten years.1−34 However, these reviews mainly focus on the design and property study of CPs for highly sensitive detection of chemical CONTENTS and biological molecules, such as metal ions, small molecules, 1. Introduction 4687 and biomacromolecules (DNA, RNA, and proteins). 1.1. General Introduction of Water-Soluble ConIn the past 5 years, plenty of new water-soluble conjugated jugated Polymers 4687 polymers (WSCPs) have been designed and synthesized by 1.2. Synthesis and Properties of New WSCPs 4687 many groups all over the world, and advances in biological 1.3. Mechanisms of WSCPs for Diagnosis, Imagapplications of these WSCPs have been made. These studies ing, and Therapy 4692 have focused on highly sensitive diagnosis of pathogenic 2. Diagnostics with WSCPs 4697 microorganisms and tumor cells and detection of disease2.1. Diagnostics of Microbial Infection 4697 related biomarkers. Beyond sensing, fluorescence imaging in 2.2. Diagnostics of Tumor 4701 vitro (e.g., cell level) and ex/in vivo (e.g., animal level) have 2.3. Detections of Disease-Related Biomarkers 4702 also been successfully achieved. Besides, these materials have 3. Imaging with WSCPs 4709 also attracted much attention for biomedical applications, such 3.1. Fluorescence Imaging of WSCPs In Vitro 4709 as monitoring drug delivery and release, gene delivery, drug 3.2. Fluorescence Imaging of WSCPs Ex/In Vivo 4718 screening, and antimicroorganism and anticancer therapy. It has 4. Biomedical Applications of WSCPs 4721 opened the door for new functional studies of WSCPs for 4.1. Drug Delivery and Release 4721 imaging, diagnosis, and therapy. Therefore, it is necessary to 4.2. Gene Delivery 4722 introduce the latest progress in this field to the chemical 4.3. Drug Screening 4723 community. In this review, we restrict our discussions to 5. Photodynamic Therapy Using WSCPs as LightWSCPs for imaging, diagnosis, and therapy, and the progress in Harvesting Complexes 4725 the recent 5 years (from 2007 to 2011) will be specifically 5.1. Anti-Microorganism Activity of WSCPs 4725 discussed. 5.2. Anti-Tumor Activity of WSCPs 6. Concluding Remarks Author Information Corresponding Author Notes Biographies Acknowledgments References

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1.2. Synthesis and Properties of New WSCPs

Structurally, WSCPs are generally composed of two important components: (i) π-conjugated backbones, which determine the main optical properties of WSCPs, such as absorption and emission spectra, light-harvesting ability, and quantum yield (QY); (ii) charged side-chains, such as cationic quaternary ammonium groups, anionic carboxyl groups, sulfonic groups, and phosphate groups, which endow WSCPs sufficient capability to dissolve in aqueous solution for their further interactions with biomacromolecules, microorganisms, or cells. Although a large number of WSCPs have been devised and synthesized during the past few decades, no significant change has been made to the primary backbone structures and terminal charged groups. On the basis of the difference of backbone structures, CPs can be classified into several types, namely,

1. INTRODUCTION 1.1. General Introduction of Water-Soluble Conjugated Polymers

Conjugated polymers (CPs) are characterized by a delocalized electronic structure that exhibits efficient coupling between optoelectronic segments. Excitons can be efficiently transferred to lower electron/energy acceptor sites over long distances to superquench the fluorescence of CPs or to amplify the signals © 2012 American Chemical Society

Received: July 15, 2011 Published: June 6, 2012 4687

dx.doi.org/10.1021/cr200263w | Chem. Rev. 2012, 112, 4687−4735

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Table 1. Typical Polymerization Methods for the Preparation of WSCPs

synthesized by Liu and Bazan43 and anionic PFP derivative 9 subsequently synthesized by Wang and co-workers exhibited a distinct emission color change from blue to green;44 the BTcontaining polyfluorenyldivinylenes (PFVs) 10 and 11 synthesized by Liu and co-workers displayed an emission color change from green to red.45,46 By incorporating greenemitting exciton-trapped anthryl units into anionic PPEs, Satrijo and Swager reported another type of WSCP (12) that exhibited aggregation-enhanced energy transfer. Upon analyteinduced aggregation, the polymer displayed a remarkable blueto-green fluorescence color change. Taking advantage of this unique property, the sensing of multicationic amines was realized.47 In 2008, Liu and co-workers designed a series of cationic porphyrin-containing conjugated polyfluoreneethynylenes (PFEs, 13). The incomplete intramolecular energy transfer from fluoreneethynylene to porphyrin units resulted in dual emission of blue and red. They utilized these polymers to achieve the selective detection of Hg2+ ions.48 Because of the limited types of backbones and terminal charged groups, it is difficult to develop a new class of WSCPs. As a result, researchers focus their studies on modifying or conjugating the pendent chains with a variety of recognition elements (e.g., sugar, small-molecule ligand, and antibody) to

poly(fluorene-co-phenylene) (PFP), poly(p-phenylenevinylene) (PPV), poly(p-phenyleneethynylene) (PPE), polydiacetylene (PDA), and poly(thiophene) (PT). The commonly employed reactions for the synthesis of WSCPs are listed in Table 1, known as palladium-catalyzed coupling reactions (Suzuki,35 Heck,36 and Sonogashira37), Wessling reaction,38 topopolymerization reaction,39 and FeCl3 oxidative polymerization.40 Besides the reactions mentioned above, other routes are also explored to synthesize special WSCPs. In general, Wittig− Horner coupling reaction is used to obtain E-alkenes by reacting aldehydes or ketones with stabilized phosphorus ylides (phosphonate carbanions). Wang and co-workers utilized this reaction to produce a new cationic polymer 7 that contains rigid fluorene units and flexible diene units in the backbone.41 By using this reaction, poly(fluorene-co-vinylene) (PFV) was also obtained.42 In terms of new WSCPs, introduction of 2,1,3-benzothiadiazole (BT) components into fluorene-based polymers is a typical example. In such molecules, aggregation-induced intramolecular fluorescence resonance energy transfer (FRET) from fluorene units to BT sites can take place in the presence of oppositely charged species. For example, under aggregation conditions, the BT-containing cationic PFP derivative 8 first 4688


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selective binding with concanavalin A (ConA) and bacteria.49,50 Similarly, to selectively identify microorganisms and/or ConA,

overcome the existing limitations. Bunz and co-workers functionalized PPEs with α-mannose (14−16) to achieve the 4689


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Liu and co-workers synthesized α-mannose and β-glucose containing PTs and PFPs (51−53);51,52 Nagy, Wang, and coworkers prepared a Gal-α1,4-Gal disaccharide-functionalized PDA 55.53 Besides, Liu and co-workers also synthesized mannose-substituted polyfluorene and BT-containing neutral

conjugated oligomer, between which FRET can be induced, to achieve the specific detection of ConA.54 Other than sugars, Bunz and co-workers functionalized PPEs with folate (17) to target cancer cells with overexpressed folate receptor (FR).55 Park and co-workers and Sim and co-workers reported biotin 4690


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and antibody-containing PDA, respectively.56−58 Multidentate ligands can also be used to functionalize WSCPs. The iminodiacetic acid (IDA)-containing PPE 18 synthesized by Moon et al. exhibited the property of chelating metal ion Ga3+.59 By utilizing the Gd3+-chelating PFP 19 and PPE 20, both Wang and co-workers60 and Gillies and co-workers61 realized the application of WSCPs in magnetic resonance imaging (MRI). In addition to recognition elements, regulation components are also employed to enrich WSCPs. By grafting PFPs with lipids (21), Wang and co-workers obtained an amphiphilic carrier for gene delivery.62 Although CPs are modified with charged groups to make them water-soluble, the intrinsic hydrophobic backbones are still prone to aggregate and interact with hydrophobic substrates (e.g., 96-well plate made of PS, proteins with large hydrophobic domains and lipid components in cells), resulting in decreased QY and unexpected nonspecific binding. To overcome these drawbacks, poly(ethylene glycol) (PEG) sidechains are more and more frequently introduced into CPs to improve water solubility and QY, reduce undesirable aggregation, and eliminate nonspecific interactions with biomacromolecules (e.g., proteins) or cells. By utilizing click reaction, Liu and co-workers grafted a red-fluorescent CP with dense PEG side-chains to construct a novel “molecular brush”. Further functionalization of PEG-g-CP with folate made probe 22 extremely specific to the targeted cancer cells.63 More recently, taking advantage of Grignard metathesis polymerization, Bazan and co-workers reported the synthesis of a monofunctionalized neutral WSCP 23 with uniform molecular weight (MW) and excellent polydispersity ( T and 50 for rs1800469: C > T) were analyzed and discriminated (Figure 15). In the presence of each pair of allele-specific primers, if the sample was heterozygous (e.g., C/ T or G/T), FRET signals were positive for both primers; if the sample was homozygous (e.g., C/C, T/T, or G/G), the FRET signal was only positive to the corresponding specific primer

Figure 15. (a) Genotyping results for a SNP site (rs1800469: C > T) using the WSCP-based SNP genotyping method. (b) Fluorescent image of PCR products of eight Chinese individuals by using WSCP under UV light. Reproduced with permission from ref 135. Copyright 2009 Macmillan Publishers Ltd. 4703


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complementary target oligonucleotide.142 Subsequently, to integrate assays with a commercial laser source (408 nm) and further improve the detection sensitivity, Liu and co-workers constructed a microarray consisting of PNA-mobilized polystyrene (PS) beads for the detection of SNP by using cationic poly(fluorenyldivinylene-alt-1,4-phenylene) (PFVP) 69 (absorption maximum at 430 nm).143 The emission spectrum of 69 overlaps with the absorption spectrum of Cy5, ensuring the possibility for FRET to occur from PFVP to Cy5. The fabrication of the array was to self-assemble PS beads (500 nm in diameter) into the microwells on silicon substrates, followed by linking NH2-terminated PNA probes to the NHSmodified PS spheres. Specific hybridization of Cy5-labeled complementary ssDNA targets with PNA probes increased the negative charge density of PS beads, which promoted the cationic PFVP to keep in close proximity with Cy5 so as to favor the FRET process (Figure 16). For the specific Cy5-

nitrogen of the adenine purine ring. In mammals, it almost exclusively occurs within a CpG dinucleotide context. The abnormal hypermethylation of CpG is found to result in many diseases, especially human malignancies. In the development of cancers, hypermethylation commonly occurs at CpG islands (clustered CpG sites) in the promoter region of tumor suppressor genes and subsequently suppresses the transcription of the corresponding genes, leading to the aberrant protein expression and finally cancer formation and progression. It is believed that DNA methylation is correlated with almost all types of cancers. As a result, identification of abnormal DNA methylation is very useful for early cancer diagnosis and epigenetic therapy. In addition, the site-specific analysis of CpG methylation might be able to specifically discriminate various cancer types and provide some deep insights into the relationship between cancer and methylation. Building on the princlple of SNP detection, a protocol for site-specific CpG methylation detection has been established by Wang’s group.144,145 In this protocol, a methylation-sensitive restriction endonuclease (HpaII) was introduced to specifically digest unmethylated recognition sites (situation A) that was directly derived from a small amount of genomic DNA, while leaving the methylated DNA intact (situation B). Subsequent nested PCR amplification will exclusively incorporate Fl-dNTPs into the PCR products for situation B but not for situation A. Upon addition of polymer 1, distinct FRET from polymer 1 to Fl was observed for situation B. The reported study also investigated the methylation statuses of three cancer suppressor genes (p16, HPP1, and GALR2 promoters) from five cancer cell lines, HT29, HepG2, A498, HL60, and M17 (Figure 17; higher E values indicate higher degree of methylation). These results afford extremely significant correlation information between cancers and susceptibility genes, which is very useful for early cancer diagnosis. In addition to SNP and methylation detection, other alterations with respect to DNA, such as DNA mutation and DNA lesion, have also been investigated. Aneja et al. studied DNA mutation detection of B. subtilis bacteria using a FRETbased method.146 The perfect hybridization between Fl-PNA and ssDNA target can mediate efficient FRET from PFP to Fl via electrostatic attraction. With the increase of mutation number, the FRET efficiency gradually decreased with sufficient sensitivity to detect up to 4 base mismatches. Besides DNA sequence alterations, other mutation types can also be analyzed by using WSCPs. In 2009, He and co-workers utilized

Figure 16. Three-dimensional image of polymer 69-sensitized Cy5 emission from complementary PNA and Cy5-labeled ssDNA in the microwells. Reproduced with permission from ref 143. Copyright 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

labeled DNA targets, the signals from the sensitized Cy5 could be detected as low as 10−17 M by CLSM. By taking the volume (300 μL) of the DNA target into consideration, the absolute amount was calculated to be at the zeptomole level. The ultrahigh sensitivity was ascribed to the large surface area provided by PS beads and the light-amplification property of PFVP. In contrast, even when the concentration of the singlebase mismatch ssDNA reached up to 10−8 M, no signals were detected because of the extremely unstable character of mismatched PNA/DNA duplexes, exhibiting the high selectivity. By employing unlabeled ssDNA, the assay could also be broadened for label-free detection of DNA by directly monitoring the fluorescence emission of PFVP. DNA methylation is a process to incorporate a methyl group to the 5-position of cytosine pyrimidine ring or the number 6

Figure 17. Schematic representation of WSCP-based DNA methylation detection and the quantitative analysis of DNA methylation statuses in different cancer types. Reproduced with permission from ref 145. Copyright 2010 Macmillan Publishers Ltd. 4704


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the LOD was 10 ng·mL−1. However, in contrast with cells, the response signals to proteins were relatively weak due to their smaller sizes. To further improve the detection sensitivity so as to meet the clinic requirement (95% killing efficiency toward P. aeruginosa under irradiation with white light for 1 h (Figure 48). Similarly, the produced singlet oxygen and successive other ROS were responsible for the light-activated antibacterial activity of WSCPs.

Figure 48. (a) CLSM image of a microcapsule cluster 10 min after introduction into a solution of P. aeruginosa kept in the dark. (b) Interior image of a microcapsule cluster, showing bacteria entrapped within the cluster and killed after 1 h of exposure to white light. Reproduced with permission from ref 204. Copyright 2009 American Chemical Society. 4725


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In the following investigation, Whitten and co-workers compared the differences regarding the light and dark biocidal activity between cationic PPEs (47, 125, and 126) and a cationic PPE alternative (127, where a phenyl ring was replaced by thiophene).124 Both solutions of free polymers and suspensions of physisorbed polymer SiO2 microspheres (5 μm) were investigated. On the basis of the diverse photophysical properties, different biocidal activities were shown. For PPE (47, 125, and 126)-supported colloids mentioned above, light-activated biocidal activity against P. aeruginosa and C. marina was remarkable and little killing effect was displayed in the dark for a short time. Even over prolonged incubation, only a slow dark destruction was observed. Differently, polymer 127 exhibited outstanding dark biocidal activity against P. aeruginosa with >95% killing efficiency and little enhanced antimicrobial performance under light irradiation. The authors explained the efficient dark biocidal activity of the thiophene polymer as its highly lipophilic structure and its antimicrobial QA groups to destroy the outer membranes. The low light toxicity was ascribed to the high aggregation both in solution and on the microspheres, which gave rise to the poor capability in the generation of singlet oxygen and other ROS. The compared results account for the mechanisms of dark killing and demonstrate that the aggregation of polymers is harmful to light-activated biocidal activities, providing new insights into the regulation of killing mode.

Figure 49. Schematic presentation of mixtures of 1,2-dioleoyl-snglycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) liposomes with polymers 127 and 47.

air−water interface were selected as the model anionic lipid membranes. Experimental data demonstrated that compared to zwitterionic lipid membranes consisting of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids, the dark-toxic 127 showed selective binding and insertion into anionic PG lipid membranes as a result of electrostatic attraction, which was responsible for the main driven force to associate with negatively charged bacteria so as to favor the subsequent bacterial membrane disorganization. In addition, the increased membrane fluidity led to the improved conformational conversion of 127 in both model lipid membranes and more insertion amount into lipid monolayers. Moreover, the numbers of 127 that inserted into lipid bilayers generally rose with the increase of the thickness of lipid bilayers. After demonstrating the antimicrobial activity of PPEs, Whitten and co-workers synthesized four “end-only” functionalized oligo(phenylene ethyneylene)s (OPEs, 128−131) and investigated their capability in bacterial killing.117 Except anionic OPE 131 (due to Coulombic repulsion), the other three cationic OPEs exhibited efficient light-induced biocidal behavior (under 365 nm irradiation) toward both Gramnegative E. coli and Gram-positive S. epidermidis and S. aureus, where OPE 130 presented nearly 100% killing efficiency as a result of the highest sensitized ability to generate singlet oxygen. In contrast to Gram-negative bacteria, the biocidal activity against Gram-positive bacteria was better, which gave rise to the more complicated cell wall compositions and protective mechanisms of Gram-negative microorganisms. Taken together, the biocidal process of OPEs was proposed. Driven by the electrostatic and hydrophobic interactions, cationic OPEs initially displayed strong association with negatively charged bacterial membrane (presumably penetration). Under irradiation, the generation of singlet oxygen and/ or other ROS at the interface of OPE−bacteria destroyed the normal cell metabolic activities, ultimately resulting in bacterial damage and death. More recently, Whitten and co-workers explored the dark and UV light-activated biocidal activity of several symmetrical and asymmetrical cationic OPEs (132 and 133) against Gramnegative E. coli and Gram-positive S. epidermidis and S. aureus.209 All OPEs exhibited efficient dark toxicity, and the biocidal effects were positively related to the conjugation length, concentration, and incubation time. However, the symmetric oligomers 132 possessed stronger antibacterial activity than that of the asymmetric 133. For light-activated killing, the structure−activity relationship between S-OPEn(H) and OPE-n series was obviously different, where 132−1 presented the highest biocidal effect in the 132 series as a result of the large singlet oxygen QY, whereas 133−3 mediated the

Whitten and co-workers conducted a series of experiments to get further insights into the biocidal mechanism of PPE-based cationic CPs. They first investigated the interaction of PPEs with negatively charged phosphatidylglycerol (PG) lipid membranes.207 In this study, three model anionic lipid membranes were utilized to simulate bacterial cell membranes, including unilamellar lipid vesicles (i.e., liposomes) in aqueous solution, lipid bilayer-coated silica microspheres, and lipid monolayers at the air−water interface. The chosen polymers were the pronounced dark-toxic polymer 127 and the strong light-toxic polymer 47. Upon electrostatic and hydrophobic interactions of both cationic PPEs with anionic lipid membranes, the enhanced fluorescence and evident FRET from PPE to rhodamine incorporated in the lipid membrane were observed, indicating the strong binding and insertion of cationic PPE into the anionic lipid membranes to transform the aggregated polymers to a more separate and planar conformation. However, in contrast with 47, the thiophenecontaining 127 exhibited stronger affinity by generating larger fluorescence enhancement and more rapid and efficient insertion into lipid membranes, which further accounted for the excellent dark antimicrobial activity (Figure 49). The observed different interactions with lipid membranes between the two polymers lie in the different molecular structures, where 47 has longer and bulkier pendant QA groups than 127. In the next place, lipid headgroup charge and membrane fluidity were considered as other factors to further probe the antimicrobial mechanism of PPE 127.208 In this study, small unilamellar vesicles (liposomes) and lipid monolayers at the 4726


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membranes. Facilitated by the strong electrostatic driven forces, other cationic PPEs and OPEs exhibited pronounced membrane-disruption selectivity toward bacterial-membrane mimics over mammalian cell-membrane mimics. For OPEs, the longer the molecules were, the easier the bacterial membrane was destroyed. Therefore, phospholipid composition of vesicles strongly influences the interaction between PPEs/OPEs and lipid membranes. The disclosed structure− property relationship of PPE/OPE further establishes the basis for exploiting selective biocidal agents toward bacteria over mammalian cells. To combine the fluorescent property of WSCPs and the antimicrobial activity of gold NPs, Moggio and co-workers fabricated gold NP/PPE composites by blending bromobenzenethiol passivated gold NPs and PPE (49) with flexible thioester chains.90 The proposed assembly mechanism was Au−S coordination and π−π interactions between the bromobenzenethiol components and PPE backbones. The resultant particles were uniformly dispersed and still maintained moderate fluorescent property both in solution and in solidstate film in spite of the quenching effect of gold NPs. Upon contact with P. variotii fungus, the composite films exhibited a quenched fluorescence response, indicative of the affinity of 49 toward microorganisms. Unexpectedly, only a slight antimicrobial effect of 1.84% reduction was observed for E. coli and no antimicrobial effect against P. variotii was found, which was presumably ascribed to the inefficient accessibility of bacteria to contact with the 49-coated gold NPs. Further improvement was necessary to keep the antimicrobial activity of gold NPs and the fluorescence property of WSCPs. On the basis of the identical expectation of simultaneous detection and destruction of microorganisms, Moggio and co-workers fabricated a silver NP/49 composite.211 The resultant nanocomposite possessed strong emission, which indicated that no marked energy transfer from 49 to silver occurred. Therefore, the nanocomposite film could be used for the detection of P. variotii fungi. Indeed, the film immersed in the suspension of P. variotii fungus displayed a distinct fluorescence signal reduction as a result of the deposition of P. variotii on the film to quench the fluorescent PPE. Most importantly, the nanocomposite film exhibited an attack and damage behavior toward the mycelium of P. variotii fungi, resulting in empty and fragmented cells due to the local direct contiguity of the silver NPs with P. variotii fungi (Figure 50).

strongest killing efficiency in the 133 series due to the efficient membrane disruption ability and singlet oxygen-sensitizing ability. Correspondingly, the proposed light-induced antibacterial mechanism was attributed to the combination of bacterial membrane disruption and the interfacial or intracellular generation of singlet oxygen or other ROS. Nevertheless, singlet-oxygen generation still seemed to be the main factor to account for the biocidal activity.

Because of the different phospholipid compositions, mammalian membrane (e.g., erythrocyte) generally displays nearly neutral surface charge, whereas bacterial membrane (e.g., E. coli) is highly negatively charged. Building on the simulated model bacterial and mammalian membranes (in the form of large unilamellar vesicles), Whitten and co-workers further investigated the membrane-perturbation capability of several cationic PPEs (47, 95, 125, and 127) and OPEs (129, 130, 133, and 134) with different side-chains to better understand how these antimicrobial molecules interact with membranes.210 Their results indicated that no destructive effect toward lipid membranes was observed in anionic 95 and the shortest cationic 133−1 as a result of electrostatic repulsion or unbound attribute and the insufficient length to span the entire membrane, respectively. Because of the high charge density and hydrophobic alkyl chains of 47 and the comparable linear length of 129 and 130 to insert into the lipid bilayer, polymer 47 and two “end-only” functionalized OPEs (129 and 130) are able to perturb both model mammalian and bacterial

Figure 50. Laser confocal micrographs of 49/Ag NP films after immersion in the P. variotii suspension. (a) In mycelium, fragmentation is observed, and (b) a mycelium fragment in which pores can be observed. Reproduced with permission from ref 211. Copyright 2010 Elsevier. 4727


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which was facilitated by the good light-harvesting ability and the large 2P absorption cross section of PFP.213 Subsequently, Xu and co-workers prepared photosensitizer-doped CPNs with a size of ∼50 nm. The obtained CPNs were composed of PFEMO (135) and tetraphenylporphyrin (TPP), between which an efficient energy transfer from 135 to TPP could be induced. Under 2P-excitation conditions (800 nm), a 21-fold enhancement emission in TPP was obtained. Simultaneously, the enhanced 2P singlet-oxygen generation in the photosensitizer-doped CPNs was directly observed (Figure 52).120

Previous biocidal studies mainly focused on simple bacterial systems or artificial biological membranes, and none of the specific association in a mixed biological system is reported due to the intricate surface situations. Very recently, Wang and coworkers reported a multifunctional cationic PPV-1 (polymer 2) for simultaneously accomplishing three functions: selective recognition, imaging, and killing of bacteria over mammalian cells.212 In a mixture of bacteria and mammalian cells, polymer 2 exhibited selective electrostatic binding toward bacteria as a result of the higher negative charges on bacterial surface and further lit them up (Figure 51). On the basis of the differential

Figure 52. Schematic illustration of the formation of CPNs for 2Pexcitation singlet-oxygen generation. Reproduced with permission from ref 120. Copyright 2011 American Chemical Society.

On the basis of the fact that 2P cross sections of organic molecules can be significantly improved by extending the conjugation length and coplanarity, Xu and co-workers further improved the 2P optical cross sections of WSCPs at 800 nm by incorporating ethynylene (polymer 136) and vinylene (polymer 137) bridges into the backbones of polymer 1.214 Facilitated by the efficient FRET from WSCPs to photosensitizer Rose Bengal, an up to 85-fold enhanced 2P-excitation emission of Rose Bengal was demonstrated. Besides, McNeill and co-workers also fabricated several TPP-doped CPNs (including PFO 85, PFPV 87, and PDHF 90, with a size of ∼50 nm) that exhibited high excitation cross sections of 1P excitation (10−15 to 10−12 cm2) and 2P excitation (∼106 GM) and further demonstrated the enhanced singlet-oxygen generation (QY = ∼0.5 for TPP-doped PHDF-CPNs).121 The above studies pave the groundwork for the potential application of WSCP-based energy-transfer systems in enhanced PDT.

Figure 51. CLSM images of Jurkat T cells and Gram-positive B. subtilis incubated with PPV-1 (a) and Jurkat T cells and Gram-negative Ampr E. coli incubated with PPV-1 (b). Left: bright field images, right: fluorescent images. Reproduced with permission from ref 212. Copyright 2011 Wiley-VCH Verlag GmbH & Co. KGaA.

binding of PPV-1 toward bacteria and mammalian cells, the selective killing of bacteria was successfully achieved, where mammalian cell viability was basically unaffected. Further investigation indicated that the antimicrobial activity of PPV-1 arose from two collaborative components: (i) dark-toxic QA groups and (ii) light-toxic PPV-1 main backbone. The proposed light-activated biocidal effect originated from a small amount of singlet oxygen, other ROS, and/or the direct interaction between PPV-1 triplet and the biological targets on bacterial cell membrane. Although the biocidal efficiency toward Gram-negative E. coli under white light is somewhat modest (9-fold enhancement of HP emission by 1P excitation (380 nm) and 30-fold enhancement by 2P excitation (800 nm) were observed,

Wang and co-workers first reported a PT−porphyrin-based energy-transfer system for improved light-activated killing of bacteria.119 The energy-transfer pair was composed of an anionic PT donor (138) and a cationic porphyrin acceptor (TPPN). By virtue of electrostatic interactions, 138 and TPPN formed tight complex particles (size = 1−4 μm) with net 4728


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and simultaneous apoptosis imaging.215 In this study, five cell lines, including two normal (fibroblast and HPF) and three cancer (A498, A549, and HepG2) cells, were utilized. Cell viability analysis demonstrated that PMNT was basically biocompatible to all cell lines with the exception of A498 cells, indicative of the potential to be selective renal cancer therapy. Upon incubation with A498 cells, PMNT was able to enter into the cells in a passive diffusion manner and localize in the cytoplasm. Following that, cell apoptosis was induced by the internalized PMNT, which was confirmed by the upregulated caspase-3. Moreover, the increased expression of caspase-3 was dependent on the incubation time and PMNT concentrations. Of note, with the aid of irradiation light at 455/ 70 nm, cell apoptosis caused by PMNT could be accelerated to take place only within several minutes. Importantly, PMNT itself could be used for monitoring the apoptosis process by selectively staining apoptotic cells with a pattern of dense yellow clusters (Figure 54), avoiding the extra addition of apoptosis-specific imaging agents. However, the specific target site of PMNT and the selective mechanism were still unclear.

positive charges on the surfaces. Under white-light irradiation, efficient energy transfer (Dexter mechanism) from 138 to TPPN followed by ISC to the triplet of TPPN resulted in the sensitization of oxygen molecules to produce singlet oxygen (Figure 53a). Owing to the excellent capability of light

Figure 54. Fluorescence images of A498 cells grown with PMNT for 48 h. Reproduced with permission from ref 215. Copyright 2010 Royal Society of Chemistry.

To further improve the antitumor efficiency of WSCPs, Wang and co-workers designed another cationic porphyrincontaining PTP (139) that simultaneously possessed the abilities of imaging and therapy.97 The obtained polymers intrinsically existed in the form of NPs with an average diameter of 350 nm. Under light irradiation, the cytotoxic singlet oxygen could be sensitized via the intermolecular energy transfer from PT backbones to porphyrin units. Importantly, in contrast to PT alone and porphyrin alone, 139 displayed significantly enhanced ability to sensitize singlet oxygen. Two types of cancer cells, including A549 and A498 cells, were chosen as the models to investigate the light-activated killing ability of 139. As expected, upon irradiation at 470 nm for 30 min, an obvious decrease in cell viability for both cells was observed (Figure 55). However, the control groups (PT alone, TPN alone, and 139 without illumination) were basically not toxic. Moreover, the constructed multifunctional molecule still kept the ability of imaging apoptotic and necrotic cells with a distinguishable nucleus-staining pattern. To attain the objective of selectively recognition and killing of cancer cells, another electrically neutral folate-functionalized PT (140) was synthesized to target FR-overexpressed KB cells. After incubation for 24 h, a specific internalization was found in KB cells over NIH 3T3 cells (FR-negative). Cell viability analysis indicated that the light-induced cytotoxicity of 140 was selective against KB cells with over 80% cell damage, whereas for NIH 3T3 cells above 70% cells survived. As a control, no evident dark toxicity was observed for both cells. This work demonstrates the

Figure 53. (a) Schematic antibacterial mechanism of 138/TPPN complexes. (b) Biocidal activity of 138/TPPN complex toward E. coli in the dark and under white-light illumination for 5 min. Reproduced with permission from ref 119. Copyright 2009 American Chemical Society.

harvesting and light amplification of 138, the produced singlet oxygen was more efficient than the situation by direct excitation of the sensitizer TPPN. The positively charged 138/TPPN complexes exhibited efficient association with Gram-negative E. coli and Gram-positive B. subtilis driven by electrostatic and hydrophobic interactions. Subsequently, white-light-activated singlet oxygen produced at the interfaces of 138/TPPN complexes and bacteria led to the direct bacterial inactivation by membrane disruption. Even for 5 min of irradiation with white light at a fluence rate of 90 mW, the system still displayed obvious biocidal activity with a killing efficiency of ∼70% against E. coli and >90% against B. subtilis (Figure 53b). 5.2. Anti-Tumor Activity of WSCPs

In addition to the antimicroorganism activity, the antitumor activity of WSCPs has aroused researchers’ great interests and is beginning to emerge as a new research hotspot. In 2010, facilitated by the multifunctional cationic PMNT (polymer 6), Wang and co-workers successfully achieved cancer cell killing 4729


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as a result of the presence of huge development space for researchers to better settle the issues mentioned above and explore more novel applications by designing and synthesizing novel WSCPs. Looking ahead, we speculate that WSCPs will display functions in more sophisticated and more in-depth biological systems. From the perspective of cell or animal level, body fluid-compatible systems with the integration of multiple functions (i.e., multifunctional systems), such as simultaneous imaging with high specificity and tissue penetration (NIR probe or 2P excitation), diagnosis with high accuracy, and therapy with less side effects, will be designed to further develop their practicality in biomedical and even clinical applications (e.g., early diagnosis and therapy of cancers). From the perspective of molecular level, targeting intracellular recognition and regulation events, such as interfering cellular signaling pathways by controllable photoinactivation of specific proteins and regulation of gene expression by exogenous stimuli, will possibly be another application of WSCPs to further explore the intricate cell biological process. There is still good potential for WSCPbased approaches to be incorporated into routine biological assay protocols in the near future. In the end, we sincerely hope that this tutorial review is able to help readers form an overall framework of the applications of WSCPs in imaging, diagnosis, and therapy; we also more hope that a large number of innovative ideas can be inspired by this review.

Figure 55. Merged images of A498 cancer cells under phase-contrast bright field and fluorescence field for PTP and EB before and after 30 min irradiation. Reproduced with permission from ref 97. Copyright 2011 Wiley-VCH Verlag GmbH & Co. KGaA.

concept of photocontrollable targeted cell damage with both imaging and therapy functions. It should be noted that, in spite of the efficient antitumor activity of WSCPs, selective killing is still lower toward cancer cells over normal cells. That issue needs to be resolved by designing more novel systems with high specificity and low side effects.

AUTHOR INFORMATION Corresponding Author

6. CONCLUDING REMARKS In this review, we mainly describe the progress of WSCPs in imaging, diagnosis, and therapy during the past 5 years (2007− 2011). By virtue of the unique light-harvesting ability of CPs, numerous exciting advancements have been successfully realized. Specifically, we include a comprehensive overview of the applications of WSCPs in (i) diagnosis of pathogenic microorganisms, tumor cells, and diseases-related biomarkers, (ii) fluorescence imaging in vitro and ex/in vivo, (iii) drug delivery and release, gene delivery, and drug screening, and (iv) light-assisted antimicroorganism and antitumor therapy. Relative to previous usages of WSCPs in chemical/biological sensors, the studies shown in this review, from another point of view, upgrade the depth and breadth of the applications of WSCPs and demonstrate a powerful vitality of WSCPs in biological and biomedical areas. In spite of the encouraging progress, limitations that prevent these WSCPs from further development and practical applications still exist, including undesirable self-aggregation and interactions with hydrophobic substrates, uncertain MW and varying QY between batches, low QY of most NIR WSPCs, low killing selectivity toward cancer cells over normal cells, impotent function in biological fluids, and potential toxicity for in vivo use. Despite these remaining challenges, a very bright future of WSCPs can still be expected

*Email: [email protected]. Notes

The authors declare no competing financial interest. Biographies

Chunlei Zhu obtained his B.S. degree in Chemistry from Jilin University in 2008. He then moved to Institute of Chemistry, Chinese

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Academy of Sciences, to begin his graduate study under the supervision of Professor Shu Wang. His current Ph.D. thesis focuses on the design and synthesis of novel water-soluble conjugated polymers for their biomedical applications, including microbial pathogen detection, cell imaging, drug screening, and antimicroorganism/antitumor therapy.

Fengting Lv received her B.S., M.S., and Ph.D. degrees from School of Chemistry and Materials Science of Shaanxi Normal University in 2002, 2005, and 2008, respectively. During the period of pursuit of her Ph.D. degree, she spent one year at Michigan State University as a joint Ph.D. student. She moved to Institute of Chemistry, Chinese Academy of Sciences, in 2009 as a postdoctoral fellow in Professor Shu Wang’s group. In 2011, she joined the research team as an assistant professor. Her current research interests focus on the design of novel water-soluble conjugated polymers for cell imaging and biosensor applications.

Libing Liu received his B.S. and M.S. degrees from College of Biological Sciences at China Agricultural University, respectively, in 2000 and 2003. He received his Ph.D. degree from Institute of Radiation Medicine, Academy of Military Medical Sciences, in 2007 in the field of biochemistry and molecular biology. He then moved in 2007 to Institute of Chemistry, Chinese Academy of Sciences, to carry out postdoctoral research in Professor Shu Wang’s group. He joined thereafter the research team, and in 2009 he became associate professor. His current research interests focus on the design of novel water-soluble conjugated polymers for cell imaging, gene regulation, and biosensor applications.

Shu Wang received his B.S. degree from Department of Chemistry, Hebei University, in 1994 and Ph.D. degree in organic synthesis at Department of Chemistry, Peking University, in 1999. Following two years of postdoctoral research at Institute of Chemistry, Chinese Academy of Sciences, he moved to Institute of Polymers and Organic Solids, University of California at Santa Barbara, to continue his postdoctoral research. In 2004, he became a professor of Institute of Chemistry, Chinese Academy of Sciences. His current research interests include design, synthesis, and properties of light-harvesting conjugated polymers, biosensors, and chemical biology.

Qiong Yang received her B.S. degree from Department of Clinical Medicine, Jinzhou Medical College, in 1998 and M.S. degree in Medical School, Jilin University, in 2001. She received her Ph.D. degree in 2004 and then worked until 2007 in the Institute of Genetics and Cytology, in Northeast Normal University. Following two years of postdoctoral research in UPMC, Pittsburgh University, she joined Institute of Chemistry, Chinese Academy of Sciences, as an associate professor. Her current research interests focus on tumor diagnosis and cell imaging.

ACKNOWLEDGMENTS The authors are grateful to the National Natural Science Foundation of China (Nos. 20725308 and 21033010) and the Major Research Plan of China (No. 2011CB932302, 2011CB935800, and 2011CB808400). REFERENCES (1) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. Rev. 2000, 100, 2537. (2) Bunz, U. H. F. Chem. Rev. 2000, 100, 1605. (3) Liu, B.; Bazan, G. C. Chem. Mater. 2004, 16, 4467. (4) Ho, H. A.; Bera-Aberem, M.; Leclerc, M. Chem.Eur. J. 2005, 11, 1718. 4731


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