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Recent studies have shown a great interest in conjugated polymers because of their ability to be used for creating bioimaging tools or biosensors linking spectral signal and different biological processes directly. By employing the efficient light harvesting properties or the conformation sensitive optical properties of the CPs, the detection schemes of these sensors can be achieved through fluorescence resonance energy transfer (FRET) between the CP and an acceptor molecule. Due to their intrinsic merits, conjugated polymer based fluorescent nanoparticles have received wide interest in bioimaging as an alternative to conventional fluorescent probes. This review article focuses on the recent development of polymer encapsulated polymer nanoparticles in bioimaging applications, especially fluorescent bioimaging and biosensors. Specific requirements of various imaging tasks are satisfied by obtaining polymer nanoparticles with desired optical properties and surface functional groups through emulsion or nanoprecipitation. Advanced fluorescent probes for in vitro and in vivo bioimaging applications are made possible by fine-tuning of the conjugated polymer structure and polymeric encapsulation matrix.
Nature always provides us the most favorable solutions to give one inspiration when aiming for more sensitive sensory systems. For the need of monitoring of life, the evolution has designed complex living organisms and provided them with a large variety of ultimate chemical sensors. Specifically tuned to interplay between receptor derived selectivity and analyte sensitive signaling cascades, the biosensory system itself is a finely display result from the evolution. It is obvious that in order to be successful, the requirement of strong and specific analyte binding and an easily observable recognition event must be fulfilled by the design of artificial sensory systems.
On one hand, conjugated polymers (CPs), being able to directly linking between spectral signal and different biological processes, have proven to be playing an important role in coupling analyte receptor interactions into observable responses. Molecular probes which are sensitive to very minor perturbations can be achieved by combining the electro-optical properties of conjugated molecules and the characteristics of polymers . Conjugated electrons provide connection for the potential binding sites along the polymeric backbone and the entire assembly is affected by any perturbation of transport properties of the conjugated system. Therefore, even a small change leads to large signal amplification due to the collective features of the CP . An application of CPs for colorimetric detection of biological targets was reported by Charych and colleagues  in 1993 for the first time to our knowledge. Because of the induced conformational change of the polymer backbone, the technique was based on a ligand-functionalized CP (polydiacetylene) which can signal receptor-binding into a chromatic transition called biochromism. The analyte specificity of this first generation of CPs, however, was dependent on a ligand attached to the polymer side chains, thus, the detection and recognition event became a function of the nature and characteristics of the polymer chains. As side-chain functionalization of the CP requires advanced and extensive purification of a large number of monomeric and polymeric derivates, this became a major drawback for this first generation of CPs. Using optical absorption as source of detection, the sensitivity of the first generation of sensors was much lower compared to other sensing systems for biological processes .
On the other hand, fluorescent techniques have been playing a significant role in cellular imaging to understand the function and mechanism of target biological species, and also to provide important information for early stage cancer diagnosis and therapy. Fluorescent materials with molecular imaging functionality, especially small fluorophores, fluorescent proteins and semiconductor quantum dots (QDs) have been most extensively employed for bioimaging. They are not only of great importance for biology and pathophysiology, but also of practical interest for clinical diagnosis and therapy [5-7]. They have durable high brightness, sufficient water-dispensability, good biocompatibility and ease of bioconjugation, which are essential properties for optimum deciphering and tracking of physiological behaviors and functions with maximum spatial resolution and minimal perturbation to biological systems. However, small fluorophores and fluorescent proteins usually have low photobleaching thresholds that limit their effectiveness in long-term and three-dimensional imaging , while QDs generally comprise intrinsically toxic elements such as cadmium and selenium which can release and toxic to both cell cultures and live organisms [9, 10]. The intrinsic disadvantages of those conventional fluorescent materials lead researchers to discover and explore new fluorescent probes to improve their performance [11s].
The aim of this review is to give the reader an insight into the expanding application field of CPs for imaging of different biological events. The utilization of CPs especially polymer encapsulated CP NPs for in vitro and in vivo bioimaging applications will be discussed in detail. The applications of polymer encapsulated CP NPs in a diverse range of areas, such as non-specific cellular imaging, targeted in vitro/in vivo cellular imaging and other bioimaging, are to be discussed in detail. In the summary, the advantages and limitations of using CP NPs for bioimaging are covered.
Chemical structure and property of conjugated polymers
Conjugated polymers (CPs) have attracted intense attention based on their property of conductivity, photoluminescence, electroluminescence, or light-induced charge generation. An important advantage of CPs is their potential to exhibit collective properties which are sensitive to very minor perturbations, allowing CPs couple analyte receptor interactions into observable responses[1, 2].
The common structure feature of conjugated polymers is a fully conjugated polymer backbone which provides an extended delocalized pi-electron system and thus makes their electrical and optical properties realizable. The absorbed light energy could be effectively emitted as fluorescence light due to the rigid polymer backbone, instead of being relaxed radiationless with more flexible structures. CPs could be classified as water-soluble CPs (WSCPs) and water-insoluble CPs based on whether they have charged side-chains to be dissolved in aqueous solution. Another kind of newly developed CPs are conjugated polymer nanoparticles (CP NPs) which have nanometer size and many unique advantages. But CP NPs are different from quantum dots (QDs) as CP NPs do not have size-dependent photophysical properties and fluorescence blinking phenomenon[5, 6].
The first generation of CPs for sensory systems was dependent on a specific ligand attached to the polymer side chains to detect ligand-receptor interactions and achieve the analyte specificity[2, 7]. However, these CPs have the drawbacks of covalent attachment of the ligand to the side chain and low sensitivity. Therefore, luminescent ionic derivatives of CPs, which can be called as the second generation of CPs, were developed, based on multivalent non-covalent interactions between a synthetic polymer and a natural polymer (the CP and the biomolecule). These sensors can be classified into two categories based on their detection types. One type makes use of the effective light harvesting capacity of CPs and the transferring of the collected energy from conjugated chain to an adjacent acceptor. In the first type, there are two kinds of acceptors: a quencher acceptor induces the loss of fluorescence from the conjugated chain, while the fluorophore acceptor has efficient fluorescence resonance energy transfer (FRET). The other type is based on the changing of conformation and geometry of the CP chain caused by biological processes, which induces the observable alteration of the emission and absorption properties of CPs.https://lh3.googleusercontent.com/7jXIPne3F-ID2NDtJuyRBlNEQzxku3OV4OLGVHbftYtcwWAGekoExnCZy8BbYEiizVuKchwd8BbRmK8crpGKPNQcKsyB3se1PSBlcXAnupIgavjpfLfQ-sgU
Figure 1 Two detection systems of CPs for DNA hybridization detection
Applications of CP in biomedical imaging
Because CP NPs have been widely used for a variety of bioimaging areas such as non-specific and targeted cellular imaging, our discussion here is mainly focused on applications of CP NPs in bioimaging. CP NPs not only have been extensive used for in vitro and in vivo cellular imaging, but also have been involved in biological metal ion detection, intracellular pH value and temperature sensing by utilizing their unique fluorescence response to the environment change.
Non-specific cellular imaging
Fluorescence-based cellular imaging is an important approach to understand the bio-molecular interactions and cellular processes such as gene expression, protein transport, signaling, and regulatory processes . Due to advantages on ï¬‚uorescence brightness, photostability and low toxicity, conjugated polymer nanoparticles (CP NP) is a potential candidate for non-specific cellular imaging. For bare hydrophobic CP NP, in vitro cellular imaging applications of CP NP were initially conducted by a nonspecific labeling method, such as endocytosis. Research with PFBT and in J774A.1 cells  has been carried out on the mechanism behind the cellular uptake of CP NP. Results show a high efficiency of CP NP uptake with no significant cytotoxic or inflammatory effects and it suggests that macropinocytosis is the main mechanism behind the uptake. The non-specificity of macropinocytosisis leads to the non-specificity of the CNP uptake.
Also, CP NPs could gain hydrophilic characters by introducing extra parts like iron ligands. Composites built in this way have been widely used in the cancer research. In one research, hydrophilic CP NP was developed by binding the poly(DL-lactide-coglycolide) (PLGA) nanoparticles (NPs) with CP through solvent extraction/evaporation technique. The efficacy of this functional CP was tested by incubating with breast cancer cells named MCF-7. Results showed a high uptake efficiency and low toxicity with this hydrophilic CP NP. Generally, the uptake mechanism of this hydrophilic CP NP is through macropinocytosis or receptor-dependent endocytosis approaches. Actually, this CP NP construction strategy can not only apply to bioimaging but also apply to develop high efficient and specific cancer drug delivery media.
Specific cellular imaging.
Compared with non-specific cellular imaging, specific cellular imaging is of more importance in the applications like the detection and research of cancer cells and its intrinsic mechanisms. Due to the exceptional brightness and nontoxic feature, polymer dots have great potential in the specific cellular imaging. The challenges for applying CP NP in specific cellular imaging includes the low quantum yields of CP NP fluorescence brightness in the red and near infrared (NIR) and the difficulty to deliver the CP NP-based probes to the diseased tissues in vivo. In a research for tumor-targeting CP NP, a blending method was implemented to settle the low quantum yield problem. A visible-light-harvesting polymer (PFBT) which serves as the donor and a deep-red emitting polymer (PFDBT5) which serves as the acceptor was blended together. For this composite particle, the intra-particle energy transfer can lead to the complete quenching of the donor part while high fluorescence from the acceptor alone. The quantum yield could be as high as 60%. Chlorotoxin (CTX), which has a strong affinity with tumors of neuroectodermal origin is selected as a tumor-targeting ligand. The composite particle shows strong binding specificity with malignant brain tumors.
Another specific cellular imaging application is bioorthogonal labeling. The introduction of bioorthogonal chemistry has allowed the real-time study of biomolecules in living cells without toxic effects on cell .In one research, probe was constructed by covalently linking functional molecules to CP NP for click chemistry-based bioorthogonal labeling on cellular targets. Probes built in this way could selectively bind with newly synthesized proteins or glycoproteins in mammalian cells which were metabolically labeled with bioorthogonal chemical reporters. This functional CP NP could assist the research in biology research.
In vivo application
In addition to the in vitro cellular targeting applications of CP NPs, their potential in in vivo imaging was also investigated as fluorescent probes. For example, research has been carried on conjugating the PSMA encapsulated P9/P12 hybrid NPs to chlorotoxin (CTX) and PEG, and thus, their ability to target tumor and circulation in vivo can be increased. As shown in Figure 2A, the ND2: SmoA1 mice had P9/P12 NP-CTX conjugates accumulating at the brain tumor regions. Thus, the P9/P12 NPs' targeting ability to malignant brain tumors had been confirmed. On the contrary, P9/P12 NPs without CTX conjugation has not shown obvious tumor site accumulation in brain. The accumulation of conjugates in the brain tumor was achieved within 24 hours and the signal intensity remained steady for 48 hours during the 72 hours analysis using real-time investigation of the circulation profile of the P9/P12 NP-CTX conjugates. In Figure 2B, however, obvious non-specific accumulation in liver and kidney during the tested period was shown from the conjugates.
Figure 2 A) Ex vivo fluorescence imaging of healthy brains in wild-type mice and medulloblastoma tumors in ND2: SmoA 1 mice. Each mouse was injected with either P9/P12 NP (top), or P9/P12 NP-CTX (middle). Control: no injection (bottom).
B) Biophotonic images of resected livers, spleens, and kidneys from wild-type (middle) and ND2:SmoA 1 (bottom) mice receiving P9/P12 N-CTX injection. Control: no injection (top).
Single particle tracking
Single particle tracking has found a rapidly expanded use within the life science due to an increased demand to understand the spatial dynamics of bio-molecular interactions at the molecular-size spatial scale without ensemble averaging, such as analyzing the molecular organization of biological membranes, molecular transport, and the motion of motor proteins.[6, 9, 10]
Conjugated polymers nanoparticles (CP NPs) make the single particle tracking more easily accessible with satisfactory resolution. Wu et al. developed conjugated polymer dot nanoparticles with well controlled size distribution and high quantum yields (as high as 40%). They also demonstrated that the fast emission rate of CPs and highly packed chromophores could result in high emission rates and minimal "blinking" behavior under single particle imaging conditions, indicating their potential for high speed super-resolution single particle tracking in live cells and highly sensitive assays.  Later, Yu et al. evaluated the highly fluorescent CP NPs for nanoscale two-dimensional (2D) and three dimensional (3D) tracking applications. By imaging single CP NPs with an inverted fluorescence microscope equipped with CCD and analyzing trajectories of fixed and freely diffusing particles, respectively, they obtained a theoretical particle tracking uncertainty of less than 1nm and lateral tracking uncertainty of 1-2nm. Another research by Yu et al. studied tracking single charge carries in CP NPs. Using video rate fluorescence microscope, fluctuations in the fluorescence intensity and nanometer-scale displacements in the fluorescence centroid of single CP NPs were observed. This phenomenon was due to the hopping motion of hole polarons and could be used to measure polaron displacement.
The ability to probe the organizational and dynamic characters like PH and temperature value of single cells play an important role to understand the cellular function. For example, the local pH within a cellular organelle is a key element to understand physiological processes of these organelle due to its fundamental effect on biomolecular environment.
Förster resonance energy transfer( FRET) based nanoparticles were firstly tried by linking quantum dots with pH-sensitive dyes. FRET occurs between the quantum dot with the pH-responsive dyes once the quantum dots or dyes are excited based on the pH of environment. The pH is determined by the ratio of fluorescence intensity of the dyes and the quantum dot. However, negative effects like cytotoxicity limits the application of quantum dots based nanoparticles.
The CP NP is an ideal replacement of quantum dots on pH detection and has attract great interest recently due to extraordinary light-gathering capability and efficient intraparticle energy transfer. For example, a pH-sensitive CP NP probe was developed by binding poly(2,5-di(3', 7'-dimethyloctyl)phenylene-1,4-ethynylene) (PPE) with pH-sensitive dye fluorescein. This composite has a linear wide PH sensing range between 5 to 8 which are suitable for most biological applications.
The construction strategy of temperature-sensitive CP NP is quite similar to that of pH-sensitive CP NP. In one research, a temperature-sensitive dye named Rhodamine B(RhB) was attached with semiconducting CP NP. The RhB has the characteristics that its emission intensity decreases with increasing temperature. The Pdot-RhB composite shows high temperature sensitivity and brightness. Compared with other temperature-sensitive composite, the Pdot-RhB could express ratiometric temperature sensing under a single wavelength excitation with a wide linear temperature sensing range .
Also, CP NP has been developed to detect biologically relevant ions like Cu2+, Fe2[18,22] and biomolecules[23, 18], such as protease which also based on the high energy transfer efficiency of CP NP.
In this review article, we have summarized the efficient light harvesting ability or the unique conformational induced optical properties of CPs and their utilization to image certain biological events. Detailed discussion has been placed on the recent development in polymer encapsulated CP NPs, because they can serve as a class of promising alternative to conventional fluorescent materials to meet the requirements of in vitro and in vivo bioimaging. It is also indicated by several studies that CP NPs are of very low cytotoxicity, and this is beneficial to their applications in bioimaging. With the rapid development in materials and biological engineering, polymer encapsulated CP NPs along with other new conjugated polymers will serve as a great inspiration for new research opportunities in bioimaging.