AIEgen–lipid structures: Assembly and biological applications

Aggregation‐induced emission (AIE) molecules possess notable advantages outperforming traditional aggregation caused quenching (ACQ) materials on various aspects. They are rapidly developed these years. More and more AIE luminogens (AIEgens) are designed to possess multifunctions such as the abilities of near‐infrared two‐photon absorption and reactive oxygen species (ROS) generation, which could be used for deep tissue imaging and photodynamic therapy. The AIEgens exhibit great potential in biological application field. However, despite the photophysics stability and ROS generation ability in aggregated states are favorable conditions, their applications in biological field are retarded by uncontrolled size, single imaging mode, low targeting efficiency, and also poor biocompatibility and dispersibility in physiological environment. The combination of AIEgen and lipid is a straightforward, promising, and intensively used way to solve the above problems. Due to the special amphipathic property of lipid, which results from a hydrophilic head and hydrophobic tail structure, there are various possibilities of combination modes between lipid and AIEgen. Even a little procedure or condition change during the synthesis process will impact the structure of obtained product, which can further influence its application. Herein, we summarize the synthesis methods of different AIEgen–lipid compounds with diverse structures and properties, as well as their biological applications in this contribution, which has not been presented before, being aimed at serving as a synthesis and application reference for these promising AIEgen–lipid compounds applied in biological region.


INTRODUCTION
The concept of aggregation-induced emission (AIE) was first put forward by Tang et al. in 2001, [1,2] which described a phenomenon that AIE luminogens (AIEgens) displayed no or weak fluorescence in dilute solutions but strong emission in the aggregated states. [3,4] The mechanism is illustrated as that once the molecules aggregate, the process of nonradiative relaxation is blocked due to the restriction of intramolecular vibrational and rotational motions, thus the radiative decay process inducing strong fluorescence is activated. [5][6][7] The structures and properties of AIEgens are quite different from the traditional aggregation caused quenching (ACQ) or their aggregates exhibit some limitations in multifunctional biological research on account of their large size, low targeting efficiency, poor biocompatibility, and stability in physiological fluids. [15,16] To overcome these defects, some assembled AIEgen-based multifunctional materials have been constructed, [17] such as AIEgen-silica, [18,19] AIEgen-BSA, [20,21] AIEgen-chitoson, [22] and AIEgen-lipid. [23] Among various types of clinically used formulations, lipidbased carriers hold a big share for their incomparable advantages, such as excellent biocompatibility, [24,25] negligible optical interference, [26] diverse modifiable groups, [27] high water-solubility, and stability. [16,28] Besides, the hydrophobic tail and hydrophilic head sections endow lipid amphiphilic property, which is diversely used as a delivery platform for different properties of drugs. [29,30] That means, lipid can carry hydrophobic materials inserted in the bilayer, hydrophilic materials in the interior core, and amphiphilic ones partitioned at the surface. Because of the entirely different performances of AIE molecules in different states, the manifestations can be justified by different combination modes between AIEgen and lipid. The lipid surface is easily modified with intended ligand for special targeting purpose and its interactions with some hydrophobic molecules present firmer effect than most encapsulation methods, which is very beneficial for the long-time tracing in vivo. [31] Some environmental response characters, for example, disruption property by the enzyme in lysosome, and some thermalsensitive properties are widely used in intelligent response system for drug relieving. [32,33] Moreover, for some AIE photosensitizers (PSs), their generation rates of ROS are greatly enhanced by the combinations, because the outside polymer layer protects them from interacting with the polar solution environment, which is beneficial for more effective ROS generation. [34] Owing to the delicate structures of lipids and the rapid development of AIE molecules with diverse structures and properties, the combination compounds of AIEgen and lipid various from each other when adjusting a little procedure during the synthesis process, the obtained AIEgenlipids will possess distinct properties. Thus, by controlling the synthesis methods, the acquired AIEgen-lipids could be used for different applications.
A series of AIEgen-lipid molecules and nanoparticles have been reported with multifunctional properties used in biological applications. However, to our knowledge, there is no comprehensive summary focusing on the AIEgen-lipid materials, especially the synthesis methods, which will greatly influence the performances of obtained products in this delicate system. Recently, a great deal of novel AIEgen-lipids have been designed and reported by some groups including ours, which are of great significance for the following investigating directions and applications of AIE molecules. Therefore, we are motivated to systematically summarize the synthesis methods, structures, and biological applications of AIEgen-lipids, including the "AIEgen-like-lipid" structures in recent years.

MULTIFUNCTIONALIZATIONS OF AIEGEN AND LIPID
Before the combinations, AIEgen and lipid molecules are usually designed and modified with multifunctions. Some excitation-driven boron complexes are designed and applied for their high sensitive stimuli-responsive luminescence. [35] Since two-photon absorption has plenty of advantages over one-photon, a series of AIEgen molecules have been designed with the capacity of two-photon absorbance through enhancing the conjugation extent, extending the effective conjugation length, improving the donor-accepter intensity or increasing the planarity and symmetry of molecule. (MesB) 2 DTTPS, a two-photon excited dye with strong red fluorescence was developed, the dot exhibited a large twophoton absorption cross section (TPAC) of 3.43 × 10 5 GM, and was successfully applied in cell and blood vascular imaging. [36] FTTDNP owned a large two-photon absorption across section (TPAC) value of 2.3 × 10 6 GM and bright farred/near-infrared emission. [16] The AIEgen molecule can be endowed with the ability of ROS generation through delicate molecule design for enhancing the intersystem crossing (ISC) process to be used for photodynamic therapy (PDT). One straightforward way is attaching heavy atom to fluorophore, however, the cytotoxicity of heavy atoms to normal tissues restricts its biological applications. [37][38][39] By separating highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) distribution, the value of ΔE ST would be reduced, thereby accelerating the ISC process, which results in the high ROS generation rate. [40,41] Incorporating donor and acceptor moieties onto AIEgen molecule is another efficient and widely used strategy, as well as the way of lengthening the distance between donor and acceptor. [42] More recently, conjugated-polymer-based PSs were found to have a better ability of ROS production than that of small-molecule analogs. [43][44][45][46] The AIE nanoparticle encapsulated by lipid have been proved more effective than bare AIEgen in ROS generation as the amphiphilic matrix protects the functional materials from exposing to oxygen and water invasion. [47,48] All these methods can be used to design and develop AIE-PSs and further used in the lipid system for the photodynamic anticancer therapy.
Also, the multifunction of lipid is simple to obtain because of its easy modification property with some functional molecules. The usually used target molecule is folate in the form of DSPE-PEG-folate, which can be mixed with other lipids into a liposome. It can be internalized into the cells due to specific interaction between lipid-folate and folate receptor overexpressed on the cancer cell membrane, which would promote the endocytosis progress. [16] HIV-1 Tat peptide is used to accompany with DSPE-PEG-Mal (maleimide), which can enhance the endocytosis amount of nanoparticles. For example, the collected Tat-Gd-AIE dots displayed greatly increased cell internalization efficiency and dualmodal imaging functions. [49] Tang et al. reported the fabrication of another AIE-Tat nanoparticle based on NPPITBT-TPE for long time tracing of the osteogenic differentiation of mouse. [25] The AIEgen-lipid compound has been proved to have many excellent characteristics. They are illustrated as follows: (1) High stability: for the obtained AIEgen-lipid of FTTDNP, no obvious precipitation was observed even after a storage period for 5 months; [16] (2) good light penetrability: the maximum absorption and emission spectra of FTNP and bare AIEgen nanoparticle were similar, [26] some nanoparticles with different lipid ratios possessed similar optical properties; [16] (3) controlled size: the nanoparticle size can S C H E M E 1 Summarized different self-assembled structures of AIEgen-lipid complex be controlled by the membrane pore size of extruder or sonication condition; [34] (4) the nanoparticle also presents low cytotoxicity, which is essential for in vivo bioimaging applications. [50]

COASSEMBLY AND STRUCTURES OF AIEGEN AND LIPID
It is well known that AIE molecules are highly soluble in common organic solvents but poorly soluble in water. They will disperse evenly or present aggregated states in various solutions. Utilizing this property, AIEgen-lipid compounds of diverse characteristics are obtained and applied in different ways. Since the interactions between them have great relations with the synthesis methods, detailed process are summarized. The synthesis methods and structures are summarized into four kinds as shown in Scheme 1, the properties of compounds differ from each other because of their different structures.

The core-shell structure by nanoprecipitation method
The detailed synthesis process of nanoprecipitation method is described as following: An organic solution containing AIEgens and lipids is poured into water to form a water/organic mixture solvent, and then followed by sonication with a microtip probe of sonicator. The obtained solution is then stirred for a certain period to ensure the evaporation of residual organic solvent. Some functional lipids, for example, DSPE-PEG-folate is also added in the first dissolved process by organic solvent. The AIEgen-lipid complex usually forms a kind of core-shell structure by this method (Scheme 1A).
The combination of AIE material and lipid was first reported by Liu and coauthors in the year 2011. [26] In this work, AIE chromophore T1 possess the ability of two-photon absorbance, the AIEgen was encapsulated in the lipid with an obvious core structure. Then, the authors also reported the lipid-folate encapsulated FTTDNP of two-photon absorption and bright farred/near-infrared emission. [16] As shown in Figure 1A, the nanoparticle was spherical with a diameter about 53 nm, and the large emission range appeared from 550 nm to 850 nm, which was a benefit for fluorescence imaging.
Tang et al. then reported the fabrication of another AIEgen-Tat NP (nanoparticle) based on NPPITBT-TPE for long time tracing of the osteogenic differentiation of mouse. [25] Then, a lipid nanopartilce based on (MesB) 2 DTTPS, a two-photon excited dye with strong red fluorescence was studied, and was successfully applied in cell and blood vascular imaging, 3D blood vasculature was visualized clearly upon excitation of the two-photon laser. [36] With the development of AIEgen studies, an increasing number of AIE PSs were designed and TPETCAQ was reported with enhanced ROS generation rate and red-shift emission, it was encapsulated by DSPE-PEG-Mal with TPETCAQ NP in the core and functionalized with HIV-1 Tat peptide as shown in Figure 1B. [34] The solution results indicated that TPETCAQ NP could generate ROS more effectively than TPETCAQ aggregates, and the cytotoxicity assay showed a much lower value of halfmaximal inhibitory concentration (IC50) than that of Ce6 and ICG. Also, the in vivo experiments were conducted and TPETCAQ NP presented promising potential in imageguided PDT, which promote the development of more AIE PSs for clinical applications.
The product results from a nanoprecipitation method and is a kind of nanoparticle that with an AIE aggregation in the aqueous core and encapsulated by lipid layer on the surface. Then, the AIE core will emit fluorescence and is used for imaging, the outside lipid layer is always modified with some targeted molecules to enhance the detected efficiency of nanoparticle. As compared to intricate chemical modification method to get valid imaging probe, polymeric nanoparticle formation of AIEgen is a more facile and versatile strategy. Some AIEgens with photosensitive performance can generate ROS, which will transfer across the lipid layer and reach the aimed region for damaging cells. However, due to the dense packing of AIEgens and limited oxygen dissolved in the core solution, which encapsulated in the nanoparticle formation process, the generation rate of ROS is somehow restricted by this method. Besides, owing to the short life time of ROS, the amount will be reduced during the transfer process. [51] Modified combination methods of AIEgen and lipid were further developed by the researchers.

The inserted structure of AIEgen into liposomes
The AIEgen-lipid conjugate was then developed by researchers to solve the problem of comprised ROS generation by nanoprecipitation method. Briefly, it is a new lipid product that combines AIEgen with lipid into a AIEgen-lipid molecule by chemical reaction; it could be used as nanoparticle, which is usually written as AIEsome, or just applied in its molecule form, and both states have their special features, which could be used in different applications. The AIEsome is prepared with the synthesized AIEgen-lipid conjugate, Same as the nanoprecipitation method, the obtained AIEgen-lipid conjugate and other aimed lipids are mixed and dissolved in organic solvent at first, then the solution is treated with a stream of nitrogen gas or dried under vacuum to form a lipid film, then phosphate buffer solution or water is added for rehydration, freeze-thaw cycles, and mini extruder are used for the uniform AIEsome, whose particle diameter is determined by the pore size of extruder membrane. Because of the full exposure to environment oxygen and the looser packing structure, this kind of AIEsome has much higher ROS generation efficiency than the core-shell structure under the same concentration of AIE molecules.
Liu et al. reported a AIEgen-lipid conjugate with compound 9 using this method and a uniform spherical nanoparticle was obtained with an extruder. [52] As presented in Figure 1C, a black ring less than 10 nm could be clearly observed on the surface, which indicated the structure of AIEgen inserted in the bilayer of liposome. AIEgen was inserted in the bilayer of lipid, thus resulting in the restriction of intramolecular motion, which could induce its fluorescence emission according to the mechanism of AIE. According to the ROS generation experiment, the ROS gen-eration rate of AIEsome was much higher than that of conventional PS and the AIEgen nanoparticle with core-shell structure. That was because of the looser packing and higher surface area of the AIEgen-lipid conjugate liposome, which could result in better oxygen diffusion and exposure, and this assumption was further demonstrated by an air supplement experiment.
Other works used the synthesized AIEgen-lipid conjugate directly as the individually dispersed AIEgen could also generate fluorescence when it was entrapped in a tight space. [53] In a recent research, an AIEgen-lipid conjugate TL with a donor-acceptor structure was used for rapid labeling of neutrophils and long-time tracing. [54] The binding ability of TL was investigated by a liposome model, the fluorescence intensity of TL was greatly enhanced after adding liposomes, which simulates the membranes structure of neutrophils, demonstrating the successful combination of TL and liposome. Further investigation was conducted with mitochondria (Mito) ( Figure 1D). [55] After the incubation of Mito and AIEgen-lipid, the obtained Mito-AIEgen-lipid was collected by centrifugation. The AIEgen-lipid was successfully inserted on the surface of the Mito membrane according to the bright circular fluorescence signal by confocal detection and the black circle due to the higher electron density of AIEgen in TEM image. The encapsulated and inserted AIEgen-lipid structure has obvious emission for biological imaging, while all of the molecules emit during the whole process. For some photosensitize AIEgen, the untarget part also suffers the risk of damage when not protected from the laser irradiation. It is a long term for the metabolism of these materials and not convenient in the practical use. More methods have been tried to deal with this issue.

The "imbedded" structure by thin-film hydration
Stimulated by the above two synthesis methods and AIEgenlipid properties, some works were then performed using another physical encapsulation method, thin-film hydration method, which is just like a combination of the above nanoprecipitation and AIEgen-lipid conjugate methods. Some different parts from them is that the AIE molecule is monodispersed and imbedded in the bilayer of lipid by physical effect and does not emit fluorescence (Scheme 1C). The detailed synthesis process is that, first, the AIE molecule and lipid are dissolved with organic regent in a flask with large volume, using this kind of container is very important for the successful product by this method because the evenly monodispersed AIEgen is requested.
Then the organic solvent is removed under rotary evaporation treatment at room temperature, a thin-film is formed after that. Liposomes are obtained by the lipid hydrating with PBS buffer under a certain temperature. Then mini-extruder and polycarbonate membrane are used in the following extrusion for uniform spherical particles with determined size. The obtained nanoparticle has characteristics as follows: (1) the AIE molecule is almost monodispersed and imbedded in the bilayer of the mixed lipid in a nonrestricted molecular rotation state, the AIEgen liposome has weak or no emission in solution; (2) when the lipid layer is disrupted by some surfactants or enzymes, the monodispersed AIE molecules would aggregate and have strong emission. Taking advantage of these properties, some intelligent response systems could be developed.
The first representative work by this method was reported with the AIE dye TPE-BICOOH as shown in Figure 2A. [56] To demonstrate that the AIE dyes were located in the hydrophobic bilayer, cell-sized liposomes were prepared using the above method except for the procedure of extrusion, which was substituted by occasional vortex. A fluorescent ring structure collected by confocal instrument was clearly seen, which proved that the hydrophobic AIE dyes were indeed in the bilayer instead of the hydrophilic interior. A direct way to demonstrate the monodispersed state of AIEgen monomer in the lipid bilayer was the changes of fluorescence intensity of AIEgen-lip nanoparticle solution and bare AIEgen solution dissolved in organic solvent. The results showed that the intensity was almost no change by the imbedded treatment. The reason was that the AIE dye was kept as monomer distributed in the bilayer and stayed in free rotation state, which induced weak emission.
The following investigation of a two-photon excited AIE PS was conducted by our group with bis(pyrene) (BP) as presented in Figure 2B. [32] Specially, the concentrationdependent tendency was detected and the fluorescence intensity of AIEgen-lipid nanoparticle was increased with the enhanced AIEgen concentration, demonstrating that aggregation come up with high density of AIEgen monometer inserted in the limited space. Further, the ROS generation rate was detected by ABDA, and its UV absorbance incubated with BP@liposome presented little inducement upon the irradiated process, while the pure BP nano aggregation showed a faster decrease under the same condition, demonstrating a lower ROS production rate of the imbedded AIEgen-lipid in accordance with the fluorescence intensity. A strong emission was presented once the destructive reagent of triton X-100 was added according to the fluorescence spectra detection results. As the enzymes in cell could disrupt the lipid, the same effect was also detected after an incubation for 30 h with MCF-7 cells. Thus, this system could be used in the PDT, which could reduce the phototoxicity to normal tissues induced by PS reagent, and turn on the cytotoxicity when it took up into cells and reassembled into an AIE aggregation. It has significant meaning in the PDT progress and is a promising method that would liberate the patients from dark protections during the therapy. Since the turn on reactivity of this method is heavily involved with the incorporated AIEgen concentration, the addition amount should be carefully groped and designed. On

The AIEgen-like-lipid by coupling reaction
The AIEgen-like-lipid was always synthesized through chemical reactions, such as McMurry coupling reaction, click reaction, and some treatments of deprotections, and so on. By selectively changing the numbers, lengths, and positions of hydrocarbon chains, a new AIEgen conjugate could be obtained with some property changes on optics, electronics, or self-assemble way. The like-lipid is comprised of long alkyl chains as hydrophobic "tails" and hydrophilic "heads," and these amphiphiles have the ability to reassemble into micelle or liposome structure (Scheme 1D).
In a related work reported by Lu et al., four kinds of TPE amphiphiles ( Figure 3A) were synthesized with different long hydrocarbons as hydrophobic parts and triazole- [12]aneN 3 as hydrophilic moieties. [57] It was designed to be used as gene vector and responsible for the delivery and tracing. Because of the amphipathic property, the AIEgen-like-lipid molecules emitted no fluorescence in binary solvent, after the adding of HCl solution, THF was added as a poor solvent, and evident enhancement of emission intensities were detected. Because of the AIE emission of the nanoassemblies, the critical micelle concentrations was evaluated by recording their fluorescence changes. Further, the four compounds were mixed with calf thymus DNA to study their interactions, and the results indicated that lengthening the hydrophobic chain promoted their interactions with DNA. Moreover, a longer alkyl chain resulted in stronger binding effect and a saturated chain contributed to a stronger binding strength compared with an unsaturated one. Some other studies like "phosphole-lipids" also occupied an important part in AIEgen-like-lipid section ( Figure 3B). [58] Chemical modification would make a great impact on the optoelectronic properties for their special structural and electronic characterizations. Auaternization is a key force for the self-assembly of this like-lipid and the benzyl moiety greatly influences its optical transitions. Various substitutes of the dodecyl chains and chain length and attachment patterns resulted in different self-assembly progresses and electronics. The control process of on/off AIEgen could take place through modulating the donor strength of benzyl moiety, and its 4-position could be utilized as a controller for crystalline and amorphous transitions. Herein, by changing these functional parts of the molecule, targeted phospholelipid could be designed and synthesized for diversity applications.

As probes and sensors
The products of AIEgen-lipids are widely used in the biological field, and an indispensable part is using as probes for special organelles and cells imaging. Most of them are twophoton imaging materials and usually with targeting effect and response activity for their AIE emission properties. One of the representative and interesting work of "chameleon" fluorescent probes was reported by Tang group, which was used for the tracing of lipid droplet (LD) and lysosome interplay. [28] TPA-BTTDO, a red fluorogenic molecule with AIE ability, was synthesized and its emission wavelength presented sensitive changes along with different polarities of the environment. TPA-BTTDO-lipid NPs were incubated with Hela cells to study the long-time changes in cells; meantime, Lysotracker and BODIPY were co-staining, respectively. After 2 h, emission could be detected in lysosome by the fluorescence colocalization with Lysotracker; after 4 h, enhanced emission was collected in the overlapped region of BODIPY, while reduced intensity was observed in lysosome, which indicated that the NPs were transferred from lysosome to LDs. The AIEgen-lipid NPs exhibited red emission in lysosomes, while cyan fluorescence in LDs, which was owing to the different polarities. Molecules with donor-accepter structures could present similar phenomena. The long tracing process and consumption of LDs were clearly visualized by TPA-BTTDO NPs. Another intriguing work reported recently was about a Mito probe, which was membrane potential (MMP)-independent and also could be applied for long-term visualization. Other AIEgen-lipid conjugates like ECPI-12 and IVPI-12 were two AIEgen molecules with long lipophilic aliphatic chains, as described in the AIEgen-like-lipid part; they target Mito by means of electrostatic interaction and could be detected by two-photon laser in deep tissues with low signal to noise ratio (Figure 4A). [59] Because of the stable staining effect, the two molecules could realize real-time mitophagy tracking in situ.
Probes were also used to label some special cells. For example, bone marrow-derived mesenchymal stem cells (BMSCs) were targeted imaging by core-shell AIEgen-lipid NP with Tat modified on the surface, superior tracking ability, and high cell labeling efficiency were observed in a serious of detections [25] . Neutrophils could be rapidly labelled by AIEgen-lipid conjugate for long-term monitoring and inflammation imaging in mice. [54] The labeling method was also used to track the process of autonomous chasing and clearance of pathogens. Normally neutrophils moved randomly in the test solution and suddenly shifted to the added E. coli when the E. coli was added and its morphology was observed presenting distinct changes during the process. The obtained results indicated the labeling method is effective and appropriate to track the behavior of neutrophil without damage of bioactivity and chemotaxis ability.
An AIEgen-lipid nanoparticle of molecule P2 based on tetraphenylethene was prepared by reprecipitation method for intracellular Fe 3+ detection as a sensor ( Figure 4B). [60] The selectivity of this NP toward various metal ions was detected, once Fe 3+ was added, the fluorescence intensity was sharply reduced, while it showed no obvious change upon the addition of other metal ions. In a certain concentration range, the AIE emission intensity presented a good linear dependence versus Fe 3+ concentration, with an R 2 value of 0.995. Moreover, its sensing effect of intracellular Fe 3+ was also Other biological detection such as acetylcholinesterase was laso reported. A method utilizing the interaction effect of AIEgen and lipid was developed to detect the amount and activity of acetylcholinesterase (AChE) ( Figure 4C). [61] The AIEgen based on TPE possessed negatively charged sulfonate units, and myristoylcholine possessed positively charged ammonium unit, they would form a heteroaggregate by the electrostatic interactions, and then fluorescence emitted from AIEgen molecules. As myristoylcholine could be hydrolyzed by AChE, the aggregation would be disassembled and the fluorescence would be reduced in the presence of AChE. Lineweaver-Burk plot for the hydrolysis of myristoylcholine was detected, the obtained R 2 was 0.999 in the presence of AIEgen compound. Therefore, a convenient fluorometric method for AChE activity was established based on the interaction of AIEgen compound and lipid.

Applications of in vivo imaging
The AIEgen-lip structures are widely used in bioimaging, especially those two-photon excited materials, which can be used to observe the blood vasculature. Two-photon imaging technique presents a great deal of advantages than one-photon in many aspects, such as high signal to noise ratio, enhanced penetration depth, and good spatial selectivity. [62,63] A series of two-photon excited AIEgen molecules has been designed and proved to have large TPACs by molecular design strategy of extensive π-conjugated molecules, [64,65] However, this method contains multiple synthesized routes and the concentration quenching property restricts its application. Tang reported a silole prepared by a simple synthesized route, it emitted red fluorescence by the interaction between silole (donor) and thiophene (acceptor). The AIEgen molecule was combined with lipid by nanoprecipitation and the nanoparticle of (MesB)2DTTPS was used in the blood vessel imaging. [36] After the treatment of intravenous injection of the obtained dots, the blood vasculature of muscle was collected by CLSM, a continuous red emission in vessels could be clearly detected. Moreover, the blood vascular network at ear could also be visualized with a penetration depth over 100 μm ( Figure 5A). Three-dimensional reconstructed image and long-time observation of the blood vasculature could be easily presented by this two-photon material with excellent biocompatibility.
Another important application of the AIEgen-lipid material is for the imaging and diagnosis of tumor tissue, a problem that has been plaguing people for a long time. AIEgen-lipid nanoparticles with different target and imaging molecules have been designed and displayed more opportunities for the clinical application in tumor imaging. The combination of lipid can enhance the biocompatibility, internal circulation time, and the target efficiency. A chelation of gadolinium (III) was realized by DTPA, which conjugated the amine groups of DSPE-PEG-NH 2 first. [49] With the targeting effect of Tat, dual-model imaging of fluorescence and MRI contributes to synergistic benefits of high sensitivity and penetration depth. Moreover, the two-photon property of some AIEgens also offers a distinct spacial resolution, low background interference, and good penetration depth, which are advantageous for deep tissue imaging. Some AIEgen-lipid nanoparticles presented in situ response character due to the degradation of lipid. As shown in Figure 5B, [5] , fluorescence signal was collected immediately after the injection of TPE-LPs, indicating that nanoparticles were rapidly distributed the whole body in short time. The quantitative data collected in the tumor region showed a maximum emission intensity at the time point of 2 h after the tail intravenous injection. Then fluorescence faded over time because of the scavenging action of body, while the signal in tumor region still existed for a long time. As a control, the tumor site treated with TPE-COOH presented weak or no fluorescence signal.

Drug delivery system
The AIEgen-lipid NP provides a platform for the delivery of other therapeutic materials, such as chemical drug and gene, and combined therapy thus can be achieved with an enhanced efficiency. Just like the loading ways of AIEgens, the hydrophilic ones can be loaded in the core of liposome and the hydrophobic ones can be inserted in the bilayer, different drugs can be effectively carried to the targeted part avoiding the clearance of immune system with the help of some special liposome. The hydrophobic chemotherapy agent paclitaxel (PTX) was inserted in the bilayer of liposomes, [66] while water soluble doxorubicin hydrochloride was encapsulated in the middle core, [67] they were transferred by the liposome with AIEgens to the targeted region, along with the fluorescence labelled by luminophor.
Sometimes, the liposome carrying AIEgens could change the state of phosphors from monodiperse to aggregation. By controlling the load amount and combination mode, the AIEgen-lipid NP with AIEgen monometer imbedded in the bilayer obtained by thin-film method emits no or weak fluorescence before and during the delivery process. When they are taken up by cells, diverse enzymes in lysosomes result in the degradation of lipids and the AIEgen monometers are released from the bilayer, further aggregated owing to the hydrophobic property and emit strong fluorescence. [32], [56] For some AIE PSs, whose ROS generation amount increased along with the fluorescence intensity, this strategy could avoid the ROS damage to normal tissues immediately after the injection. When the NP arrives the targeting part by special recognition, lipid will be degraded, and AIEgen aggregates with emission and ROS generation, which can kill the cancer cells and destroy the tumor structure. For the current clinical application of PDT, patients are demanded to stay in home after the injection of PSs, the period will last from 1 week to more than 1 month, patients tolerate the longtime darkness as well as some side-effects around normal tissues. The promising way can liberate them from keeping in the dark during and after the PDT because of the targeting effect and the environmental response aggregation of AIE PSs, which own strong imaging fluorescence and damage effect of ROS.

Photodynamic therapy (PDT)
Always, the tumor-bearing model was obtained by subcutaneously injection of cancer cells. When the tumor reached a certain size, mice were treated with different materials by tail intravenous injection. A period after the injection, the tumor site was treated with laser irradiation and it was white light of 300 mW/cm 2 in the TPETCAQ NP system. [34] 4T1 cancer cells could be used for detecting the therapy effect due to their bioluminescent signals. After 14 days, weak signal indicated the efficient treatment, while the control group emitted enhanced signals during the monitoring process. The tumor size data collected every the other day also presented a same tendency as shown in Figure 5C. As for the laser group, tumor sizes of the mice only treated with irradiation presented no obvious decrement comparing to the control group. Moreover, hematoxylin and eosin (H&E) results showed clear nucleus dissociation and necrosis in the tumor tissue of TPETCAQ NP + light group, while not easy to be found in other groups. The investigation verified the superior PDT effect of TPETCAQ NP.
Other works involved the combination therapy and presented high synergistic effect of tumor ablation. As reported, although inadequate ROS generation of PDT could not thoroughly kill the cancer cell, it would promote the permeability of cell membrane and facilitate the cellular uptake progress of anticancer agents. [68,69] The AIEgen-PS, TPCI was imbedded in the bilayer of lipid with a chemotherapy reagent paclitaxel (PTX) to enable a TPCI/PTX@Lipo NP, aiming to treat mouse with late-stage/large PC3 prostate tumor. [66] The nanoparticle was demonstrated to induce cell apoptosis by mitochondrial pathway and tumor with large size value of about 200 mm 3 was almost completely ablated and presented an exceptional anticancer efficacy ( Figure 5D). Compared with the sole therapy method, the antitumor efficiency of combined effect was presented as 30 times higher. The effective elimination of tumors distinctly testify the enhancement anticancer effect of chemotherapy on PDT. Moreover, no apparent weight changes were observed and the mice life were extended after the treatment of TPCI/PTX@Lipo. H&E staining results displayed that obvious coagulative necrosis occurred in the tumor-treated NP and laser. At the last stage, the released TPCI from liposome binded with chromatin in nuclei and emitted fluorescence which could report the dead of cells in real time.

CONCLUSION
Comparing with traditional ACQ materials, AIEgens display promising potential in biological application. With the development of AIEgen these years, more and more multifunctional molecules have been designed and synthesized, not only for the organelle and cell labelling in vitro, but also the diagnosis and multimodel imaging guided therapy in vivo. AIEgens with characters of near-infrared emissive or multiphoton absorption are highly desirable for their deep penetration and low signal to noise ratio in vasculature and tissue, which can widen the range of clinical applications. In addition, some molecules with capacity of photoacoustic or photothermal imaging broaden the guidance mode for enhanced localization accuracy. Other AIEgens possessing excellent photodynamic and photothermal therapeutic abilities can be applied as theranostic platforms in bacteria or cancer ablation by innovative strategy. Furthermore, most recently, AIEgens used in translational applications also promote the development of their commercial prospects. All these excellent properties and applications of AIEgens accelerate the process of superior modifications of higher biocompatibility, better stability, enhanced targeting efficiency, and so on.
To further meet the requirements of practical use in physiological environment, the methods of AIEgen modified with lipid are developed and have been well studied. Because of the unique structure of lipid, its interaction with AIEgen molecule implies a number of possibilities: encapsulated in the core, inserted/imbedded in the bilayer, conjugated by chemical bond, or molecule conjugated with long alkyl chain, which is called AIEgen-like-lipid in this article. All of these formations have their own unique properties and have been broadly applied in cell imaging, vascular imaging, long-term tracking, disease diagnosis, and cancer therapy, some of them can also be used as sensors, which would quantitatively analyze the concentration of the material to be measured. The development of AIEgen molecules with superior features is still in progress and the properties of AIEgen-lipid compounds are full of infinite possibilities which could be used more efficiently in biological imaging, diagnosis, and therapy. Taking advantage of these unique properties will promote the development of clinical agent based on AIEgen and significantly enhance our life quality during the process of disease diagnosis and therapy.