Bispyrene-Based Self-Assembled Nanomaterials: In Vivo Self

Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Bispyrene-Based Self-Assembled Nanomaterials: In Vivo SelfAssembly, Transformation, and Biomedical Effects Ping-Ping He,†,‡ Xiang-Dan Li,*,† Lei Wang,*,‡ and Hao Wang*,‡,§ †

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Key Laboratory of Chemistry and Materials Science of the State Ethnic Affairs Commission and Ministry of Education, South-Central University for Nationalities, 182 Minzu Road, Hongshan District, Wuhan, Hubei 430074, China ‡ CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), No. 11 Beiyitiao, Zhongguancun, Haidian District, Beijing 100190, China § Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049, China CONSPECTUS: Self-assembled nanomaterials show potential high efficiency as theranostic agents for high-performance imaging and therapy. However, superstructures and properties of preassembled nanomaterials are somewhat compromised under complicated physiological conditions. Given the advantages of the dynamic nature and adaptive behavior of self-assembly systems, we propose an “in vivo self-assembly” strategy for in situ construction of nanomaterials in living objects. For the proof-of-concept study of in vivo self-assembly, we developed a bispyrene (BP) molecule as a multifunctional building block. BP molecules show nonfluorescence in the monomeric state. Quantum-chemical calculations indicate that BP forms twisted intramolecular charge transfer states, which are separated into two orthogonal units, preventing the fluorescence emission. Interestingly, the typical excimeric emission of BP is observed with the formation of J-type aggregates, as confirmed by single-crystal X-ray diffraction. Packing of the BP molecules generates parallel pyrene units that interact with adjacent ones in a slipped face-to-face fashion through intermolecular π−π interactions. BP and/or its amphiphilic derivatives are capable of selfaggregating into nanoparticles (NPs) in aqueous solution because of the hydrophobic and π−π interactions of BP. Upon specific biological stimuli, BP NPs can be transformed into variable self-assembled superstructures. Importantly, the selfassembled BP NPs exhibit turn-on fluorescence signals that can be used to monitor the self-assembly/disassembly process in vitro and in vivo. On the basis of the photophysical properties of BP and its aggregates, we synthesized a series of designed BP derivatives as building blocks for in situ construction of functional nanomaterials for bioimaging and/or therapeutics. We observed several new biomedical effects, e.g., (i) the assembly/aggregation-induced retention (AIR) effect, which shows improved accumulation and retention of bioactive nanomaterials in the regions of interests; (ii) the transformation-induced surface adhesion (TISA) effect, which means the BP NPs transform into nanofibers (NFs) on cell surfaces upon binding with specific receptors, which leads to less uptake of BP NPs by cells via traditional endocytosis pathway; and (iii) transformation of the BP NPs into NFs in the tumor microenvironment, showing high accumulation and long-term retention, revealing the transformation-enhanced accumulation and retention (TEAR) effect. In this Account, we summarize the fluorescence property and emission mechanism of BP building blocks upon aggregation in the biological environment. Moreover, BP-derived compounds used for in vivo self-assembly and transformation are introduced involving modulation strategies. Subsequently, unexpected biomedical effects and applications for theranostics of BP based nanomaterials are discussed. We finally conclude with an outlook toward future developments of BP-based self-assembled nanomaterials.

1. INTRODUCTION

physiological conditions, which is believed to be accompanied by structural changes including dissociation, aggregation, transformation, etc. due to the intrinsic dynamic nature of self-assembly systems.5−8 We propose a new strategy of “in vivo self-assembly” for the in situ construction of self-assembled nanomaterials in vivo.9,10

Self-assembled nanomaterials with diversified structures and functionalities exhibit promising applications in bioimaging and therapeutics.1−3 Compared with small molecules, selfassembled bioactive nanostructures show high stability in vivo, prolonged half-life, high payloads, and variable surface chemistry to achieve desirable performance.4 However, the superstructures and well-designed properties of preassembled nanomaterials can be compromised under complicated © XXXX American Chemical Society

Received: August 7, 2018

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DOI: 10.1021/acs.accounts.8b00398 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research

Scheme 1. Schematic Illustration of in Situ Self-Assembly and Transformation Based on Bispyrenes for Cancer Diagnosis and Therapy

Figure 1. (a) Molecular structure of BP. (b) Contour plots of the LUMO and HOMO in the first excited state of BP in the monomer state. (c) Measurements of the BP molecular packing by single-crystal X-ray diffraction. (d) TEM image of the self-assembled BP nanoparticles. (e) Characterization of fluorescence spectra of BP in the mixed DMSO/H2O solutions. The inset shows an emissive image of BP nanoparticles under 365 nm excitation. Reproduced from ref 11. Copyright 2013 American Chemical Society. (f) Two-photon fluorescence of BP across 1600 μm mock tissue. (g) Schematic of the self-assembly of BP-conjugated cyanine dye (BP−Cy) into nanovesicles. (h) TEM image of the morphology of the BP−Cy nanovesicles. Reproduced with permission from ref 15. Copyright 2015 Royal Society of Chemistry.

B


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Accounts of Chemical Research Table 1. Strategy for Self-Assembly of BP in Vitro and in Vivo component polymer carrier

BP

function encapsulation of BP and delivery into living objects

in situ self-assembly

regulator

assembly process

pH pH

carrier dissolution and BP assembly carrier swelling and BP disassembly

temperature

carrier dissolution and BP assembly

enzyme

cleavage of BP from the carrier and assembly

purpose imaging of the lysosome monitoring of the pH of the endocytic process monitoring and modulation of protein oligomerization detection of the activity of the enzyme

Figure 2. (a, b) Schematic illustrations of (a) BP-dispersed micelles and (b) release of BP from micelles and aggregation of BP in lysosomes. (c) Fluorescence spectra and (inset) images of BP-dispersed PbAE micelles at different pH under 365 nm irradiation. (d) Spinning-disk confocal microscopy (SDCM) images of BP-dispersed PbAE micelles incubated with HeLa cells at different time intervals. Reproduced with permission from ref 20. Copyright 2015 IOP Publishing Ltd. (e, f) Schematic illustrations of the (e) molecular structure and (f) in situ self-assembly mechanism of DP−BP in living cells for autophagy detection. (g) TEM images of the structural transformation of DP−BP. (h) Zebrafish embryos 2 days after fertilization were treated with PBS or Rapa for 24 h and then stained with DP−BP for 4 h before imaging by confocal laser scanning microscopy (CLSM). Reproduced from ref 21. Copyright 2017 American Chemical Society.

“observed” by fluorescence imaging techniques. The in situconstructed self-assembled nanomaterials can be achieved by endogenous or exogenous stimuli, which show some new biomedical effects. The in situ-constructed self-assembled nanomaterials are finally introduced for biomedical applications, such as drug delivery, and bioimaging (Scheme 1).

The self-assembly occurs under specific physiological conditions, and the resultant superstructures, features, and functionalities can be modulated according to predesigned building blocks. In order to monitor the in vivo self-assembly processes, we developed as building blocks bispyrenes, which are hydrophobic molecules that self-assemble into nanoparticles (NPs) with bright emission.11 The bispyrenes and their derivatives self-assemble in aqueous solution or biological environments and construct various structures in situ via hydrophobic and π−π interactions of pyrenes.12 Importantly, the bispyrenes self-assemble into J-type aggregates, which show the typical excimer emission of pyrenes. Therefore, the in vivo self-assembly process and the resulting self-assemblies can be

2. BISPYRENES AND THEIR ADVANTAGES Pyrene is a prototypical molecule exhibiting a high fluorescence quantum yield in dilute solution.13 Pyrene shows characteristic excimer emission in concentrated solutions or the solid state.14 Together with the hydrophobicity and large π-conjugated structures, we chose pyrenes C


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Figure 3. (a) Schematic representation of thermal control over BP self-assembly on cell surfaces for monitoring and manipulating HER2 receptor clustering. (b) Molecular structure of PNIPAAm containing BP. (c) Turbidity experiments. (d) Fluorescence change of PNIPAAm containing BP measured in PBS at different temperatures (λex = 340 nm). (e) CLSM images for monitoring the morphology change of BP on SK-BR-3 cells at 40 and 35 °C. LCST denotes the lower critical solution temperature. Reproduced from ref 24. Copyright 2016 American Chemical Society.

materials to the best of our knowledge.16 Therefore, the BP NPs showed deep tissue penetration for two-photon florescence imaging up to a depth of 1600 μm in mock tissue (Figure 1f). In order to further improve the penetration depth of BP for in vivo imaging of self-assembly, we utilized a Förster resonance energy transfer (FRET) strategy to increase the emission wavelength.18 BP as the FRET donor and Nile red (NR) as the acceptor coassembled into BP/NR NPs, which had a large cross sectional area of 2.4 × 105 GM and a red emission (630 nm) (Figure 1g). Due to the strong TPA and long wavelength, the fluorescence signals of BP/NR NPs were detectable at a depth of 2200 μm in mock tissue. These results revealed that BP with excellent fluorescence properties upon self-assembly showed the potential for in vivo imaging with high resolution for observation of self-assembly.

as mother molecules for the construction of self-assembled nanomaterials. It was well-known that molecular stacking is directly correlated with the properties and functionalities of organic π-conjugated molecules in the solid state. Therefore, we designed a series of bispyrene derivatives with benzene-1,3dicarbonyl, pyridine-2,6-dicarbonyl, oxaloyl, and benzene-1,4dicarbonyl as linkers. We fortunately may control the molecular packing and fluorescence properties of bispyrene derivatives.11 The bispyrenes connected with benzene-1,3dicarbonyl (BP) emerged as the best candidates with superior fluorescence and self-assembly properties (Figure 1a). Moreover, the BP modules were functionalized with different active groups, such as carboxylic acid, amide, and bromide, which could be utilized for further reactions (Scheme 1a). 2.1. Fluorescence Features

2.2. Self-Assembly Feature

The quantum-chemical calculations and experimental results revealed the low fluorescence quantum yield of BP in solution (Φ = 1.1%) due to the twisted intramolecular charge transfer (TICT), which was confirmed by quantum-chemical calculations using Gaussian 09 (DFT/TDDFT at the B3LYP/631G(d) level) in DMSO (Figure 1b).16 BP self-assembled into stable NPs (Figure 1c,d), and the fluorescence intensities increased up to 30-fold compared with those in solution (Figure 1e), exhibiting a typical aggregation-induced emission (AIE) effect.17 BP NPs showed a large two-photon absorption (TPA) cross section (δ) of 2.8 × 105 GM, which was the highest among all of the reported two-photon contrast agent

BP with hydrophobic and π−π interactions of pyrene formed highly ordered NPs with J-type character in water. The X-ray diffraction data revealed that the two pyrene units in a BP molecule are coplanar and twisted with respect to the phenyl ring (Figure 1c). BP molecules pack through intermolecular π−π interactions, generating parallel pyrene units that interact with adjacent ones in a slipped face-to-face fashion. The shortest carbon−carbon distance between the adjacent molecules was 3.361 Å. The smallest angle of centroids of two neighboring pyrene planes was 44.58°, indicating the Jtype assembly.19 BP could induce self-assembly in water as a D


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Figure 4. (a) Molecular structure of P−BP and cartoons of P−BP NPs. (b) Schematic illustration of P−BP/NR self-assembly into NPs at neutral pH and swelling of the NPs with enhanced BP fluorescence at low pH. (c) Ratio of P−BP/NR fluorescence intensity at 418 nm to that at 635 nm (R) at various pH values. (d) CLSM images of U87 cells incubated with P−BP/NR at different times. Reproduced with permission from ref 25. Copyright 2015 Royal Society of Chemistry.

(amidoamine) (PAMAM) dendrimers through an autophagyspecific enzyme, ATG4B-responsive peptide, to form DP− BP.21 The optimized DP−BP showed weak fluorescence and could be cleaved by ATG4B to release BP residues,22 resulting in the self-assembly of BP and a 30-fold enhancement of the fluorescence signal (Figure 2e,f). This intracellular selfassembly of BP showed turn-on fluorescence and could be used for the measurement of ATG4B activity. BP residues aggregated into NPs and transformed into fibrous structures in 24 h (Figure 2g). This process based on DP−BP could occur in Rapa-treated cells and zebrafish embryos (Figure 2h) for ATG4B detection. This strategy was further utilized to evaluate autophagy and optimize the autophagy-mediated chemotherapy for tumors.23 In another case, we modulated the self-assembly of BP using a thermally responsive polymer.24 The temperature-sensitive polymer poly(N-isopropylacrylamide) (PNIPAAm), acted as the thermoresponsive backbone, which was covalently linked with HER2 targeting peptide (CGKGGMSRTMSG)-conjugated BP (Figure 3a.b). The assembly behavior of BP could be modulated by the thermally responsive polymer. The polymer exhibited a sharp aqueous phase transition upon heating above a critical temperature (Figure 3c). At 40 °C, the PNIPAAm polymers collapsed and acted as a “shield” to block the aggregation of BP. Upon cooling to 35 °C, the polymers were in their extended state, exposing BP in the aqueous solution, which subsequently aggregated with 6-fold-enhanced fluorescence (Figure 3d). When the PNIPAAm polymers containing BP were incubated with living cells and the temperature was decreased from 40 to 35 °C, Her2overexpressing cells showed overlapped green and red (Figure 3e), indicating that the BP aggregated on the cell surfaces.

template. For example, cyanine (Cy) was conjugated with BP to form BP−Cy (Figure 1g), enabling Cy to form highly ordered nanovesicles (Figure 1h).15

3. IN SITU ASSEMBLY IN VITRO AND IN VIVO 3.1. Polymer-Assisted Self-Assembly

BP is a hydrophobic molecule with large π conjugation that self-assembles into NPs in water. In order to realize in situ selfassembly in vitro and in vivo, the BP monomers were dispersed into polymeric carriers, which were delivered to the targeted region and released, resulting in self-assembly of BP in situ (Table 1). 3.1.1. Intermolecular Dispersion by Polymers. For the first example of in situ self-assembly, BP was dispersed in the hydrophobic domains of pH-responsive polymeric micelles in the form of monomers by hydrophobic interactions (Figure 2a).20 Typically, the amphiphilic poly(β-amino ester)s (PbAEs) formed micelles at pH 7.4 for encapsulation of BP. The concentration of BP was critical to maintain the monomeric state of BP in PbAE micelles for weak fluorescence. When the pH was lowered to 5.5, the fluorescence at 530 nm was dramatically increased, probably because of the dissociation of micelles and release of BP followed by self-assembly of BP (Figure 2b,c). For the purpose of self-assembly in living cells, BP-dispersed PbAE micelles were incubated with, uptaken by, and then entered the lysosomes of HeLa cells. The in situ self-assembly of BP to form NPs with turn-on fluorescence was achieved in the acidic lysosomes (Figure 2d). 3.1.2. Conjugation Dispersion by Polymers. BP could be also conjugated with polymeric carriers for dispersion. For example, BP was conjugated with fourth-generation polyE


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Figure 5. (a) Chemical structures and schematic illustration of glutathione (GSH)-based biorthogonal reaction to turn on the binary fluorescence signals in organisms. (b, c) Fluorescence spectra of nanoemitters (10 μM) with increasing amounts of GSH at emission wavelengths of 820 and 520 nm, respectively. (d) Ex vivo fluorescence images of tissues and tumors 12 h after administration of nanoemitters and PBS. Reproduced from ref 26. Copyright 2016 American Chemical Society.

Table 2. Strategy for Transformation of BP NPs into NFs in Vitro and in Vivo NP module BP

NF module pentapeptide (KLVFF)

regulating module poly(ethylene glycol) (PEG) histidine (His)

endogenous stimulus

purpose

hydrophilicity increase

NF formation (faster with longer of PEG chains)

octapeptide (RGD-YIGSR)

pH-induced hydrophilicity increase ligand−receptor interactions

tripeptide (RGD)

ligand−receptor interactions

construct a fibrous network in the tumor to host theranostics construct an extracellular matrix to inhibit tumor metastasis image and induce anoikis of cancer cells

3.2. Polymer-Assisted Disassembly

wavelength upon pH changes. When the pH of the P−BP/NR solution changed from 7.4 to 5.0, the ratio of P−BP/NR intensity at 418 nm to that at 635 nm (R) showed a standard pH calibration curve (Figure 4c) for detection of the pH in endocytic organelles (human primary glioblastoma cells), which became acidified gradually (Figure 4d).

Because of the hydrophobicity and π−π interactions of the pyrenes in BP, it was easy to obtain BP aggregates in polymeric carriers. Disassembly of BP nanoaggregates could be achieved using smart polymer carriers (Table 1). For example, we demonstrated the disassembly of BP modulated by a pHsensitive polymer nanocarrier.25 The BP was conjugated with hydrophilic poly(amino ester)s (P−BP). The P−BP selfassembled into NPs through hydrophobic interactions at neutral pH, where the BP aggregated as the hydrophobic core with green fluorescence and the polymer formed the hydrophilic shell. As the pH decreased from 7.4 to 5.0, protonation of the polymer chains induced swelling of the NPs from 41.7 nm (pH 7.4) to 138.2 nm (pH 5.0) with disassembly of BP and a change in the fluorescence from green to blue (Figure 4a,b). The pH-modulated change in fluorescence was enhanced by encapsulation of NR into the P−BP NPs to establish the P− BP/NR FRET system (Figure 4a), which showed fluorescence with a wide range of shift and ultrasensitive changes in dual

4. IN SITU TRANSFORMATION IN VITRO AND IN VIVO 4.1. Transformation from Small to Large NPs

We designed responsive molecules with two emissive units, BP and Cy, covalently linked through a disulfide bond (BP−S−S− Cy),26,27 which self-assembled into NPs along with aggregation-caused quenching (ACQ) of the fluorescence. The NPs could be activated by glutathione (GSH), resulting in the formation of GSH-bonded Cy molecules and BP residues followed by the formation of BP aggregates (Figure 5a). The diameter changed from 32.8 ± 6.3 to 67.8 ± 12.3 nm as determined by dynamic light scattering. The fluorescence intensity in the near-infrared region (820 nm) was 30-fold F


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Figure 6. (a) Schematic illustration of the morphology transformation of BP−KP from monomers to NPs to NFs. (b) Molecular structure of BP− KP. (c) TEM images of the structural transformation with time for BP−KP2 and BP−KP3. Reproduced with permission from ref 31. Copyright 2016 Royal Society of Chemistry.

(integrin) interactions (Figure 7a,b),37 which was mainly driven by the coordination interactions between RGD and metal ions (Ca2+, Mg2+).38,39 In solution, the metal ion can induce the structural transformation of BP−KR nanostructures from NPs to NFs (Figure 7c,d). When BP−KR NPs were incubated with U87 cancer cells overexpressing integrin αvβ3, they could bind the surfaces of cells and be transformed into BP−KR NFs in situ (Figure 7e). These transformable BP−KR NPs showed different biological effects from other NPs, which were uptaken by cells through endocytosis. They were transformed into NFs and adhered on the cell surfaces. This process was named the transformation-induced surface adhesion (TISA) effect. 4.2.3. Transformation for Inhibition of Tumor Metastasis. On the basis of the transformation of BP−peptide conjugates, we constructed nanofibrous networks in tumor in vivo for inhibition of tumor invasion and metastasis. We designed a Lamnin-mimic peptide, BP−KLVFFK−GGDGR− YIGSR (BP−KRY), to repair natural extracellular matrix (ECM) through the transformation from NPs to NFs in situ in tumor (Figure 8a).12 The BP−KRY NPs were peptide based nanostructures that included a BP unit, a KLVFF peptide motif, and a Y-type RGD-YIGSR motif as target motifs.40 BP−KRY NPs were transformed into an NF network as an artificial ECM (AECM) via ligand−receptor binding.41 TEM results showed that the BP−KRY NPs could be transformed into short fibers in 2 days and totally converted to long fibers on the sixth day in CaCl2 solution (Figure 8b). MDA-MB-231 cells overexpressing integrin showed irregular protrusion morphologies of the surfaces. However, the cells incubated with BP−KRY NPs clearly showed fibrous structures forming nets (Figure 8c). For the in vivo study, BP−KRY NPs could target and accumulate in the tumor site through the morphology transformation on the cell surface as an AECM. The adaptive transformation in tumor showed long-term retention for 72 h, which is much longer than the control groups (nontransformable NPs) (Figure 8d). The AECM acted as a long-term barrier, resulting in highly efficient inhibition of tumor metastasis. What is more,

increased with the formation of GSH-bonded Cy and 36-fold increased for BP aggregates at 520 nm (Figure 5b,c). The BP− S−S−Cy NPs were biocompatible and showed negligible cytotoxicity at concentrations up to 100 μM on breast cancer cells (MCF-7 cells). Therefore, the BP−S−S−Cy NPs were successfully used to detect overexpressed GSH in tumors in vivo (Figure 5d). 4.2. Transformation from NPs to NFs

BP could induce self-assembly into NPs and transform into NFs through hydrogen bonding and hydrophobic interactions of a classic pentapeptide, i.e., KLVFF,28,29 which can be modulated by hydrophobic−hydrophilic interactions, ligand− receptor interactions, and so on. The transformation exhibited some interesting biomedical effects for different applications (Table 2). 4.2.1. Hydrophobic−Hydrophilic Interaction-Induced Transformation. Natural self-assembly processes, e.g., protein folding, membrane fusion, etc.,30 represent structural transformations, encouraging us to study the transformation capabilities of self-assembled NPs. To understand the structural transformation mechanism, we designed and prepared a series of building blocks, BP−KLVFFG−PEG (BP−KP), and investigated their self-assembly processes systematically (Figure 6a,b).31,32 The KLVFF peptide was proven to be highly fibrillated by hydrogen bonds.33 Poly(ethylene glycol) (PEG) with different molecular weights (MW = 368, 1000, 2000) provided different hydrophilicities. When the BP−KP solution of DMSO was injected into water rapidly, the BP−KP NPs with diameters ranging from 35.0 ± 3.2 to 105.3 ± 23.3 nm were obtained. Hydrogen-bonding interactions further drove an in situ morphology transformation into NFs (Figure 6c).34 The longer the hydrophilic chain was, the faster was the transformation. The experimental results indicated that the hydrophilic−lipophilic balance (HLB) affected the self-assembly process35 and the resultant morphology of BP−KP.36 4.2.2. Ligand−Receptor Interactions Induced Transformation. We designed and studied the transformation of BP−KLVFF−RGD (BP−KR) by ligand (RGD)−receptor G


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Figure 7. (a) Schematic illustration of the transformation of BP−KR from NPs to NFs by ligand−receptor interactions. (b) Molecular structure of BP−KR. (c) Structural transformation of BP−KR in the absence/presence of the Ca2+ from NPs to NFs at 4 days by TEM. (d) Fluorescence spectra of BP−KR in the absence/presence of metal ions in water. (e) SEM images of the BP−KR morphology transformation from NPs to NFs on U87 cell surfaces. Reproduced with permission from ref 38. Copyright 2016 Royal Society of Chemistry.

named the transformation-enhanced accumulation and retention (TEAR) effect. Small model molecules, including NR (Figure 9g), indocyanine green (ICG) and doxorubicin (DOX), could bind to NFs through hydrophobic interactions and eventually accumulate in tumor. BP−KPH NFs and the ICG group showed almost constant temperature up to 48 h, indicating that ICG can be firmly locked in the nestlike hosts (Figure 9h). Therefore, only the tumors of the BP−KPH NFs and ICG group under irradiation were cleared completely in 2 days, which showed much better therapeutic efficacy of the free ICG group (Figure 9i). The in situ morphology transformation from NPs to NFs at the tumor site could be accomplished through different stimuli, including pH, ligand, etc.44 The transformation could be highly efficient in tumor because of the EPR effect, leading to a high concentration in the specific tumor microenvironment. Furthermore, with sophisticated design, the NFs could form in different specific regions, such as vesicular and extracellular matrix, with different biological effects.

hematology and histopathology assays of a histological section showed no significant differences in pathological signs between the BP−KRY- and PBS-treated groups. 4.2.4. Transformation for Two-Step Drug Delivery. On the basis of the transformation from NPs to NFs in tumor in vivo, a new drug delivery system, i.e., a host−guest drug delivery system, was proposed in order to overcome the barrier of bioavailability (Figure 9a).10 The designed BP−KLVFF− PEG−His6 (BP−KPH) (Figure 9b)42 could self-assemble into NPs at pH 7.4 and be transformed into BP−KPH NFs after incubation at 37 °C for 2 h in pH 6.5 buffer solution (Figure 9c,d). The NFs can capture small molecules, as confirmed by the FRET mechanism with BP as the donor and NR as the receptor (Figure 9e). BP−KPH NPs were injected into mice by iv injection and transformed into BP−KPH NFs by the tumor microenvironment acidity (Figure 9f), which was further confirmed by energy-dispersive X-ray spectroscopy (EDS) through detection of iodine-labeled phenylalanine in BP−KPH. The fibrous homing nest showed a long retention time of 96 h.43 The long-term retention was much longer than the enhanced permeability and retention (EPR) effect of usual NPs, there was also a large amount of accumulation; this was H


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Figure 8. (a) Schematic illustration of the transformation of BP−KRY NPs to BP−KRY NFs to construct an artificial extracellular matrix to inhibit tumor metastasis. (b) TEM images of the morphology transformation of BP−KRY NPs in the presence of Ca2+ in aqueous solution. (c) SEM images of the structural transformation the BP−KRY NPs on cell surfaces. (d) Ex vivo fluorescence images of the biodistribution of the BP−KRY NPs in tumor. Reproduced from ref 12. Copyright 2017 American Chemical Society.

5. CONCLUSIONS Self-assembled nanomaterials have been widely used for nanomedicine. However, it is possible to transform the morphologies and their properties when interfacing the biointerfaces in vivo because of the dynamic nature of selfassembled nanomaterials. In order to give insight into what occurs with the nanomaterials in vivo, we proposed an in vivo self-assembly strategy to observe the self-assembly process in vivo and construct self-assembled nanomaterials in situ. We designed and optimized a BP molecular unit that showed enhanced fluorescence upon assembly and tended to induce selfassembly because of the strong hydrophobic and π−π interactions of pyrenes. BP units could be reversibly controlled from the monomeric state to the aggregated state. Furthermore, the BP NPs could be transformed into NFs upon coupling with KLVFF, i.e., a fibrillation pentapeptide. The in situ self-assembly was generally achieved under natural stimuli, such as enzymes, pH, and redox, in specific physiological and pathological regions for the construction of functional theranostic entities with improved blood circulation, targeting, accumulation, retention, and/or release profiles. The in situ self-assembly could be intelligent to monitor biological processes, activity of biomolecules, and disease diagnosis in vivo. The in situ transformation to construct nanomaterials showed new biomedical effects, such as the transformationinduced surface adhesion (TISA) effect, which was different from uptake by cells. The transformation-enhanced accumu-

lation and retention (TEAR) effect showed longer retention than the EPR effect of traditional NPs. Besides the morphological transformation from NPs to NFs, the modulation of nanostructures, including size, surface charge, and shape, according to the need of biomedical imaging and therapy showed promising smartness and pertinence. To date, emerging challenges along this direction have to be faced and addressed, including (i) the design principle of precursors to realize bioadaptive self-assembly and to precisely control the resultant superstructures in vivo, (ii) the development of advanced in vivo tools and strategies for characterizing the dynamic behavior of self-assembled structures, and (iii) understanding of structural evolution of exogenous selfassembled materials in the biointerface. Furthermore, the morphology and structure transformation followed by the disassembly and excretion of self-assembled nanomaterials in vivo should be paid extensive attention. Lastly, systematic evaluation of the efficacy and biosafety of self-assembled nanomaterials will greatly help clinical translation toward diagnostics and therapeutics of diseases. We believe that by collaborations with multidisplanary researchers from the fields of materials science, biology, chemistry, medicine, etc., in vivo self-assembled and transformable nanosystems will ultimately benefit medicine and public healthcare in the future. I


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Figure 9. (a) Schematic illustration of the pH-triggered structural transformation of BP−KPH from NPs to NFs. (b) Molecular structure of BP− KPH. (c, d) TEM images of the morphology transformation from NPs (c) to NFs (d) at different pH. (e) Photographs of BP−KPH NF solutions (2.0 × 10−5 M) with a gradual increase in NR from 0 to 2 μM under a 365 nm UV lamp. (f) Bio-TEM images of the morphology transformation of BP−KPH NPs in tumor slices. (g) CLSM images to observe FRET signals of BP−KPH/NR NFs and C−BP−KPH/NR NPs in tumor tissue. (h) Thermal images of MCF-7 tumor-bearing mice with or without BP−KPH NFs (2 × 10−4 M) with injection of ICG (5 × 10−5 M) for 4, 24, and 48 h under laser irradiation. (i) Quantity analysis of the time-dependent tumor volume of MCF-7 tumor-bearing mice upon different photothermal treatments. Reproduced with permission from ref 10. Copyright 2017 John Wiley and Sons.

■

AUTHOR INFORMATION

51573031), the Science Fund for Creative Research Groups of the National Natural Science Foundation of China (11621505), the CAS Key Research Program for Frontier Sciences (QYZDJ-SSW-SLH022), the Key Project of the Chinese Academy of Sciences in Cooperation with Foreign Enterprises (GJHZ1541), and the CAS Interdisciplinary Innovation Team.

Corresponding Authors

*E-mail: [email protected] (X.-D.L.). *E-mail: [email protected] (H.W.). *E-mail: [email protected] (L.W.). ORCID

Hao Wang: 0000-0002-1961-0787

Notes

Author Contributions

The authors declare no competing financial interest.

The manuscript was written through contributions of all authors. All of the authors approved the final version of the manuscript.

Biographies Ping-Ping He, a Master’s degree candidate under the guidance of professors Xiang-Dan Li, Hao Wang, and Lei Wang at South-Central University for Nationalities and the National Center for Nanoscience and Technology (NCNST), respectively. Her current research

Funding

This work was supported by the National Natural Science Foundation of China (51725302, 21374026, 51573032, and J


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interests involve in situ transformation of self-assembled nanomaterials for cancer theranostics. Xiang-Dan Li, Ph.D., is a professor in the College of Chemistry and Materials Science at South-Central University for Nationalities. Her current research interests are functional polymeric materials for use as energy, environmental, and biological materials. Lei Wang, Ph.D., is a professor in the CAS key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety at NCNST. His current interests involve the development of supramolecular building blocks and their self-assembly/transformation under physiological conditions for biomedical applications. Hao Wang, Ph.D., is a professor in the CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety at NCNST. His research interests are (i) developing polymeric assemblies under physiological conditions, (ii) studying their bioeffects and further regulating their biological behavior, and (iii) exploring peptide-based imaging probes and drug delivery systems.

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ABBREVIATIONS BP, bispyrene; Cy, cyanine; PbAE, poly(β-amino ester); DOX, doxorubicin; PAMAM, poly(amidoamine); ICG, indocyanine; AIR, assembly/aggregation-induced retention; AIE, assembly/ aggregation-induced emission; ACQ, aggregation-caused quenching; EPR, enhanced permeability and retention; TISA, transformation-induced surface adhesion; TEAR, transformation-enhanced accumulation and retention; TICT, twisted intramolecular charge transfer; TPA, two-photon absorption; FRET, Förster resonance energy transfer; PDT, photodynamic therapy; HLB, hydrophilic−lipophilic balance

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