Morphology Tuning of AIE Probe by Flash ... - ACS Publications

Subscriber access provided by TUFTS UNIV

Biological and Medical Applications of Materials and Interfaces

Morphology Tuning of AIE Probe by Flash Nanoprecipitation: Shape and Size Effects on In Vivo Imaging Mingwei Wang, Yisheng Xu, Yajing Liu, Kaizhi Gu, Jinchao Tan, Ping Shi, Dahai Yang, Zhiqian Guo, Wei-Hong Zhu, Xuhong Guo, and Martien Abraham Cohen Stuart ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08159 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Morphology Tuning of AIE Probe by Flash Nanoprecipitation: Shape and Size Effects on In Vivo Imaging Mingwei Wang,†,§ Yisheng Xu,*,†,§,║ Yajing Liu, Kaizhi Gu,‡ Jinchao Tan, Ping Shi, Dahai ┴

┴

┴

Yang,┴ Zhiqian Guo,*,‡ Weihong Zhu,‡ Xuhong Guo,*,†,║ and Martien A. Cohen Stuart† †

State Key Laboratory of Chemical Engineering, School of Chemical Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China.

‡

Shanghai Key Laboratory of Functional Materials Chemistry, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China.

┴

State Key Lab of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China.

║

Engineering Research Center of Materials Chemical Engineering of Xinjiang Bingtuan, Shihezi University, Xinjiang 832000, P. R. China

E-mail: [email protected]; [email protected]; [email protected]

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 30

KEYWORDS. Aggregation-Induced Emission, Quinolinemalononitrile, Nanoparticle Morphology, Flash Nanoprecipitation, In Vivo Imaging, Fluorescence.

ABSTRACT.

Aggregation-induced emission (AIE) imaging probes have recently received considerable attention due to their unique property of high performance in aggregated state and their imaging capability. However, the tendency of AIE molecules to aggregate into microns long and irregular shapes, which significantly limits their application in vivo, is becoming a serious issue that needs to be addressed. Here, we introduce a novel engineering strategy to tune the morphology and size of AIE nanoaggregates, based on Flash Nanoprecipitation (FNP). Quinolinemalononitrile (ED) is encapsulated inside properly selected amphiphilic block copolymers of varying concentration. This leads to a variety of ED particle morphologies with different sizes. The shape and size are found to have strong influences on tumor targeting both in vitro and in vivo. The current results therefore indicate that the FNP method, together with optimal choice of amphiphilic copolymer is a universal method to systematically control the aggregation state of the AIE materials and hence tune the morphology and size of AIE nanoaggregates, which is potentially useful for precise imaging at specific tumor sites.


2

Page 3 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. Introduction The fascinating turn-on and brightness properties of aggregation-induced emission (AIE) materials make them promising for biomedical imaging.1-13 However, AIE molecules generally aggregate spontaneously in water so that one does not have precise control over the morphology and size of AIE fluorescent materials. As is well-known, the size, shape, and orientation of nanoparticles can modulate the rate, efficiency, and endocytic mechanism of their cellular uptake.14,15 In particular, size and shape can be used to specifically targeting tumor sites, which indicates there is diagnostic potential.16,17 Therefore, the ability to systematically tune the size and morphology of AIE probes for optimum tumor uptake and targeting is most valuable.18,19 The present paper reports an attempt to develop this ability. The use of amphiphilic polymers or surfactants as AIE nanomaterials vehicles has been adopted to mainly prolong the circulation lifetimes in vivo. 20-23 However, rational tuning of the morphology and size of AIE molecules is still a great challenge. All these particles are formed by fast precipitation processes typical for hydrophobic solutes. As a rule, nucleation rates are poorly controlled since the aforementioned fabrication of AIE nanomaterials is based on traditional approaches where mixing is typically too slow. As a result, the obtained AIE nanomaterials are additive-dependent and commonly lack stability. An alternative synthetic strategy has been used to encapsulate AIE particles into polymeric core-shell carriers, and in this way successfully change the morphology from microsize rods to nanosize spheres,24 but this does not yet satisfy the need for a reliable and reproducible control of AIE particle growth. Fortunately, flash nanoprecipitation (FNP) in the presence of a suitably selected amphiphilic block polymer is such a method, owing to its ability to provide homogeneous mixing of organic solute and polymeric materials in milliseconds. The hope is that the rapid precipitation of organic solutes by FNP will


3


Page 4 of 30

block their growth and aggregation, resulting in a relatively stable “kinetically frozen” state, and that this is the basis for the possibility to modulate the size and morphology.25-30 Meanwhile, amphiphilic block polymers can be introduced to “freeze” the AIE nano/micro-structures in water by the interaction between the hydrophobic block and AIE molecules. It has been claimed that the structure of AIE nanoaggregates can be strongly influenced by taking amphiphilic block copolymers with different glass transition temperature (Tg) of the hydrophobic blocks which somehow reflects the rigidity of the polymer (lower Tg for more flexibility), although the meaning and role of Tg in this context is not very clear.31 Nonetheless, using FNP with a suitable block copolymer to steer the assembly of AIE seems promising since it is simple and probably universal. The method has been applied successfully to make drug-loaded particles in a number of cases.32-35 Finally, proper choice of the amphiphilic polymer block provides a hydrophilic shell which helps to prolong the blood circulation time of the particles. In this work, fluorescent AIE probes based on quinolinemalononitrile (ED and QM-2,18,19 Scheme S1) are employed as hydrophobic solutes, and different amphiphilic copolymers, namely dextran-b-polylactide (Dex-b-PLA, Scheme S2), dextran-b-polycaprolactone (Dex-b-PCL, Scheme S3) and dextran-b-poly(lactic-co-glycolic acid) (Dex-b-PLGA, Scheme S4) are used for stabilization. We explore if and in what way the morphologies and sizes (nano-sphere, nano-rod, and micro-rod) of AIE aggregates, and their sizes, ranging from one hundred nanometers to tens of micrometers, can be tuned by the nature and concentrations of the block copolymers. Using these particles with different sizes and shapes, the cell uptake efficiency and in vivo tumor imaging precision were systematically evaluated. The strategy of FNP method with optimal amphiphilic copolymer for fine regulation of the morphology and size of AIE nanoaggregates paves a new way towards fast, reliable, and reproducibly scaled-


4

Page 5 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Illustration of morphology and size tuning by FNP and their effects on tumor cell imaging of zebrafish. The AIE molecules are dissolved in THF in one stream simultaneously mixing with the other three streams of water, then the nanoparticles are produced. Polymer can be dissolved in one water stream to modulate the interactions between AIE molecule and hydrophobic polymer block, thus forming various morphologies and sizes: (a) bare nanoparticle; (b) nanorod with polymer shell; (c) microrod; (d) nanosphere.

up fabrication of AIE materials. We believe that this strategy will greatly promote AIE materials as biomedical imaging contrast agents in practical applications.

2. Results and Discussion 2.1. FNP: influence of preparation conditions on morphology As is well known, the inherent difficulty in using AIE fluorescent materials for practical purposes is to obtain control over their aggregation.36-38 Currently the strategy is to mainly focus on biocompatible amphiphilic copolymers as vehicles to systematically assemble nanoaggregates into desired shapes and sizes, mostly by traditional processes. To overcome the lack of control, we explore the FNP method that has been succesful in many cases as a rapid, kinetically controlled method,39-42 we use it here to first fabricate bare, uncoated hydrophobic AIE


5


Page 6 of 30

aggregates, and subsequently to encapsulate AIE with help of an amphiphilic polymer. Figure 1 describes the general approach; as FNP allows control over composition as well as mixing rate, and we also employ different amphiphilic block copolymers, we have a number of variables that can be optimized. As the hydrophilic block, we choose a polysaccharide (dextran), for reasons of biocompatibility. First, we prepared AIE particles without using any polymer by simply mixing a solution of ED in THF (solvent, stream 1) with a large amount of water (anti-solvent, stream 2-4). This yields rodlike ED nanoparticles.26,30 The typical size of these particles under these conditions is about 500 nm in length and 100 nm in diameter (Figure 2a); in contrast, particles obtained by traditional simple mixing approach, without using FNP, typically aggregate to micron size.19 The difference between the two methods of preparation is presumably due to a difference in nucleation rate; the faster mixing during FNP enhances supersaturation since the dissolved solute and stabilizing amphiphilic polymer as stabilizers rapidly mix with lots of water as an antisolvent, over a time scale shorter than the characteristic nucleation and growth time scales for the nanoparticles,26-28 leading to a higher nucleation rate, and, hence, a larger number of smaller particles. During fast nucleation, only part of the solute is used up, so that the nucleation stage is followed by a growth stage leading to the rods that we observe. Apparently, post-FNP Ostwald ripening is virtually absent under these conditions. The bare ED rods prepared by FNP can be stably preserved for about 1 week, as shown in Figure 3a in which the diffusion coefficient is utilized to reflect the relative size for different shape particles since each particle should correspond to a sphere with identical diffusion coefficient. It was found that these AIE aggregates display high photostability, implying great


6

Page 7 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


potential for long-term tracking in biomedical applications. However, it turned out that without

Figure 2. TEM of NPs morphology tuned by block copolymer at different concentrations in the aqueous stream before mixing. (a, b) without polymer; (c, d) 0.1 mg mL-1 Dex-b-PLA; (e, f) 1 mg mL-1 Dex-b-PLA; (g, h) 3 mg mL1 Dex-b-PLA; (i, j) 6 mg mL-1 Dex-b-PLA; (k, l) 10 mg mL-1 Dex-b-PLA. The concentration of ED in THF stream is fixed at 0.02 mg mL-1. The stream velocities are the same for each sample, 12: 12: 48: 48 mL min-1.

any added polymer the morphology of the bare ED particles could not be altered, even though the flow rate and water/THF ratio in the FNP were changed, so that only a modest size control could be achieved. To improve in this respect, three polysaccharide-based amphiphilic block copolymers were synthesized through a coupling reaction between amino-end capped dextran and a biocompatible polyester carrying a reactive end group (Scheme S1-S4). These polymers were all characterized by 1H NMR as presented in Figure S1-S4. As they are not soluble in THF, they were injected into the multi-inlet vortex mixer (MIVM) in the form of an aqueous solution during the FNP


7


Page 8 of 30

process, which is a somewhat different condition from that used in the studies of FNP by Prud’homme and coworkers.26,30 As shown in Figure 2, the shaded area around the particles likely indicates the successful preparation of the core-shell structure which is further evidenced by much more negative surface potential as listed in Table S1. The amphiphilic block copolymers served as stabilizers in at least two ways, namely by inhibiting aggregation due to steric repulsion, and by reducing the interfacial tension of the particles.42 As expected, the shape of the particles is observed to depend on both the type and the concentration of the hydrophobic block (Table S2).31 For PCL, we obtain spherical NPs of about 130 nm in diameter for all polymer concentrations, as shown in Figure S5. In contrast, for PLA/PLGA, we observe a change in morphology: at low polymer concentration (0.1 mg mL-1, before mixing) we obtain, like with PCL, nanospheres of about 90 nm (Figure 2c and 2d), whereas at higher concentrations there is a transition to nanorods about 100 nm in diameter and 400 nm in length (Figure 2e and 2f); the cross section and length increase with increasing polymer concentration to 500 nm and 4 – 10 microns, respectively; while with absence of ED, PLA-based copolymer alone form nanospheres of about 160 nm (Figure S6). Detailed sizes are summarized in Table S3. In contrast, the traditional self-assembly approach in the presence of amphiphilic polymers showed much larger NP size (lower diffusion coefficient), broader size distribution, less reproducibility, and lower ED encapsulation efficiency (EE %, Table S4). The difference in response between PCL- and PLA-based copolymers is remarkable. As the mixing conditions and concentrations in both cases are identical, it is very unlikely that the FNP process itself takes a different course in either case. More likely, it is some slower, post-FNP process which underlies the difference, and a plausible candidate is Ostwald ripening. In the absence of polymer, ED yielded stable nanosized rod particles; no post-FNP slow coarsening


8

Page 9 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


was observed, so that Ostwald ripening in the absence of polymer does not seem to occur. Different from the surfactant stabilization of crystals,43,44 the growth of the NP become more prominent as the polymer concentration was increased. In addition, the further examination of electron diffraction pattern from the TEM images clearly indicates a non-crystalline state of the rod NPs (Figure S7). In fact, addition of polymer to a final (low) concentration of 0.01 mg mL-1 (0.1 mg mL-1 FNP-sample, Figure 2c) does two things: (1) it affects the rate of nucleation, probably by association between the hydrophobic block and ED, so that many small nuclei appear, and (2) it covers these small particles and stabilizes them against aggregation. We note that the polymer concentration here is close to the CMC of Dex-b-PLA (about 0.0047 mg mL-1, as shown in Table S2), so that free micelles are not present. Then, further increase of the polymer implies a finite concentration of polymer micelles; provided that these micelles can solubilize ED, they can also enhance the transport rate of ED between small and large particles. If there is enough driving force, Ostwald ripening may occur which, in turn, leads to further growth of ED. That is to say, the polymer can both reduce and enhance the Oswald ripening process depending on the polymer concentration.45,46 To verify this hypothesis, extra polymer was added post-FNP to a 0.1 mg mL-1 FNP-prepared sample (spherical particles, Figure 2c) to obtain a final concentration equal to 1 mg mL-1.In line with our argument, the spherical NPs are indeed observed to transform into micro-rods (Figure S8). Moreover, further increasing the polymer concentration leads to the formation of larger micro-rods at the expense of smaller ones. Hence, we believe that free polymer induces Ostwald ripening by acting as a carrier for AIE molecules; apparently, the particles stabilized by the PLA-based polymer still have sufficient interfacial free energy to drive the ripening process, whereas the particles stabilized by the more hydrophobic PCL block lose that driving force.


9


Page 10 of 30

The generic feature of this method was tested by modulating the size and morphology of another far-red AIE probe, QM-2, again in the presence of Dex-b-PLA, as shown in Figure S9. The nanoshperes can be systematically tuned to nanorods by increasing the concentration of AIE probe and polymer. This further indicates that high concentrations of both solute and polymer favors the formation of rod-shape NPs which provides a universal method to achieve desired shape and sizes.


10

Page 11 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Diffusion coefficient (a) and normalized fluorescent spectra (b) of different NP samples: Bare Nanorod, ED rods without polymer; Nanorod, ED with 1 mg mL-1 Dex-b-PLA; Microrod, ED with 3 mg mL-1 Dex-b-PLA; Nanosphere, ED with 3 mg mL-1 Dex-b-PCL. The excitation wavelength is 430 nm. The stream velocities are same for each sample, 12: 12: 48: 48 mL min-1.

2.2. Optical properties of the NPs The structure of microrods is slightly visible under the fluorescence microscope, as shown in Figure S10, but that of nanospheres and nanorods is beyond resolution. The fluorescence spectra after encapsulation with Dex-b-PLA do not show a dramatic wavelength shift compared to bare ED rods (Figure 3b, Figure S11 and Table S5), while a small blue shift appears on the UV-Vis spectra, indicating the successful encapsulation of ED inside the polymer shell (Figure S12). The


11


Page 12 of 30

maximum emission wavelength is 554 nm which is consistent with the fluorescence profile of ED aggregates in water.19 In contrast, upon encapsulation with the more hydrophobic block PCL, a peak at 600 nm becomes more obvious indicating a water fraction in the core of around 30-40 % as we studied in previous work.19

2.3. Cellular uptake by NPs with different morphologies The cellular uptake efficiency and targeting capability of NPs with various shapes and sizes were systematically examined, as shown in Figure 4. The commonly used cervical cancer cells (Hela cells) were chosen as model cancer cell lines. All of these particles do not show apparent toxicity to cells (Figure S13). After incubation of Hela cells with different types of NPs, all of these NPs show certain cellular uptake effect even though the largest size reaches the micron level. This is becuase that 1) the elongated particles (e.g., rods) have prolonged circulation time in vivo and achieve significant accumulation in tumors than their spherical counterparts due to the overall lower curvature and difficulty to wrap large dimensions avoiding clearance from blood; 2) the axial width of these particles are usually in the range between 100 - 500 nm (Table S3). As substantially reported, the penetration of the NPs into the cancer cell in vivo can be realized through their long axis perpendicular to the cell membrane.17,47,48 It is observed that the bare ED nanorods show intense fluorescence in cancer cells (Figure 4b). The TEM images of Hela cells clearly show that aggregates have formed in the cells (Figure 4c) which is consistent with the strong aggregation-induced emission effect. These small aggregates around 2 µm are not only seen evenly


12

Page 13 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Confocal and TEM images of Hela cell incubated with NPs having different morphologies: (a, b, c) Bare ED nanoparticle without polymer decoration; (d, e, f) ED nanorod assembled with 1 mg mL-1 Dex-b-PLA; (g, h, i) ED microrod assembled with 3 mg mL-1 Dex-b-PLA; (j, k, l) ED nanosphere assembled with 3 mg mL-1 Dex-b-PCL. The CLSM images obtained from 570 to 620 nm, λex = 405 nm. Green dashed line is the cellular outlines, and the red dashed line is the nucleus outline.

distributed inside the cells, but also outside the cells. In contrast, for nanorods prepared with the amphiphilic copolymer Dex-b-PLA, the fluorescence signals in cancer cells are more distributed, and the small rods can be found exclusively in cells (Figure 4e and 4f). These rods however do not seem to have specificity to different organelles, even though most of them are observed in nuclei. As the rod size increases to the micron range, the fluorescence intensity becomes much weaker (Figure 4h) which is consistent with a strongly reduced number of rods observed in cells by TEM (Figure 4i).


13


Page 14 of 30

Interestingly, nanorods appear to have better penetration capability in contrast to the recent reported finding that the spherically shaped NPs penetrate more efficiently. This is reflected by the weak fluorescence signal as shown in Figure 4k. It can be attributed to the following reasons. One reason is that the shape influences directional diffusion and the hydrostatic forces orient the nanorods perpendicularly to the membrane, enabling the insertion of the nanorods with an axis around 50 nm, but it is more difficult for nanospheres.15-17 Another reason is that free diffusion of nanospheres through the cell membrane was seen by TEM (Figure 4l), and hence the spherical NPs were observed both inside and outside the cells.15-17 In addition, without the polymer coating, bare ED nanorods have less negative surface charge (Table S1), causing a less repulsion between NPs and cell membrane. In order to confirm the ability of nanorods to specifically target cancer cells, commonly used human embryonic kidney cells (293T cells) were also chosen as a normal cell line model for comparison with the Hela cell line (Figure S14). There is no obvious difference in the fluorescence in 293T cells when incubated with different types of NPs, indicating non-specific cellular uptake in the normal cells.


14

Page 15 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. In vivo imaging of different types of NPs fed in intact normal or cancer zebrafish. (a, b) Bare ED nanoparticle without polymer; (c, d) ED nanorod assembled with 1 mg mL-1 Dex-b-PLA; (e, f) ED microrod assembled with 3 mg mL-1 Dex-b-PLA; (g, h) ED nanosphere assembled with 3 mg mL-1 Dex-b-PCL. In each figure, top left is the bright field image and the bottom left is the merge image of bright field and green channel and red channel; (i) 3D CLSM image of (d). The CLSM images obtained from 570 to 620 nm (λex = 405 nm) for ED, and from 500 to 550 nm (λex = 405 nm) for green fluorescence protein (GFP). Red fluorescence is emitted from ED and green fluorescence arising from the GFP from the cancer cells injected into the zebrafish.

Flow cytometric studies were also conducted to evaluate the kinetics of the cell uptake process of different morphologies (Figure S15). After 48 hours incubation, the cellular uptake of ED rods without polymer shell was the fastest one, and close to 100%. Indeed without polymer protection, ED lacks the ability of long circulation. For particles with polymer coating, the rate of cellular uptake is shape dependent: that of the coated nanorods is much faster than that of the long rods and spherical NPs, but slower than that of bare nanorods. Again, this latter result is probably because the dextran coated NPs have a more negative surface charge (Table S1), causing a stronger repulsion between NPs and the cancer cell membrane, resulting in slower uptake of polymer-decorated nanorods in cancer cells.


15


Page 16 of 30

2.4. In vivo imaging in tumor The targeting ability of NPs with varying morphology and size was further examined in vivo by feeding these NPs to zebrafishes. Zebrafish was chosen as a model due to its high genomic homology with humans and its transparent body, which facilitates fluorescent observation. SMMC-7721 cancer cells labeled with Green Fluorescent Protein (GFP) were injected in the perivitelline cavity to introduce cancer cells. It is shown in Figure 5 that after 36 hours of feeding, all ED nanoparticles, without and with polymer shell, can reach most of fish body and accumulate in the perivitelline space. In the case of fish with cancer cells, most of the bare ED NPs are accumulated in the perivitelline space, but they do not specifically enter the GFP labeled cancer cells (Figure 5b). Notably, among all four types of nano/micro-aggregates, only the small nanorods decorated with dextran can specifically enter the cancer cells in zebrafishes thereby targeting them precisely without any random distribution (Figure 5d). The Pearson correlation coefficient calculated between small nanorods prepared with Dex-b-PLA and the tumor is 0.85 (Table S6), which indicates its good specificity as a probe for imaging of tumor cells; even after additional 24 h of feeding, the small nanorods still seems to be selectively localized, as presented in Figure S16. When the nanorods become longer and reach micron size, the targeting ability becomes worse which is reflected by their partial entering into the tumor cells; indeed, a large portion of the rods are observed to circulate inside the zebrafish (Figure 5f), and this comes with a lower Pearson correlation coefficient (Table S6). Interestingly, for the spherical NPs with polymer brush , the majority does not enter into the cancer cells although they accumulate in the perivitelline region (Figure 5h, Table S6). This may have two reasons: 1) the aspect ratio of


16

Page 17 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


spherical particles make them less prone to extravasate through fenestrated blood vessels in tumors than nonspherical particles; 2) the spherical NPs can easily diffuse through the cell membrane, which further supports the findings in Figure 4h and 4k. Overall, the in vivo zebrafish experiments certainly indicate an optimum cellular uptake efficacy, and the most precise targeting for the amphiphilic, polymer-decorated nanorods prepared by FNP, much better than for other type of particles.

3. Conclusion We have developed a simple and easy-to-operate method to finely control the morphology and size of AIE particles, with amphiphilic block copolymers of Dex-b-PLA or Dex-b-PCL as polymeric shell, and ED/QM-2 as the AIE fluorescent molecules. The size and morphology of ED particles can be tuned by the polymer concentration and by proper selection of the hydrophobic block. Intriguingly, as the aspect-ratio of the length and width is about 5, the nanorods show better cellular uptake and targeting capability in cancer cells, even better than nanospheres. More importantly, in a zebrafish model, the nanorod particles with polysaccharide (dextran) shell display excellent tumor targeting capability, while the nanorod particles without such a polymer shell are randomly distributed. In the light of the simplicity of the FNP method to obtain size and morphology control, imaging AIE probes turns out to be an effective tool for tumor targeting and diagnosis.

4. Experimental Section


17


Page 18 of 30

Materials. Dextran (Mn = 6 kDa) was purchased from Sigma and sodium cyanoborohydride (95%) was purchased from Acros. Phenylcarbinol (99%), sodium tetraborate (99%), D,L-lactide (99%), acryloyl chloride (97%), stannous octoate (tin(II) bis(2-ethylhexanoate) (Sn(Oct)2, 95%), triethylamine (99%),4-formyl-benzoic acid (97%), 4-dimethylamino-pyridine (99%) and dimethyl sulfoxide (DMSO, 99.8%) were purchased from J&K Chemical. Ethylenediamine was chemically pure grade and purchased from Sinopharm Chemical Reagent Co. Tetrahydrofuran (THF) was chemically pure grade and purchased from Shanghai Tianlian Fine Chemical Co. Pure water was obtained by a Milli-Q water purification system and was used in all experiments. Other reagents and solvents were used as received without any further treatment. Dex(6k)-bPLA(10k), Dex(6k)-b-PLGA(10k), and Dex(6k)-b-PCL(8k) were synthesized according to literature.49-52 The ED was synthesized according to our previous report.19 Characterization. Nanoparticle morphology was observed on a JEOL JEM-1400 TEM instrument with an acceleration voltage of 100 kV, and a JEOL JEM-2100 TEM instrument with an acceleration voltage of 200 kV. One drop of the nanoparticle solution was deposited on carbon-coated copper grid. The droplet was allowed to dry under ambient conditions. The 1H NMR spectra in deuterated DMSO were recorded on a Bruker AM 400 MHz instrument (chemical shifts relative to TMS). Gel permeation chromatography (GPC) was conducted at 40 °C in THF on a WATERS 1515 system equipped with a series of PS gel columns (HR3, HR4, HR5). Polydispersity indices (PDI) of the hydrophobic blocks were determined from Mw /Mn by GPC. The critical micelle concentration (CMC) values of the amphiphilic block copolymers were measured through HITACHI F-4500 Fluorescence spectrometer. Pyrene was used as fluorescent probe and the concentration of pyrene solution was 1*10-6 mol L-1. The pyrene excitation wavelength was 333 nm and the fluorescence intensity was measured at 372 nm and


18

Page 19 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


383 nm. The intensity ratio of the peaks at 372 nm to those at 383 nm (I372/I383) was plotted against the logarithm of the concentration to determine the CMC. Dynamic light scattering (DLS) was carried out at 25 °C with a NICOMP 380 ZLS instrument with acquisition times of 180 s. The angle of measurement was fixed at 90°. For data processing, the number distribution of particle size (PSD) was chosen for plotting, in order to avoid a dominant scattering effect of large aggregates. Average and standard deviations were obtained from three duplicates. UV-vis absorption spectra of samples were recorded on a UV-2550 UV-vis spectrophotometer. Fluorescence spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer excited at 430nm wavelength. Preparation of NPs. The AIE-NPs were prepared by the FNP method. ED (1 mg) was added into 50 mL THF, and used as organic solution (stream 1). Dextran-based block copolymer was added into pure water, used as water stream (stream 2). The organic stream was fed at a flux (12 mL min-1) equal to that of the polymer solution (12 mL min-1) and excess of pure water (streams 3 and 4, 48 mL min-1 each), into a MIVM (Figure 1) system, using two digitally controlled syringe pumps (Harvard Apparatus, PHD 2000) to yield NPs. The obtained NP suspensions were dialyzed for 8 h against Milli-Q water (3 L Milli-Q water per 20 mL NP suspension) using a Spectra/Por®6 MWCO 10kDa membrane, to remove remaining THF, and stored at room temperature. Cell Culture. The human epithelioid cervical carcinoma cell line Hela (Hela cells) was purchased from the Institute of Cell Biology (Shanghai, China). They were propagated in T-75 flasks cultured at 37 °C under a humidified 5% CO2 atmosphere in RPMI 1640 medium (GIBCO/Invitrogen, Camarillo, CA)supplemented with 10% fetal bovine serum (FBS, Biological


19


Page 20 of 30

Industry, Kibbutz Beit Haemek, Israel) and 1% penicillin−streptomycin (10 000 U mL-1 penicillin and 10 mg mL-1 streptomycin (Solarbio Life Science, Beijing, China). In vitro cytotoxicity assay. The cell cytotoxicity of NPs with different morphology towards Hela cancer cell lines were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell cytotoxicity was evaluated by Cell Counting Kit-8 (Dojindo, Tokyo, Japan) according to the factory’s instruction. Cells were plated in 96-well plates in 0.1 mL volume of DMEM medium with 10% FBS, at a density of 1*104 cells/well. After incubation for 24 h, absorbance was measured at 570 nm with a Genios multifunction-reader (Tecan GENios Pro, Tecan Group Ltd. Maennedorf, Switzerland). Each samples was measured in triplicate and used in three independent experiments. Zebrafish culture. Zebrafishes were purchased from the China Zebrafish Resource Center (Wuhan), and maintained as shown in the “Zebrafish Book”.53 Embryos were obtained by natural spawning and kept in E3 zebrafish water at 28 °C. Green fluorescent protein-labeled SMMC7721 cells (human hepatoma cell line) were transplanted into 3 dpf (day post-fertilization) zebrafish embryos, to introduce a green fluorescence-labeled tumor. For each implantation, about 400 cells were microinjected into the perivitelline cavity of each zebrafish, then zebrafish were kept in E3 zebrafish water at 28 °C. As control, zebrafishes without any treatment were also kept in E3 zebrafish water at 28 °C. Confocal Fluorescence Imaging for Living Cells. Cells were plated onto glass-bottom Petri dishes in 1.5 mL of complete culture medium and allowed to adhere for 12 h before treatment. The cells were then exposed to different NP solutions at a final concentration of 4.5 µM ED, and incubated for 4 min at 37 °C under a humidified 5% CO2 atmosphere. Fluorescence imaging was


20

Page 21 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


then performed using a confocal laser scanning microscope (CLSM, Nikon A1R system, Japan) with a 60× oil immersion objective lens. The fluorescence signals of cells incubated with NPs were collected at 595 nm, using a laser at 405 nm as excitation light source. Confocal Fluorescence Imaging for Living zebrafish. 5.5 dpf zebrafish embryos were incubated with different NP solutions at a final concentration of 4.5 µM ED for 1.5 day at 28 °C. Fluorescence imaging was then performed using a CLSM. The fluorescence signals of zebrafish incubated with NPs were collected at 595 nm using a laser at 405 nm as excitation light source, while the fluorescence signals of tumor with green fluorescence were collected at 525 nm using a laser at 488 nm as excitation source.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.xxxxx. Synthesis routine and 1HNMR spectrum of Dex-NH2, Dex-b-PLA, Dex-b-PCL, Dex-b-PLGA; zeta potential of NPs; property of amphiphilic dextran block copolymer; NP Sizes counted from TEM images and the encapsulation efficiency; TEM images of NPs formed by pure Dex-b-PLA alone; characterizations of NPs prepared by FNP and traditional self-assembly; TEM and selected area electron diffraction of NPs; TEM images of Post-addition of extra Dex-b-PLA after FNP; NPs morphologies tuned by QM-2 with Dex-b-PLA by FNP; fluorescence microscope of NPs; fluorescent spectra and normalized UV-Vis spectra of different NPs; fluorescence quantum


21


Page 22 of 30

yield of NPs; cytotoxicity of NPs with different morphology on Hela cells; confocal images of 293t cell incubated with NPs having different morphologies; cellular uptake of NPs with different morphology in Hela cells; in vivo imaging of intact normal and cancer zebrafish fed with nanorods after 36 h or 60 h feeding; Pearson correlation coefficient of different NPs with cancer zebrafish.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Author Contributions §

M. W. and Y.X. contributed equally.

Conflicts of interest There are no conflicts to declare.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (NSFC) for Science Center Programe (21676089), NSFC (21676089, 21476143), Excellent Young Scholars (21622602) NSFC/China, Distinguished Young Scholars (21325625) NSFC/China, Young


22

Page 23 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scholars (21706074) NSFC/China, Shanghai talent development fund (2017038), Oriental Scholarship of Scientific Committee of Shanghai (14ZR1409700 and 15XD1501400), and Fundamental Research Funds for the Central Universities (222201313010, 222201314029, 222201717013).

REFERENCES (1) Mei, J.; Leung, N.; Kwok, R.; Lam, J.; Tang, B. Z. Aggregation-Induced Emission: Together We Shine, United We Soar! Chem. Rev. 2015, 115, 11718-11940. (2) Ding, D.; Li, K.; Liu, B.; Tang, B. Z. Bioprobes Based on AIE Fluorogens. Acc. Chem. Res. 2013, 46, 2441-2453. (3) Li, X.; Kim, C.; Lee, S.; Lee, D.; Chung, H.; Kim, G.; Heo, S.; Kim, C.; Hong, K.; Yoon, J. Nanostructured Phthalocyanine Assemblies with Protein-Driven Switchable Photoactivities for Biophotonic Imaging and Therapy. J. Am. Chem. Soc. 2017, 139, 10880-10886. (4) Kwok, R.; Leung, C.; Lam, J.; Tang, B. Z. Biosensing by Luminogens with AggregationInduced Emission Characteristics. Chem. Soc. Rev. 2015, 44, 4228-4238. (5) Chen, S.; Wang, H.; Hong, Y.; Tang, B. Z. Fabrication of Fluorescent Nanoparticles based on AIE Luminogens (AIE Dots) and Their Applications in Bioimaging. Mater. Horiz. 2016, 3, 283293. (6) Guo, Z.; Park, S.; Yoon, J.; Shin, I. Recent Progress in the Development of Near-Infrared Fluorescent Probes for Bioimaging Applications. Chem. Soc. Rev. 2014, 43, 16-29. (7) Luo, J.; Xie, Z.; Lam, J. W. Y.; Cheng, L.; Chen, H.; Qiu, C.; Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D.; Tang, B. Z. Aggregation-Induced Emission of 1-Methyl-1,2,3,4,5-Pentaphenylsilole. Chem. Commun. 2001, 1740-1741.


23


Page 24 of 30

(8) Xu, J.; Yang, P.; Sun, M.; Bi, H.; Liu, B.; Yang, D.; Gai, S.; He, F.; Lin, J. Highly Emissive Dye-Sensitized Upconversion Nanostructure for Dual-Photosensitizer Photodynamic Therapy and Bioimaging. ACS Nano 2017, 11, 4133-4144. (9) Li, H.; Cheng, J.; Zhao, Y.; Lam, J. W. Y.; Wong, K. S.; Wu, H.; Li, B. S.; Tang, B. Z. LValine

Methyl

Ester-Containing

Tetraphenylethene:

Aggregation-Induced

Emission,

Aggregation-Induced Circular Dichroism, Circularly Polarized Luminescence, and Helical SelfAssembly. Mater. Horiz. 2014, 1, 518-521. (10) Yan, C.; Guo, Z.; Shen, Y.; Chen, Y.; Tian, H.; Zhu, W. Molecularly Precise Self-Assembly of Theranostic Nanoprobes within a Single-Molecular Framework for In Vivo Tracking of Tumor-Specific Chemotherapy. Chem. Sci. 2018, 9, 4959-4969. (11) Gu, K.; Xu, Y.; Li, H.; Guo, Z.; Zhu, S.; Zhu, S.; Shi, P.; James, T. D.; Tian, H.; Zhu, W. Real-Time Tracking and In Vivo Visualization of β-Galactosidase Activity in Colorectal Tumor with a Ratiometric Near-Infrared Fluorescent Probe. J. Am. Chem. Soc. 2016, 138, 5334–5340. (12) Li, D.; Qin, W.; Xu, B.; Qian, J.; Tang, B. Z. AIE Nanoparticles with High Stimulated Emission Depletion Efficiency and Photobleaching Resistance for Long-Term SuperResolution Bioimaging. Adv. Mater. 2017, 29, 1703643. (13) Chen, M.; Li, L.; Wu, H.; Pan, L.; Li, S.; He, B.; Zhang, H.; Sun, J. Z.; Qin, A.; Tang, B. Z. Unveiling the Different Emission Behavior of Polytriazoles Constructed from Pyrazine-Based AIE Monomers by Click Polymerization. ACS Appl. Mater. Interfaces 2018, 10, 12181–12188. (14) Blanco, E.; Shen, H.; Ferrari, M. Principles of Nanoparticle Design for Overcoming Biological Barriers to Drug Delivery. Nat. Biotechnol. 2015, 33, 941-951.


24

Page 25 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(15) Gratton, S. E. A.; Ropp, P. A.; Pohlhaus, P. D.; Luft, J. C.; Madden, V. J.; Napier, M. E.; DeSimone, J. M. The Effect of Particle Design on Cellular Internalization Pathways. P. Natl. Acad. Sci. USA 2008, 105, 11613-11618. (16) Hinde, E.; Thammasiraphop, K.; Duong, H. T. T.; Yeow, J.; Karagoz, B.; Boyer, C.; Gooding, J. J.; Gaus, K. Pair Correlation Microscopy Reveals the Role of Nanoparticle Shape in Intracellular Transport and Site of Drug Release. Nat. Nanotechnol. 2017, 12, 81-89. (17) Geng, Y.; Dalhaimer, P.; Cai, S.; Tsai, R.; Tewari, M.; Minko, T.; Discher, D. E. Shape Effects of Filaments Versus Spherical Particles in Flow and Drug Delivery. Nat. Nanotechnol. 2007, 2, 249-255. (18) Shao, A.; Xie, Y.; Zhu, S.; Guo, Z.; Zhu, S.; Guo, J.; Shi, P.; James, T. D.; Tian, H.; Zhu, W. Far-Red and Near-IR AIE-Active Fluorescent Organic Nanoprobes with Enhanced TumorTargeting Efficacy: Shape-Specific Effects. Angew. Chem. Int. Edit. 2015, 54, 7275-7280. (19) Shi, C.; Guo, Z.; Yan, Y.; Zhu, S.; Xie, Y.; Zhao, Y. S.; Zhu, W.; Tian, H. Self-Assembly Solid-State Enhanced Red Emission of Quinolinemalononitrile: Optical Waveguides and Stimuli Response. ACS Appl. Mater. Interfaces 2013, 5, 192-198. (20) Feng, G. X.; Wu, W. B.; Xu, S. D.; Liu, B. Far Red/Near-Infrared AIE Dots for ImageGuided Photodynamic Cancer Cell Ablation. ACS Appl. Mater. Interfaces 2016, 8, 21193-21200. (21) Zhu, Z.; Qian, J.; Zhao, X.; Qin, W.; Hu, R.; Zhang, H.; Li, D.; Xu, Z.; Tang, B. Z.; He, S. Stable and Size-Tunable Aggregation-Induced Emission Nanoparticles Encapsulated with Nanographene Oxide and Applications in Three-Photon Fluorescence Bioimaging. ACS Nano 2016, 10, 588–597.


25


Page 26 of 30

(22) Gao, M.; Su, H. F.; Lin, G. W.; Li, S. W.; Yu, X. S.; Qin, A. J.; Zhao, Z. J.; Zhang, Z. F.; Tang, B. Z. Targeted Imaging of EGFR Overexpressed Cancer Cells by Brightly Fluorescent Nanoparticles Conjugated with Cetuximab. Nanoscale 2016, 8, 15027-15032. (23) Gao, Y. T.; Feng, G. X.; Jiang, T.; Goh, C. C.; Ng, L. G.; Liu, B.; Li, B.; Yang, L.; Hua, J. L.; Tian, H. Biocompatible Nanoparticles based on Diketo-Pyrrolo-Pyrrole (DPP) with Aggregation-Induced Red/NIR Emission for in Vivo Two-Photon Fluorescence Imaging. Adv. Funct. Mater. 2015, 25, 2857-2866. (24) Li, Y.; Shao, A.; Wang, Y.; Mei, J.; Niu, D.; Gu, J.; Shi, P.; Zhu, W.; Tian, H.; Shi, J. Morphology-Tailoring of a Red AIEgen from Microsized Rods to Nanospheres for TumorTargeted Bioimaging. Adv. Mater. 2016, 28, 3187-3193. (25) Saad, W. S.; Prud'Homme, R. K. Principles of Nanoparticle Formation by Flash Nanoprecipitation. Nano Today 2016, 11, 212-227. (26) Johnson, B. K.; Prud'Homme, R. K. Mechanism for Rapid Self-Assembly of Block Copolymer Nanoparticles. Phys. Rev. Lett. 2003, 91, 118301-118302. (27) Liu, Y.; Kathan, K.; Saad, W.; Prud'Homme, R. K. Ostwald Ripening of β-Carotene Nanoparticles. Phys. Rev. Lett. 2007, 98, 36102. (28) Liu, Y.; Cheng, C.; Liu, Y.; Prud'Homme, R. K.; Fox, R. O. Mixing in a Multi-Inlet Vortex Mixer (MIVM) for Flash Nano-Precipitation. Chem. Eng. Sci. 2008, 63, 2829-2842. (29) Russ, B.; Liu, Y.; Prud'Homme, R. K. Optimized Descriptive Model for Micromixing in a Vortex Mixer. Chem. Eng. Commun. 2010, 197, 1068-1075. (30) Akbulut, M.; Ginart, P.; Gindy, M. E.; Theriault, C.; Chin, K. H.; Soboyejo, W.; Prud'Homme, R. K. Generic Method of Preparing Multifunctional Fluorescent Nanoparticles using Flash Nanoprecipitation. Adv. Funct. Mater. 2009, 19, 718-725.


26

Page 27 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(31) Zhu, Z. Effects of Amphiphilic Diblock Copolymer on Drug Nanoparticle Formation and Stability. Biomaterials 2013, 34, 10238-10248. (32) He, Z.; Santos, J. L.; Tian, H.; Huang, H.; Hu, Y.; Liu, L.; Leong, K. W.; Chen, Y.; Mao, H. Scalable Fabrication of Size-Controlled Chitosan Nanoparticles for Oral Delivery of Insulin. Biomaterials 2017, 130, 28-41. (33) Nikoubashman, A.; Lee, V. E.; Sosa, C.; Prud Homme, R. K.; Priestley, R. D.; Panagiotopoulos, A. Z. Directed Assembly of Soft Colloids through Rapid Solvent Exchange. ACS Nano 2016, 10, 1425-1433. (34) Zhu, Z. Flash Nanoprecipitation: Prediction and Enhancement of Particle Stability via Drug Structure. Mol. Pharmaceut. 2014, 11, 776-786. (35) Zhu, Z.; Anacker, J. L.; Ji, S.; Hoye, T. R.; Macosko, C. W.; Prud'Homme, R. K. Formation of Block Copolymer-Protected Nanoparticles Via Reactive Impingement Mixing. Langmuir 2007, 23, 10499-10504. (36) Cai, X. L.; Bandla, A.; Mao, D.; Feng, G. X.; Qin, W.; Liao, L. D.; Thakor, N.; Tang, B. Z.; Liu, B. Biocompatible Red Fluorescent Organic Nanoparticles with Tunable Size and Aggregation-Induced Emission for Evaluation of Blood-Brain Barrier Damage. Adv. Mater. 2016, 28, 8760-8765. (37) Nicol, A.; Kwok, R.; Chen, C. P.; Zhao, W. J.; Chen, M.; Qu, J. A.; Tang, B. Z. Ultrafast Delivery of Aggregation-Induced Emission Nanoparticles and Pure Organic Phosphorescent Nanocrystals by Saponin Encapsulation. J. Am. Chem. Soc. 2017, 139, 14792-14799. (38) Gui, C.; Zhao, E. G.; Kwok, R.; Leung, A.; Lam, J.; Jiang, M. J.; Deng, H. Q.; Cai, Y. J.; Zhang, W. J.; Su, H. F.; Tang, B. Z. AIE-active Theranostic System: Selective Staining and Killing of Cancer Cells. Chem. Sci. 2017, 8, 1822-1830.


27


Page 28 of 30

(39) Wang, M.; Yang, N.; Guo, Z.; Gu, K.; Shao, A.; Zhu, W.; Xu, Y.; Wang, J.; Prud'Homme, R. K.; Guo, X. Facile Preparation of AIE-Active Fluorescent Nanoparticles through Flash Nanoprecipitation. Ind. Eng. Chem. Res. 2015, 54, 4683-4688. (40) York, A. W.; Zablocki, K. R.; Lewis, D. R.; Gu, L.; Uhrich, K. E.; Prud'Homme, R. K.; Moghe, P. V. Kinetically Assembled Nanoparticles of Bioactive Macromolecules Exhibit Enhanced Stability and Cell-Targeted Biological Efficacy. Adv. Mater. 2012, 24, 733-739. (41) D'Addio, S. M.; Prud'Homme, R. K. Controlling Drug Nanoparticle Formation by Rapid Precipitation. Adv. Drug Deliver. Rev. 2011, 63, 417-426. (42) Zhu, Z.; Margulis-Goshen, K.; Magdassi, S.; Talmon, Y.; Macosko, C. W. Polyelectrolyte Stabilized Drug Nanoparticles via Flash Nanoprecipitation: A Model Study with β-Carotene. J. Pharm. Sci.-US 2010, 99, 4295-4306. (43) Zhao, Y. S.; Fu, H.; Peng, A.; Ma, Y.; Liao, Q.; Yao, J. Construction and Optoelectronic Properties of Organic One-Dimensional Nanostructures. Acc. Chem. Res. 2010, 43, 409-418. (44) Fu, H.; Xiao, D.; Yao, J.; Yang, G. Nanofibers of 1,3-Diphenyl-2-pyrazoline Induced by Cetyltrimethylammonium Bromide Micelles. Angew. Chem. Int. Edit. 2003, 42, 2883-2886. (45) Weiss, J.; Canceliere, C.; McClements, D. J. Mass Transport Phenomena in Oil-in-Water Emulsions Containing Surfactant Micelles: Ostwald Ripening. Langmuir 2000, 16, 6833-6838. (46) Ariyaprakai, S.; Dungan, S. R. Influence of Surfactant Structure on the Contribution of Micelles to Ostwald Ripening in Oil-in-Water Emulsions. J. Colloid Interf. Sci. 2010, 343, 102108. (47) Huang, C.; Zhang, Y.; Yuan, H.; Gao, H.; Zhang, S. Role of Nanoparticle Geometry in Endocytosis: Laying Down to Stand Up. Nano Lett. 2013, 13, 4546-4550.


28

Page 29 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(48) Shi, X.; von Dem Bussche, A.; Hurt, R. H.; Kane, A. B.; Gao, H. Cell Entry of OneDimensional Nanomaterials Occurs by Tip Recognition and Rotation. Nat. Nanotechnol. 2011, 6, 714-719. (49) Schatz, C.; Louguet, S.; Le Meins, J.; Lecommandoux, S. Polysaccharide-BlockPolypeptide Copolymer Vesicles: Towards Synthetic Viral Capsids. Angew. Chem. Int. Edit. 2009, 48, 2572-2575. (50) Zhao, Z.; Zhang, Z.; Chen, L.; Cao, Y.; He, C.; Chen, X. Biodegradable Stereocomplex Micelles Based on Dextran-Block-Polylactide as Efficient Drug Deliveries. Langmuir 2013, 29, 13072-13080. (51) Liu, J.; Zhang, L. Preparation of a Polysaccharide-Polyester Diblock Copolymer and its Micellar Characteristics. Carbohyd. Polym. 2007, 69, 196-201. (52) Li, B.; Wang, Q.; Wang, X.; Wang, C.; Jiang, X. Preparation, Drug Release and Cellular Uptake of Doxorubicin-Loaded Dextran-B-Poly(ε-Caprolactone) Nanoparticles. Carbohyd. Polym. 2013, 93, 430-437. (53) Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish. The University of Oregon Press: Eugene, Oregon, 1993.


29


Page 30 of 30

Table of Contents


30

Morphology Tuning of AIE Probe by Flash ... - ACS Publications

Recommend Documents