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The Blood Clearance Kinetics and Pathway of Polymeric Micelles in Cancer Drug Delivery Xuanrong Sun, Guowei Wang, Hao Zhang, Shiqi Hu, Xin Liu, Jianbin Tang, and Youqing Shen ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b02830 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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The Blood Clearance Kinetics and Pathway of Polymeric Micelles in Cancer Drug Delivery Xuanrong Sun,a,b,‡ Guowei Wang,a,‡ Hao Zhang,c Shiqi Hu,a Xin Liu,a Jianbin Tang,a Youqing Shena* a

Center for Bionanoengineering and Key Laboratory of Biomass Chemical Engineering

of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China b

Collaborative

Innovation

Center

of Yangtze

River

Delta

Region

Green

Pharmaceuticals, Zhejiang University of Technology, Hangzhou 310014, China c

College of Chemical Engineering, Zhejiang University of Technology, Hangzhou

310014, China ‡

The authors contributed equally to this work.

*Corresponding Author: [email protected]

ABSTRACT: Polymer micelles are one of the most investigated nanocarriers for drug delivery; many have entered clinical trials and some are in clinic use, but their delivery systems have not yet shown the expected high therapeutic efficacy in clinics. Further understanding their in vivo behaviors, particularly how quickly and by what mechanism polymer micelles are cleared (i.e., via micelles or unimers) once injected, is key to solving this dilemma. Herein, we hope to answer these questions for the clinically relevant polyethylene glycol-block-poly(ɛ-caprolactone) (PEG-PCL) and PEG-block-poly(DLlactide) (PEG-PDLLA) micelles. A small fraction of the hydrophobic chain ends was conjugated with a pair of fluorescence resonance energy transfer (FRET) dyes, Cy5 and Cy5.5, and used to fabricate FRET micelles whose FRET efficiency was correlated to the percentage of polymer chains in the micelles, the micelle degree. In vitro, serum proteins induced PEG-PCL micelle dissociation to some extent; mouse serum or blood surprisingly did not induce micelle dissociation but once with shear applied by a microfluidic channel caused most PEG-PCL micelles dissociated. After intravenous 1

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administration in mice, the PEG-PCL or PEG-PDLLA micelles were quickly sequestered into the liver as unimers and the micelle degree in the blood quickly decreased to about 20%. The FRET-imaging experiments showed that in blood vessels the micelles quickly dissociated into unimers, which were found associated with albumin in blood and in liver. Thus, it is concluded that, upon intravenous injection, the shear and the bloodborne proteins (particularly albumin) induced the most (~80%) PEG-PCL and PEG-PDLLA micelles to quickly dissociate into unimers, which were sequestered by Kupffer cells, while intact micelles were difficult to be cleared. These micelles were able to arrive tumors and were very stable with cell membrane, but dissociate gradually inside cells. These findings on in vivo micelle fate and the clearance mechanism are directional for the rational design of polymer micelles for improved therapeutics; particularly, improving micelle stability in blood is the prerequisite for surface functionalizations such as introducing targeting ligands.

KEYWORDS: Fluorescence resonance energy transfer; cancer drug delivery; polymeric micelle; micelle disassembly; micelle stability; micelle clearance pathway

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Block copolymer micelles have been explored as one of the main nanocarriers for cancer nanomedicine aimed at delivering drugs to tumors1-7 via the enhanced permeability and retention (EPR) effect.8 These nanocarriers have the advantage of a stealth shell-hydrophobic core structure, which is capable of encapsulating a variety of water-insoluble drugs without altering their chemical structures.3,

6, 7

One of such

successful examples is polyethylene glycol-block-poly(D,L-lactide) (PEG-PDLLA) micelles, which have been extensively used as nanocarriers for various drugs.9 PEGPDLLA loaded with paclitaxel (Genexol-PM) is already used in clinics in several countries and is under clinical trials in the US and Europe.10 However, as with other nanomedicines, these micellar nanomedicines only reduce some adverse effects of the drugs; they do not show the expected therapeutic advantages over free drugs.11, 12 Thus, further understanding the in vivo behaviors of polymer micelles is key to solving this dilemma and improving the therapeutic efficacy.13 To efficiently deliver drugs from an intravenous injection site into tumor cells to realize high therapeutic efficacy, a micelle needs go successively through at least five cascade steps: circulation in the blood compartments, accumulation and penetration in tumor, and finally cellular internalization followed by intracellular drug release, which is named the CAPIR cascade.14 The micelle’s chemical and physical properties, particularly stability, dictate its ability to accomplish this cascade and thus its therapeutic efficacy.1, 15 Obviously, high stability in blood is the prerequisite for drug delivery16 because once a micelle loses its integrity in the blood, it will immediately release the carried drug into the bloodstream and fail its mission, let alone such functions as tumor targeting via the EPR effect or active targeting through tumorbinding ligands.17 Micelles are in thermodynamic and kinetic equilibrium with their amphiphilic unimers.18 Once administered into the bloodstream, micelles are immediately diluted and mixed with blood cells, plasma proteins and other components, shifting the equilibrium toward dissociation into unimers.18-20 Previous in vitro studies already showed that a fraction of micelles would lose their integrity21 in serum-mimicking conditions22,

23

and even immediately transform their structures24 and release their 3


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payloads upon interaction with lipoproteins.25 Some recent in vivo studies focused more on the multiple dynamic processes occurring inside the micelles. For instance, poly(ethylene oxide)-block-polystyrene (PEO-PS)26 and PEG-PDLLA micelles22 were found to rapidly release encapsulated probes upon i.v. administration, so-called premature drug release, but it is unknown whether this release was due to drug loading on the core-shell interface or micelle dissociation. Self-assembled lipidic nanoparticles were found to rapidly exchange their components with plasma proteins and progressively dissociated following tumor accumulation.24 The stability of micelles in contact with the cell membrane is also important as they are expected to be internalized into cells and then release the drug to overcome membrane-associated drug resistance.1, 14

However, while intact polymeric micelles are reported to be taken into cells via

endocytosis followed by intracellular release of drug molecules,5, 27 micelles were also reported to dissociate on the cell membrane, thus releasing the contents extracellularly.28



Scheme 1. Schematic illustration of polymeric micelle trafficking and fate upon intravenous administration related to the basic questions, i.e., what happens with micelles in the blood? Does liver sequester the integrated micelles or unimers? Do micelles or unimers accumulate in the tumor? Despite these intensive studies of polymer micelles as carriers, three basic 4


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questions still remain unanswered (Scheme 1): 

Once injected into the blood, how quickly and to what extent will the micelles dissociate?



What is the mechanism of the micelle blood-compartment clearance? Does the blood clearance of micelles cause their dissociation or dissociation cause clearance? That is, are micelles cleared as integrated micelles or as unimers?



Do micelles or unimers accumulate in the tumor? The answers to these three key questions can direct our further design of micellar

drug delivery systems for high therapeutic efficacy. For instance, it is usually taken for granted that micelles are mainly cleared by the reticuloendothelial system (RES) as a whole; great efforts have, thus, been made to engineer micelle surfaces to evade RES clearance or install ligands on the shell to enhance their tumor targeting.1, 29-31 However, if micelles quickly dissociate to unimers upon injection, these efforts would end up meaningless. Herein, we tried to answer these questions for the clinically relevant micelles of PEG-PDLLA9 and PEG-PCL32 using FRET.23, 28, 33, 34 Importantly, the FRET dyes were conjugated to the polymers’ hydrophobic ends to avoid any release of the dyes.

RESULTS AND DISCUSSION PEG-PCL-, PEG-PDLLA-, and PEG-PS-block copolymers with terminal primary amine groups were synthesized and characterized as described in the Supporting Information Table S1 and confirmed by 1H-NMR spectra (Fig. S1-S3). The extensively used near-infrared dyes,35, 36 Cy5 (Ex: 640 nm; Em: 680 nm) and Cy5.5 (Ex: 680 nm; Em: 720 nm), were selected as a FRET pair because blood and living tissues have very weak autofluorescence in this tested region and thus their fluorescence can be measured directly without any separation. The dyes were separately conjugated to the ends of their hydrophobic blocks (Fig. 1a) to prevent dye release from the micelles, which was found in physical encapsulation. Only 1% of the polymer chains were conjugated with the dyes to minimize effects of the dyes on micelle properties. The micelles fabricated from the block copolymers containing 1% polymer-Cy5 and 1% polymer-Cy5.5 were 5


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found to have a high FRET efficiency. Once excited at 640 nm, the micelles containing PEG-PCL-Cy5/PEG-PCL-Cy5.5 in deionized water had strong Cy5.5 fluorescence at 720 nm with weak Cy5 fluorescence at 680 nm, indicating a strong FRET occurred (blue curve in Fig. 1b). The FRET efficiency (R)—calculated from the intensity ratio IA/(ID + IA), where IA and ID were the fluorescence intensities at 720 nm and 680 nm, respectively—was 0.80. Once acetone was added to the solution to dissolve the micelles, the FRET peak at 720 nm almost disappeared and the calculated FRET efficiency was 0.23 (Fig. 1c). Thus, these FRET micelles were used for the following studies.

Figure 1. Design and working principle of the FRET micelles. (a) PEG-PCL with its PCL end conjugated with a FRET pair of Cy5 (donor) or Cy5.5 (acceptor). (b) The fluorescence spectra of the PEG-PCL-FRET micelles in aqueous solution (blue, 1.0 mg/mL) or diluted with 10 × acetone (red) (1%PEG-PCL-Cy5 and 1%PEG-PCL/Cy5.5 (molar)). (c) The formation of micelles of PEG-PCL/PEG-PCL-Cy5/PEG-PCL-Cy5.5 turns on the FRET fluorescence, while their dissociation turns off the FRET fluorescence and turns on the Cy5 fluorescence. (d) The size distribution of the PEGPCL-FRET micelles.

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The PEG-PCL-FRET micelles at 1 mg/ml of the polymer in deionized water were stable for more than three months with an average diameter of 43 nm (Fig. 1d). The fluorescence quenching method was used to confirm if any of the dyes were exposed to the outer layer of the micelles or released. Gold nanoparticles (AuNP, 10 nm) can quench a dye’s fluorescence if within about 8 nm.37 As shown in Fig. S4, adding AuNP to the PEG-PCL-FRET-micelle solution did not affect the fluorescence at all, suggesting that the dyes were buried in the micelle core and not released at all. For a block copolymer with a CMC of 10 μg/mL in deionized water at 1 mg/mL, theoretically 99% polymer chains assemble as micelles and less than 1% of chains stay in solution as unimers. Thus, a FRET efficiency of 0.80 is an indication that almost all the polymer chains were in micelles, yielding 100% micelle degree. In acetone, all the micelles dissolve into unimers. Thus, a FRET efficiency of 0.23 indicates no polymer chains associating as micelles, 0% micelle degree. Assuming that the percentage of polymer chains as micelles (referred to as micelle% or micelle degree) is linearly proportional to the FRET efficiency

33

, the micelle degree of a block copolymer in

solution could be calculated from its FRET efficiency. Similarly, the FRET efficiencies of the PEG-PDLLA-FRET and PEG-PS-FRET solutions were measured and their calibration curves of the micelle degrees were established. For an amphiphilic copolymer in solution at concentrations above its CMC, its unimers are in equilibrium with its micelles.38 Upon diluting the PEG-PCL-FRET solution at 1 mg/ml, its FRET intensity at 720 nm initially decreased, so did the FRET efficiency and the calculated micelle degree (Fig. 2a). Surprisingly, the micelle degree did not decrease to zero. Rather, it leveled off at about 60%, at and even far below the CMC (≈ 4.67 μg/mL). PEG-PDLLA-FRET micelles had the same trend (data not shown). PEG-PS micelles, known to be highly stability owing to the high glass transition temperature (107 ºC) and strong hydrophobicity of the PS block,39 were used as a control for comparison. Indeed, dilution of the PEG-PS-FRET solution did not change its FRET efficiency and thus all the chains were in micelles even at very low concentrations (Fig. 2a). 7


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Figure 2. The integrity of PEG-PCL and PEG-PS micelles upon dilution or the presence of proteins or serum. (a) Concentration-dependent micelle degrees of PEGPCL-FRET and PEG-PS-FRET upon dilution. Time-resolved micelle degree of the FRET micelles in the solutions of protein (b) VLDL (1.0 mg/mL), CM (1.0 mg/mL), HDL (2.0 mg/mL) or LDL (2.0 mg/mL), or (c) BSA (40 mg/mL) or α,β-globulin (13 mg/mL) or (d) in fresh mouse blood or serum. The polymer concentration was 1 mg/mL. (e) The transmission electron microscopy (TEM) images of PEG-PCL-FRET (upper row, e1-5) and PEG-PS-FRET (bottom row, e6-10) micelles upon dilution or incubation with BSA. Concentration: 1 mg/mL micelles (e1,6), 0.1 mg/ml micelles (e2,7), 0.01 mg/ml micelles (e3,8). The micelles (0.1 mg/mL) were incubated with 4 mg/mL BSA for 1 h (e4,9) or 4 h (e5, 10). (Scale bar=100 nm, magnification 50k ). (f) The micelle degree relative to the initial value versus the cycles of the micelle in fresh mouse blood passing through a microchannel as a measure of the shear induceddissociation. Polymer concentration, 0.1 mg/mL; microfluidic channel, 40 μm width 8


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and height, 3 cm long; flow rate, 0.3 mL/h. (Scale bar=100 µm).

The stability of PEG-PCL-FRET micelles in the presence of the major blood-borne proteins and lipoproteins was further tested by adding these components to the micelle solution. α,β-Globulin (15 mg/mL), albumin (40 mg/mL), LDL (2.0 mg/mL), HDL (2.0 mg/mL), VLDL (1.0 mg/mL) or CM (1.0 mg/mL) at the same concentrations found in blood were separately added to the FRET–micelle solutions (1 mg/mL) and their FRET efficiencies and micelle degrees were determined (Fig. 2b, c). The presence of globulin or albumin did not induce dissociation of PEG-PS-FRET micelles, but did induce PEGPCL-FRET micelles to dissociate by about 18% or 13% in 24 h. Similarly, adding lipoproteins LDL or VLDL to the PEG-PCL-FRET solution decreased the micelle degrees by about 20% but HDL and CM only caused 8% micelle dissociation. Very surprisingly, fresh blood or serum from the ICR mice did not induce micelle disassociation (Fig. 2d). This is different from the finding that serum decreased the FRET efficiency of the dye-loaded micelles, which may be caused by dye release due to partitioning rather than micelle disassociation.40 TEM images were acquired to better understand the influence by both diluting and BSA on micelles stability. Upon dilution, the number of micelles of both PEG-PCLFRET (Fig. 2, e1-3) and PEG-PS-FRET (Fig. 2, e6-8) decreased. At 100-fold dilution, the PEG-PCL-FRET micelles became heterogeneous (Fig. 2, e3), indicating dissociation and re-assembly, while PEG-PS-FRET remained uniform. After incubated with BSA for 1 h or 4 h, the PEG-PCL-FRET micelles were dilated and surrounded by BSA proteins (Fig. 2, e4,5) and the morphologies changed. In contrast, the PEG-PSFRET micelles were stable and their morphology did not change upon dilution or incubation with BSA (Fig. 2, e6-10). As opposed to the static stability, micelles in blood flow are under strong fluidic shear. A microfluidic channel with square cross section (40 μm width and height by 3 cm) was employed to mimic a blood vessel to probe the shear stress-effects on micelle integrity. The shear, τ, was approximated from the equation used for straight microchannels, τ = 7.1 μQ/a3,41 where μ is the blood viscosity with an average of 7.1  9


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10−4 Pa·s,42 Q is the flow rate in a range of 0.1 mL/h–10 mL/h, and a is the height and width of a square channel. The FRET-micelle solution in mouse blood was continuously passed through the microfluidic channel using a syringe pump at a flow rate of 0.3 mL/h to obtain a shear stress of 6.54 Pa, which is relevant to the value of instantaneous high shear stress encountered in mouse blood vessels.43 The solution flowed through it for about 0.57 s. The polymer solution was mixed with fresh mouse blood at the polymer concentration of 0.1 mg/mL to mimic the initial blood concentration in mice after iv injection of 0.2 mL of the micelle solution containing 0.2 mg (assuming 2 mL total blood; dose, 10 mg/kg). The relative micelle degree to the initial micelle degree at 0.1 mg/mL as a measure of reduction was calculated from the FRET values. As shown in Fig. 2f, after five times through the microchannel, the micelle degree of PEG-PS micelles decreased by 12% while that of PEG-PCL-FRET decreased by 63%, and more cycles through the microchannel further decreased the micelle degree. For comparison, at static condition the integrity of PEG-PCL-FRET or PEG-PS FRET micelles in fresh mouse blood remained unchanged. This result indicates that shear stress did induce dissociation of micelles, even the ultrastable micelles with the glassy PS core. The effect of the cell membrane on the integrity of the micelles was analyzed using confocal microscopy at the cellular level. Labeled PEG-PCL micelles were cultured with HepG2 cells and their images and fluorescent spectra were record. The single-dyelabeled micelles, PEG-PCL-Cy5 and PEG-PCL-Cy5.5, or PEG-PS-Cy5 and PEG-PSCy5.5 were first incubated with cells to validate their spectra recorded by the confocal microscopy. As shown in Figs. S5 and S6, the fluorescence spectra recorded in the cells agreed well with the dyes’ spectra. PEG-PCL-FRET micelles were then cultured with HepG2 cells at 37 °C for different time intervals and the fluorescence spectra at different sites on the cell membrane or inside cells were recorded at excitation of 640 nm. As shown in Fig. 3 a-g, the cell membrane had a weak fluorescence at 680 nm but a strong FRET peak at 720 nm at different incubation times (blue curves) with calculated micelle degrees all above 80%. However, the randomly selected intracellular sites showed a strong FRET peak at first 0.5 h and 1 h (red lines in Fig. 3 a,b), but after incubated for 10


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2 h or longer (6 h), almost no FRET peak but only the Cy5 fluorescence at 680 nm was found at different sites inside cells (red lines in Fig. 3 c,d). For comparison, the cells incubated with PEG-PS-FRET micelles for different times had strong FRET fluorescence at all sites inside the cells and on their membrane at 30 min, 1 h or 2 h, indicating that the micelles remained intact when cultured with cells (Fig. 3e-g). At 6 h Cy5 fluorescence at 680 appeared in few intracellular sites, indicating some micelle dissociation, but the calculated overall micelle degree was still above 75% (Fig. 3 h). The overall fluorescence spectra of PEG-PCL-FRET cultured with HepG2 cells were also recorded at timed intervals (Fig. S7). The whole cells showed a weak FRET peak with a calculated micelle degree from 50.6% at 2 h to 46.6% at 24 h.

Figure 3. Confocal fluorescence images and the fluorescence spectra of representative sites of HepG2 cells incubated with PEG-PCL-FRET (left two columns) and PEG-PS-FRET micelles (right two columns) for different times. The cells were cultured with PEG-PCL-FRET micelles (a-d) or PEG-PS-FRET micelles (eh) for 0.5 h, 1 h, 2 h or 6 h; representative sites on the cell membrane (1 and 3, blue lines) and intracellular sites (2 and 4, red lines) were randomly selected and their 11


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fluorescence spectra excited at 640 nm were recorded. The spectra of the single-dye labeled PEG-PCL and PEG-PS controls are shown in Figures S5 and S6. Polymer dose, 0.2 mg/mL.

PEG-PCL-FRET micelles in PBS (200 μL, 1 mg/mL, 10 mg/kg) were injected into a mouse (~ 20 g) via the tail vein. The polymer concentration in the blood was determined using the Cy5.5 fluorescence according to the calibration curve and the FRET fluorescence spectra was measured accordingly (Fig. 4a). The polymer concentration in the blood rapidly decreased to 22% of the injected dose after 1 h injection and further decreased to 3.7% after 24 h. Clearly, the FRET peak intensity quickly decreased while the Cy5 fluorescence peak increased relative to the FRET peak. The calculated FRET efficiency, R, decreased from the initial vale 0.83 to 0.57 at 15 min postinjection, 0.47 at 30 min, and 0.33 at 24 h. Accordingly, the calculated micelle degree was 47% at 15 min, and 21% at 3 h and finally 20% at 24 h (Fig. 4b). A very similar trend was found in PEG-PDLLA-FRET with different PDLLA block lengths (2K–3K and 2K–7K) (supporting Information Fig. S8). These results indicate that, once injected, the polymers were cleared very quickly from the bloodstream and the micelles immediately underwent dissociation. We first hypothesized that rapid clearance of the PEG-PCL-RFET lowered the chain concentration in blood and thereby caused micelle dissociation; thus, increasing blood-polymer concentration would increase the micelle degree. The clearance kinetics and micelle degrees at escalated doses of PEG-PCL-FRET were examined (Fig. 4c). The clearance of PEG-PCL-FRET at 80 mg/kg dose was still very quick similar to that of 10 mg/kg, but the 250 mg/kg dose indeed very significantly increased the polymer blood concentrations at all the time points; for instance, the blood still retained 40% and 35% of the injected dose at 2 h and 7 h postinjection. Surprisingly, the elevated blood polymer concentrations initially increased micelle degrees in the first hour (48.5% compared to 28.3%) but the later values had no significant difference from those of the 10 and 80 mg/kg doses. Thus, the higher blood concentration of the polymer chains did not prevent micelle dissociation and the majority of the polymer chains still existed as 12


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unimers.

Figure 4. Blood clearance and integrity of micelles i.v. injected in mice. (a) The blood clearance and the fluorescence spectra (inserted), and (b) the corresponding FRET efficiency calculated as Icy5.5/(Icy5.5 + Icy5) (blue curve) and the percentage of the injected polymers as micelles (micelle degree, Micelle%) (red curve) of the PEG-PCLFRET micelles after i.v. injection (10 mg/kg, n = 3). (c) The blood clearance and micelle degree of i.v.-administered PEG-PCL-FRET at different doses. (d) The blood clearance and micelle degree of PEG-PCL-FRET at 10 mg/kg i.v.-administered 10 min after predosing 50 mg/kg PEG-PCL. The mice were first i.v.-administered PEG-PCL micelles at 50 mg/kg 10 min before the injection. (e, f) The blood clearance and micelle degree of PEG-PCL-FRET and PEG-PS-FRET (both 10 mg/kg) after the mice were pretreated with three i.v. injections of GdCl3 at 15 mg/kg at 48 h, 24 h, 6 h before the injection of PEG-PCL (or PS)-FRET. Data are reported as the mean (±SD) for triplicate samples (n = 3).

RES macrophages and Kupffer cells are known to be responsible for sequestration 13


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of foreign substances from the blood.44 We therefore proposed that predosing with unlabeled PEG-PCL in the bloodstream would saturate these cleaners and therefore would slow the blood clearance and increase the micelle degree of PEG-PCL-FRET. The mice were first i.v. injected with 50 mg/kg of PEG-PCL and 10 min later PEGPCL-FRET at 10 mg/kg was injected (Fig. 4d). Indeed, predosing PEG-PCL significantly increased the PEG-PCL-FRET concentration at each time point compared with the injection of PEG-PCL-FRET at 10 mg/kg alone, for instance, from 31.7% to 69.7% at 0.5 h and from 8.9% to 19.9% at 6 h. The micelle degrees slightly increased, from 28.2% to 40.8% at 0.5 h and 19.7% to 31.7% (significant difference) at 6 h. The blood clearance and micelle-degree profiles of the stable PEG-PS-FRET micelles were also determined for comparison (Fig. 4e). In contrast to the rapid clearance of PEG-PCL-FRET micelles, those of PEG-PS3K-FRET dosed at 10 mg/kg were retained in the blood at much higher concentrations at all the times points. There was still 60% at 6 h and even 40% at 24 h after the injection of PEG-PS3K-FRET in the blood circulation and the corresponding micelle degrees were also high, still about 60% at 24 h post-injection. The micelles with shorter PS length, PEG-PS1.5K-FRET, had a faster blood clearance and lower micelle degrees at all the time points. The micelle degree was 47% at 2 h and 33% at 24 h. Comparison of these results with those of PEGPCL-FRET indicates that the micelle stability determines the blood clearance. Kupffer cells are macrophages resident in liver known to be responsible for clearance of foreign substances in blood.45 Blocking their sequestration would offer insight into the clearance mechanism of taking up micelles or unimers. GdCl3 is a specific Kupffer-cell blocking agent that inhibits the phagocytosis of liver macrophages and selectively eliminates the large macrophages in the periportal zone of the acinus.46 Thus, mice were pretreated with GdCl3 prior to the injection of the PEG-PCL-FRET solution (Figure 4f). Clearly, the GdCl3 pretreatments significantly slowed the clearance of PEG-PCL-FRET from the blood. At all the time points, the polymer blood concentrations greatly increased; there was still 30.2% of the injected dose at 17 h postinjection compared to only 4% in the mice without the GdCl3 pretreatments. Again, very surprisingly, given the high polymer blood concentrations the micelle degrees did 14


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not significantly change at any of the time points (Fig. 4f). The same trend was found with PEG-PS3K-FRET (Fig.4e).

Figure 5. The in vivo imaging (a,c) and biodistribution (b,d) of PEG-PCL (or PS)FRET i.v. injected in tumor-bearing nude mice without (a,b) or with (c,d) GdCl3 pretreatment. a) Mice were i.v. injected with PEG-PCL-FRET at 10 mg/kg and imaged at 0.5 h, 2 h and 6 h and then sacrificed and dissected, and b) the polymer concentration in each tissue was analyzed. c) Mice were i.v. administered with GdCl3 at 15 mg/kg at 48 h, 24 h, 6 h prior to the injection of the polymer solution and then d) analyzed as in (b). Data are reported as the mean (SD) for triplicate samples. The imaging was taken by the FRET fluorescence (Ex/Em = 640 nm/720 nm).

The corresponding biodistributions of these FRET micelles after i.v. injection were tracked by in vivo (at 0.5, 2 and 6 h postinjection) and ex vivo (at 6 h postinjection) fluorescence imaging (Fig. 5a). PEG-PCL-FRET was rapidly cleared from the blood and mainly accumulated in the liver. In contrast, the fluorescence of PEG-PS-FRET spread over the whole body due to its high blood concentration; it accumulated little in the liver but more in the spleen. The corresponding concentrations of the polymers in the tissues at 2, 6 and 24 h postinjection were determined by their Cy5.5 fluorescence according to the calibration curve (Fig. 5b). In agreement with the fluorescent imaging results, PEG-PCL-FRET 15


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was mainly sequestered in the liver, about 36% dose/g at 2 h after administration and 20.5% dose/g liver even at 24 h. About 15% dose/g of the polymer was in spleen and much less distributed into other organs and tumor. In contrast, PEG-PS-FRET had little in the liver, less than 5% dose/g liver at all times, but about 12% dose/g in spleen and distributed evenly in other organs and tumor at different times. The GdCl3-pretreatment effects on the biodistribution were further investigated (Fig. 5c,d). The in vivo fluorescent imaging showed that PEG-PCL-FRET spread widely in the chest and upper abdomen of the GdCl3-pretreated mice at 0.5 h and 2 h. This was very different from the GdCl3-untreated mouse, whose liver was lit up only (Fig. 5c vs. 5a). The ex vivo fluorescent imaging of the anatomized organs of the mouse at 6 h postinjection found that PEG-PCL-FRET had much weaker fluorescence in the liver but much brighter in spleen than that in the GdCl3-untreated mice. By comparison, the GdCl3 pretreatment did not significantly alter the fluorescence biodistribution of PEG-PS-FRET. These observations were confirmed by the quantitative analysis shown in Fig. 5d. A significant observation is that the GdCl3-pretreatment substantially reduced the liver concentration of PEG-PCL-FRET, from 35% dose/g to about 2.5% dose/g at 2 h postinjection and not higher than 5% dose/g thereafter. However, the treatment did not change the liver concentration of PEG-PS-FRET but led to its gradual accumulation into the spleen, reaching about 30% dose/g after 24 h. The micelle degrees of the polymers in liver and tumors were further analyzed using confocal fluorescence microscopy (Fig. 6). The FRET spectra of the randomly selected sites showed that the liver sections had very weak FRET fluorescence with an R of about 0.47, while the tumor sites had strong FRET fluorescence with an R of 0.73, corresponding to micelle degrees of 43% and 88%, respectively (Fig. 6a, b). In contrast, the FRET spectra of PEG-PS-FRET micelles in the liver and tumor sections both had strong FRET fluorescence with an R of 0.645 and 0.692 (Fig. 6c, d), corresponding to 73% and 81% micelle degrees.

16


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Figure 6. Confocal fluorescence images and FRET spectra of PEG-PCL-FRET (a,b) and PEG-PS-FRET (c,d) in liver (a,c) and tumor (b,d). Mice were i.v. injected with 10 mg/kg PEG-PCL-FRET. After 2 h, they were terminated and dissected. The section slices were 10 m thick, the excitation was at 640 nm and the fluorescence images and spectra were recorded at 640–740 nm. At least five sites were randomly selected to record the spectra. All the spectra were similar, two of which are shown.

Upon injection into the bloodstream, micelles are immediately surrounded by blood components, particularly albumin. We hypothesized that albumin might host the hydrophobic PCL block of PEG-PCL in its hydrophobic pocket and thus facilitate PEGPCL micelle dissociation. This role was also probed using FRET. Mouse albumin was labeled with Cy5 (MSA-Cy5). The mixture solution of MSA-Cy5 with PEG-PCL(or PS)-Cy5.5 had almost no FRET after standing still or gently stirring for 0–30 min, but the FRET peak gradually showed up after 1 h (Supporting Information Fig. S9). MSACy5 (100 mg/kg) and 5 min later the PEG-PCL (or PS)-Cy5.5 solution (10 mg/kg) was injected into the bloodstream of a mouse. The blood microvessels in the abdomen skin were imaged for FRET fluorescence at 720 nm excited at 640 nm (MSA-Cy5) and the corresponding fluorescence spectra of the bloodstream were recorded using confocal microscopy (Fig. 7). At 5 min post-injection of PEG-PCL-Cy5.5, the FRET fluorescence of the blood was clearly visible, became stronger after 10 min and gradually dimmed out thereafter (Fig. 7, a0–a4). The FRET ratio between MSA-Cy5 and PEG-PCL-Cy5.5 was 0.26 at 5 min and then increased to 0.66 at 10 min and 0.73 17


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at 60 min even though the fluorescence was already very weak at this time (Fig. 7, b1– b4). For comparison, under the same conditions, the blood microvessels injected with PEG-PS-Cy5.5 had a much dimmer FRET fluorescence at all time points. Their fluorescence spectra showed a weak FRET peak (Fig. 7c1–c4,d1–d4).

Figure 7. The in vivo FRET between the Cy5.5-labeled micelles and the Cy5labeled albumin (MSA-Cy5) in mouse blood. Confocal FRET fluorescence images of a mouse vein and the corresponding spectra at timed intervals after injection via the tail vein of PEG-PCL-Cy5.5 (a,b) or PEG-PS-Cy5.5 (c,d). The mice were first i.v. injected with MSA-Cy5 (2 mg/mice) 5 min prior to the micelle injection. The excitation was at 640 nm and the fluorescence spectra were recorded and imaged at 640–730 nm. PEG-PCL-Cy5.5 or PEG-PS-Cy5.5 dose: 10 mg/kg.

The FRET fluorescence of MSA-Cy5 and PEG-PCL(or PS)-Cy5.5 sequestered into the livers of the mice in Fig. 7 was then analyzed using confocal microscopy (Fig. 8). At all the tested sites in the liver MSA-Cy5/PEG-PCL-Cy5.5 had a strong FRET peak, suggesting that they were sequestered together in the liver. In contrast, in the liver of 18


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the mice administered with MSA-Cy5/PEG-PS-Cy5.5, few sites had strong FRET peak; most had only MSA-Cy5 fluorescence.

Figure 8. Confocal FRET fluorescence images and spectra of the livers of the mice administered with PEG-PCL-Cy5.5 (a) or PEG-PS-Cy5.5 (b) and MSA-Cy5. Mice were injected via the tail vein first with MSA-Cy5 (2 mg/mice) and 5 min later PEGPCL-Cy5.5 or PEG-PS-Cy5.5 (10 mg/kg). After 2 h, the mice were sacrificed; the livers were dissected and sectioned into 10 m thick slices; the images and the spectra were taken with excitation at 640 nm. The spectra of at least five randomly selected 20-point sites were recorded at 640-730 nm.

Fluorescence and FRET technologies are frequently used to study the stability and integrity of micelles.28, 40, 47, 48 Dyes were generally loaded by physical trapping in the micelles’ hydrophobic cores as drugs are loaded. The drawback of this method is that the intrinsic partitioning of the dye in the micelle core and the solution and its components—for instance, the proteins in the medium, in the serum or on the cell membrane—makes it hard to differentiate micelle disassembly or dye release,33 particularly in the body. Thus, in this study, the FRET dyes were separately conjugated to the chain ends of the hydrophobic blocks of the block copolymers to exclude any dye release. These dye-conjugated block copolymers formed micelles with the two dyes tightly buried in the hydrophobic core, as confirmed by the inquenchability of the dye 19


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fluorescence by gold nanoparticles (Fig. S4). Furthermore, we found that the block copolymer containing 1% of Cy5- and 1% Cy5.5-conjugated block copolymers had the highest FRET efficiency and thus the micelles were all made at this composition. Furthermore, the low percentage of the dye-conjugated polymer chain in the micelles precludes the potential influence of dye conjugation on the micelle properties. In addition to FRET micelles of the clinically relevant polymers PEG-PCL and PEGPDLLA, known-stable micelles with a glassy core from PEG-PS49 were also fabricated as a control. Their sizes and zeta-potentials were kept similar, approximately 50 nm and −3 mV. The in vitro stability of the micelles was first assessed. As expected, dilution led PEG-PCL micelles to gradually dissociate to about 60% micelle degree, but unexpectedly, further dilution, even by 1000 fold to concentrations well below the micelles’ CMC, did not further decrease the micelle degrees, suggesting the kinetic stability of the micelles.50 Blood-borne proteins, particularly lipoproteins can interact with micelle surfaces 51-53

and thereby may influence micelle integrity depending on the polymer structures

25, 33

and the type of proteins.25 Lam et al. found that serum albumins and

immunoglobulin gamma have moderate effects on the integrity of the nanoparticles whereas lipoproteins lead to complete nanoparticle dissociation.25 Cheng et al. reported that α-,β-globulins were the prime factors responsible for disassembly of PEG-PLA micelles.28 We found that albumin, α,β-globulin, VLDL and LDL, at the concentrations similar to theirs in the blood, induced about 20% micelle disassembly after 24 h incubation, while the HDL and CM had little effect on micelle integrity (Fig. 2b, 2c), probably because of the large size of chylomicron (>100 nm) and HDL’s compact structure.53 These results lead to an immediate intuition that the blood or serum, which contains all of these proteins, would be more effective in inducing micelle dissociation; very unexpectedly, the micelle degree of PEG-PCL-FRET remained unchanged in serum and blood even after a prolonged incubation (Fig. 2d). This result suggests that it was the interaction of the unimers with the proteins that shifted the equilibrium to the dissociation of the micelles. In blood these proteins are known to carry hydrophobic 20


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cargos including triacylglycerols and cholesterol. Thus, their occupied hydrophobic pockets could no longer harbor these hydrophobic blocks of the unimers. However, in the purified proteins, these hydrophobic components are removed and thus the pockets can accommodate the unimers’ hydrophobic blocks, driving the equilibrium to micelle disassembly. Furthermore, it was recently found that proteins adsorbed on the PEG chain bushes on the nanoparticle surface could further penetrate the PEG layer to the underlying surface.54 Adding MSA-Cy5 to the PEG-PCL-Cy5.5 micelle solution gradually induced FRET (Fig. S9), indicating that the protein may gradually penetrate into the PEG shell of PEG-PCL micelles and interact with the hydrophobic cores, inducing dissociation. For comparison, dilution and adding the proteins did not induce significant dissociation of PEG-PS-FRET micelles (Fig. 2a–c, e). Blood flow in arteries and capillaries produce strong frictional force between blood and endothelium wall, wall-shear stress (WSS).55 Shear-stress-produced drag forces in vascular narrowing was found to induce disassembly of microscale nanoparticles55 and vesicles.56 Thus, not surprisingly, the shear stress applied by the microchannel induced micelle dissociation (Fig. 2f). The integrity of PEG-PCL decreased to less than 20% micelle degree after passing through the microchannel 20 times. As a reference, PEGPS micelles with glassy cores were less sensitive to the shear, remaining near 80% micelle degree after 20 trips through the microchannel. Thus, shear stress promotes more dissociation of PEG-PCL micelles due to their fluidic cores.57 This is in agreement with previous studies that shear stress could impact micelle stability.58, 59 Note that the induced dissociation of the micelles in the microchannel and the bloodstream may not be entirely attributed to shear stress. Other factors such as reflow and turbulence may also contribute significantly. We are conducting a more detailed study of micelle stability. After i.v. injection, PEG-PCL and PEG-PDLLA micelles were rapidly cleared and the micelle degree quickly decreased to near 20% (Fig. 4). One would immediately questions causality: Did fast clearance from the blood cause low polymer concentrations, which consequently induced dissociation and thus the low micelle degrees? Or, did the micelle dissociation into unimers induce fast clearance due to 21


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exposure of the unimers’ hydrophobic segments? The related question is whether the clearance was mainly via the micelles or the unimers. The intuitive and general explanation is the first one. If so, increasing blood polymer concentration would increase the micelle degree in the blood. However, increasing PEG-PCL blood concentrations by high dosing (Fig. 4c) or predosing (Fig. 4d) did not lead to significant increases in the blood micelle degree. Note that the result was not associated with the accelerated-blood-clearance (ABC) phenomenon observed upon repeated injection of pegylated liposomes due to IgM secreted into the bloodstream after the first dose60 as, here, the second injection was just 5 min later. Furthermore, PEG-PCL-FRET was mainly sequestered into the liver, up to 35% injected dose/g of tissue (Fig. 5a,b); even though the spleen also had a higher concentration than other tissues its small size (about of 1/10th of the liver in weight) made it contribute little to the blood clearance. As a comparison, the very stable PEG-PS3K-FRET remained greater than 60% micelle degree in the bloodstream and had a much less liver sequestration (Fig. 5). Thus, the blood clearance was much slower (Fig. 4e) than that of PEG-PCL-FRET. The pretreatments with GdCl3 to inhibit phagocytosis of liver Kupffer cells46 slowed the blood clearance of PEG-PCL-RET and greatly elevated the blood concentrations at all time points (Fig. 4f) due to the inhibited liver sequestration (Fig. 5c,d) but, surprisingly, did not significantly enhance the micelle degree at different times (Fig. 4f). The GdCl3 pretreatments had the same effect on the PEG-PS3K micelles (Fig. 4e). These results indicate that intact micelles were actually not easily captured by Kupffer cells; it was the unimers that were quickly recognized and captured due to their exposed hydrophobic segments. This conclusion was further proven by the low micelle degree of PEG-PCL-FRET in liver (Fig. 6a) but the strong FRET of MSACy5/PEG-PCL-Cy5.5 (Fig. 8a). What was the driving force inducing quick PEG-PCL dissociation? As evidenced in Fig. 7, within 10 min, the injected PEG-PCL-Cy5.5 had already formed FRET complexes with MSA-Cy5. This is much quicker than in the static solution (Fig. S9) and is consistent with shear-facilitated dissociation (Fig. 2e). For comparison, the stable PEG-PS-Cy5.5 micelles formed low FRET with MSA-Cy5 (Fig. 7) and remained 22


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mostly as micelles in the liver (Fig. 6b; Fig. 8b). Therefore, the blood-borne proteins and shear exerted by microvessels induced rapid dissociation of PEG-PCL-FRET micelles. Under shear in microvessels, PEG-PCL micelles with soft cores deformed and easily exposed the hydrophobic core surface, further facilitating adsorption of bloodborne proteins to form PEG-PCL unimer–protein complexes and thus the micelles to dissociate into unimers. It can be concluded that Kupffer cells sequestered unimers rather than micelles; once injected into the bloodstream, the shear and the blood-borne proteins drive PEGPCL micelles to quickly dissociate into unimers, which are then sequestered by Kupffer cells. The low micelle degree of PEG-PCL-FRET in the bloodstream were not due to the blood clearance of polymers, but quick micelle dissociation. Another basic question is whether PEG-PCL micelles can reach the tumor and even tumor cells as intact micelles for drug delivery. Fig. 6 showed that PEG-PCL chains in the tumor were mostly as micelles. Furthermore, we found that PEG-PCLFRET on the cell membrane were as intact micelles. After internalization into the cells, they dissociated into unimers (Fig. 3), indicating that micelles indeed can ship cargos into tumors and their cells. This difference from the previous observation that micelles would quickly dissociate on the cell membrane is probably because, in that study, the dye was physically loaded in the micelles; therefore, it was not due to the micelle dissociation but dye release due to the partition with the cell membrane.28

CONCLUSIONS PEG-polyester micelles are currently used excipients for micellar delivery of cancer drugs. Understanding the stability of polymeric micelles after injection into the blood is critical for rational micelle design as effective drug carriers. Here we show that once i.v. injected, dilution, blood proteins and shear together cause most micelles (>80%) to dissociate into unimers within an hour, which are quickly sequestered by Kupffer cells into the liver. Kupffer cells hardly capture intact micelles. Furthermore, the micelles indeed can arrive in tumors and remain intact until internalization into the cells. This finding indicates that functionalization of micelles with fluidic cores like 23


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PEG-PCL ones by introducing targeting ligands will not be effective since the majority of the micelles quickly dissociate in the bloodstream; micelle stabilization is the priority.

MATERIALS AND METHODS Materials. ε-Caprolactone (CL), stannous 2-ethyl-hexanoate (Sn(Oct)2), dicyclohexyl carbodiimide

(DCC),

N-hydroxysuccinimide

(NHS),

triethylamine

(TEA)

poly(ethylene glycol) monomethyl ether (mPEG2K, Mn ~ 2000), cysteamine and pyrene were purchased from Aladdin Chemical Reagent Co. (Shanghai, China).D, L-Lactide (DLLA) was purchased from Energy Chemical Reagent Co. (Shanghai, China). NHydroxy-succinimide (NHS)-functionalized Cy5 (Cy5-NHS) and Cy5.5 (Cy5.5-NHS) were purchased from GE Healthcare (Piscataway, NJ). Gadolinium chloride (GdCl3) was purchased from Alfa Aesar (Stevenage, UK). Water Soluble gold nanoparticles (AuNP: 10 nm (Citrate)) were purchased from Strem Chemicals Inc. (Newburyport, MA). Paclitaxel was supplied by Chengdu Lanbei Plant Chemical Science and Biotechnology Co. Ltd (Chengdu, China). Mouse serum albumin (MSA), bovine serum albumin (BSA), α,β-predominant globulins, low-density lipoprotein (LDL), high density lipoprotein (HDL), very-low-density lipoprotein (VLDL) and chylomicrons (CM) were purchased from Sigma-Aldrich (St. Louis, MO). Other reagents were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Blood samples are obtained as gifts from the Second Affiliated Hospital of Zhejiang University. PEGPCL and PEG-PDLLA were synthesized and characterized according to the reported method for CL or LA ring-opening polymerization initiated by PEG-OH (2K) in the presence of stannous octoate (0.05 wt %).61 Measurements. Gel permeation chromatography (GPC) measurements were performed on a gel permeation chromatographer equipped with a Schambeck SFD GmbH RI2000 detector and a pair of Shodex KF-402.5 HQ and KF-404 HQ columns. THF was used as eluent at a flow rate of 0.3 mL/min at 40 °C. A series of polystyrene standard samples were used to generate calibration curves. The size and distribution of micelles were measured by dynamic light scattering (DLS) on a Zetasizer Nano ZS 24


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particle sizer (Malvern Instruments Ltd., Westborough, MA). Each measurement was performed in quartz cuvettes three times. The results were processed with Dispersion Technology Software version 5.1. Fluorescence spectra of the micelles were measured on a multifunctional Spectra Max M2/M2e microplate reader (Molecular Devices, San Jose, CA) with an excitation at 640 nm (Cy5) or 680 nm (Cy5.5). The critical micelle concentration (CMC) was measured by monitoring pyrene’s fluorescence emission spectrum.62 Synthesis of PEG-block copolymers with terminal primary amine groups (Supporting Information Scheme S1–S3). PEG-PCL or PEG-PDLLA having a terminal hydroxyl group at the PCL or PDLLA end was synthesized according to the literature.61 The obtained polymer (1 g) and glutaric anhydride (0.14 g) were dissolved in 10 mL THF with 2 mL pyridine and stirred at room temperature for 24 h. The polymer with a terminal carboxylic acid group (PEG-PCL-COOH) was obtained after three rounds of precipitation. PEG-PCL-COOH (1 g), DCC (0.4 g) and NHS (0.3 g) were dissolved and stirred in 20 mL THF. After the solution was stirred at 40 °C for 12 h and then cooled to room temperature, ethylenediamine (0.1 mL) was added and the solution was stirred at room temperature for another 24 h. The solution was filtered and the filtrate was precipitated in a large excess of diethyl ether twice to give PEG-PCL or PEG-PDLLA with a terminal amine group at the hydrophobic ends (PEG-PCL-NH2 or PEG-PDLLANH2). PEG-PS diblock copolymers were synthesized by atom-transfer radical polymerization of styrene using mPEG 2-bromoisobutyrate (PEG-Br) as macroinitiator according to the reported method (Supporting Information Scheme S3).63 The PS block was controlled to be 2 or 3.5 KDa. The polymers were purified by passing an activated Al2O3 column to remove the catalyst and then precipitation in n-hexane. The PEG-PS diblock copolymer was dried in a vacuum oven overnight at room temperature. The terminal bromo-group in the PS block was reacted with cysteamine to introduce the terminal primary amine group. Briefly, PEG-PS (1.1 g) and cysteamine (0.5 g) were dissolved in DMF and stirred at room temperature overnight for 12 h in 25


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the presence of a catalytic amount of DBU. The polymer was precipitated in ether and then dissolved in dichloromethane. The solution was washed with brine with 0.1 M HCl, and then dried over magnesium oxide and precipitated in ether. Labeling the copolymers with fluorescent dyes. Cy5 or Cy5.5-labeled copolymers were synthesized as follows. Briefly, a solution of Cy5-NHS or Cy5.5-NHS in DMSO (1 mg/mL) was added gradually into a solution of the copolymer in DMF (1 mg/mL) while stirring. The weight ratio of the fluorescent dye to the copolymer was kept at 1:100 (w/w). The solution was stirred for 12 h in the dark and the polymer was precipitated in ether. To remove the unconjugated fluorescent dye, the copolymer was dissolved in THF and dialyzed in the dark against deionized water/THF (v/v 1/9) until no fluorescence was detected in the dialysate. The resultant Cy5- or Cy5.5-conjugated polymers (Polymer-Cy5 or Polymer-Cy5.5) were lyophilized in a freeze dryer. The content of the dye in each polymer was determined using their fluorescence intensity against the corresponding standard curve. FRET-micelle fabrication. A typical fabrication procedure is as follows. PEG-PCL, PEG-PCL-Cy5 and PEG-PCL-Cy5.5 (total 10 mg, 1% polymer chain containing Cy5, 1% polymer chain containing Cy5.5) were dissolved in 0.1 mL DMF. The solution was slowly dropped into 10 mL DI-water with stirring. The DMF was removed by dialyzed against DI water until no DMF was detected. The FRET micelles from other polymers were made similarly. Fluorescence spectra and FRET ratio calculation. The fluorescence spectra of FRET micelles were measured using a fluorescence spectrometer at an excitation wavelength of 640 nm with a slit width of 2 nm, integration time of 0.1 s and increment of 0.5 nm. Emission spectra were collected from 640 to 740 nm. The FRET ratio was calculated from the intensities of the fluorescence at 680 nm and 720 nm. Fluorescence quenching experiment. The fluorescence quenching by gold nanoparticles was carried out according to the method reported in the literature.64 The AuNP solution (10 nm, 1 mg/mL, 10–160 µL) was added to the polymeric micelle 26


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solution (1 mg/mL, 1 mL) in a cuvette using a microsyringe and mixed with stirring. After 30 seconds, the fluorescence emission of the solution was recorded with excitation at 640 nm. Determination of FRET efficiency of micelles under shear. A simple straight microfluidic channel with square cross section to mimic microscopic blood vessels was fabricated by soft lithography.65 Briefly, negative photo resist SU-8 (MicroChem, Newton, MA) was spin-coated on 4 inch silica wafers (Silicon Inc. Boise Idaho) and exposed under UV light (Ominicure S2000 curing system) through a transparent photomask (CAD/ART Services, Bandon, OR) of 20,000 dpi resolution. The photoresist master was developed in 1-methoxy-2-propanol acetate (Sigma-Aldrich, St. Louis, MO) and baked in an oven at 135 °C overnight. Poly(dimethylsiloxane) (PDMS) (Sylgard 184, Dow Corning, Midland, MI) solution was poured on the photoresist master, degassed for one hour and cured overnight at 70 °C. The cured PDMS replicas were peeled off the master wafers and punched with a 20 G needle to make inlet holes. Treated by oxygen plasma, the PDMS replicas were bound to glass slides to make the intact microfluidic devices. The channel was 3 cm long and had equal cross-sectional width and height, 40 μm. The microchannel was connected to a syringe pump (Model KD100, KD Scientific, New Hope, PA). The PEG-PCL-FRET micelles were dissolved in fresh mouse blood in heparin containing tubes (1 mg/mL). The solution was loaded in the syringe pump passed from the microchannel at flow rates ranging from 0.1 mL·h−1 to 10 mL·h−1 at 37 °C. The aliquot eluting from the microchannel was collected and its fluorescence intensity excited at 640 nm or 680 nm was recorded and the FRET ratio was calculated. Each experiment was run in triplicate. Cell culture. HepG2 cells, a human hepatic cancer cell line, were obtained from American Type Culture Collection (Rockville, MD). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 and grown continuously in DMEM medium supplemented with 10% heat-inactivated FBS (Genom Biological Technology Co., Ltd. Hangzhou, China), 100 unit/mL penicillin and 100 μg/mL streptomycin. 27


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Animals. Female ICR strain mice (6–8 weeks old) and BALB/c nude mice were purchased from the Animal Center of Zhejiang Academy of Medical Sciences. The use of animals was approved by the Animal Ethics Committee of Zhejiang Academy of Medical Sciences. Mice were housed in approved animal-care facilities on a 12 h light/dark cycle and given ad libitum access to food and water. Fluorescence imaging and spectra recording of micelles cultured with cells. For high-resolution imaging of the cells cultured with FRET micelles, 1105 HepG2 cells in 1 mL of growth medium were seeded into a glass-bottomed dish (Nest Biotechnology Co., Ltd., Wuxi, China) and incubated for one day to allow cell adherence and confluence. Before each experiment, the cells in 0.9 mL of culture medium without FBS were supplemented with 100 μL of micelles (1 mg/mL) and incubated at 37 °C for the timed intervals. Before imaging, the cells were washed with PBS buffer. The cells cultured with single-dye-labeled micelles (PEG-PCL-Cy5 or PEG-PCL-Cy5.5) were used to validate their fluorescence spectra recorded by the confocal microscopy. The fluorescent imaging was performed on a LSM710NLO confocal microscope (Zeiss, Oberkochen, Germany). A 633-nm HeNe laser was used to excite Cy5. The fluorescence spectra of randomly selected sites on the cell membranes or intracellular sites were recorded. The images were then separately taken from the Cy5 or Cy5.5 fluorescence channel. Raw images in lsm format were converted to 8-bit tiff files with pseudocolors for display. Only brightness and contrast were optimized, and no other image processing was used. Assessment of overall micelle degree of micelles cultured with cells. PEG-PCLFRET (100 µg/mL polymer) were incubated with HepG2 cells (24-well plates, 105 cells/cm2, 500 µL of serum-free media per well) for timed intervals. At predesignated time, the cells were washed once with an acidified saline (0.5 M NaCl, 0.2 M CH3COOH, pH 2.5) and twice with phosphate-buffered saline (pH 7.4), and then detached by EDTA, transferred to Eppendorf tubes, and pelleted by centrifugation. The cells were resuspended in PBS solution and transferred to polypropylene 96-well plates. Their emission fluorescence spectra excited at 640 nm were recorded using a 28


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SpectraMax M2/M2e microplate spectrofluorometer. The FRET ratio was calculated from the fluorescent intensities at 680 nm and 720 nm and the micelle integrity was calculated accordingly. Measuring in vivo fluorescence and FRET fluorescence of micelles in mice. The FRET-micelle solution in PBS (200 µL, 1 mg/mL, 10 mg/kg) was injected into ICR strain mice via the tail vein. The blood was sampled (200 µL) from the eye socket vein at timed intervals and stored in heparin-containing Eppendorf tubes. The plasma was separated from the blood by centrifuge at 5,000 rpm for 10 min at 4 °C, and its fluorescence spectra excited at 640 nm or 680 nm were separately recorded. The concentration of the micelles remaining in the blood was calculated from the fluorescence of Cy5.5 and their FRET ratio was calculated from the spectra excited at 640 nm. In vivo and ex vivo fluorescent imaging on a tumor-bearing mouse model. The BALB/c nude mice were subcutaneously inoculated with HepG2 tumor cells (2×106 cells) on their right flanks. When the tumors reached about 100 mm3, the mice were injected with 200 µL of FRET micelles at 10 mg/kg via the tail vein. Whole-body optical imaging was performed at 0–6 h post injection on a Kodak In-Vivo FX Professional Imaging System (New Haven, CT) equipped with fluorescent filter sets (excitation/emission, 640/700 nm). The camera was set with the maximum gain, 2 × 2 binning, 1024 × 1024 pixel resolution, and an exposure time of 30 s. Prior to imaging, the mice were anesthetized by intra-abdominal injection of 1% pentobarbital sodium (45 mg/kg). The mice were sacrificed 6 h after injection. Major organs and tumors were excised and washed with 0.9% saline. Their ex vivo images were taken using the system as described above. Confocal fluorescent imaging liver and tumor sections. The BALB/c nude mice bearing subcutaneous HepG2 tumors were injected with FRET micelles via the tail vein at a single dose of 10 mg/kg. At 2 h post-treatment, the mice were sacrificed. The tumor and the liver were excised and embedded in Tissue-Tek OCT compoundTM, frozen in liquid nitrogen, and sectioned into 10 µm thick slices. 29


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The fluorescent imaging of the tissues was performed on a LSM710NLO confocal microscope. A 633-nm HeNe laser was used to excite Cy5. The fluorescence spectra of randomly selected sites of the tissues were recorded. The images were then separately taken from the Cy5- or Cy5.5-fluorescence channel. Raw images in lsm format were converted to 8-bit tiff files with pseudocolors for display. Only brightness and contrast were optimized, and no other image processing was used. Biodistribution of polymers. BALB/c nude mice inoculated with subcutaneous HepG2 tumors (~ 70 mm3) were injected with 200 µL of FRET micelles at 10 mg/kg via the tail vein. Each group (n = 4) was sacrificed at 2 h, 6 h or 24 h after the injection. Major organs and tumors were excised and washed with 0.9% saline, and then weighed and homogenized in medium containing 1 mg/mL unlabeled PEG-PCL. The polymer concentrations in the solutions were calculated from the Cy5.5-fluorescence intensity excited at 680 nm according to the established calibration curve. Real-time FRET fluorescence imaging of micelles with serum albumin in blood vessel. The nude mice bearing subcutaneous HepG2 tumors were anesthetized using 1% pentobarbital sodium solution. The skin along with the abdominal midline was fixed on a microscope slide using Histoacryl (3M VetbondTM, USA). Attention must be paid not to break the major blood vessels. Under the LSM710NLO confocal microscope, one clear blood vessel was identified. The Cy5 labeled-mouse serum albumin (MSACy5, the labeling molar rate is about 2.05%) (100 µL, 20 mg/mL) was injected into the mouse via the tail vein. Five minutes later, the PEG-PCL-Cy5.5 or PEG-PS-Cy5.5 micelle solution (200 µL, 1 mg/mL) was injected via the tail vein. At timed intervals, the 640–730 nm fluorescence spectra of the blood excited with an 633-nm HeNe laser were recorded and the corresponding images were taken from the 720 nm fluorescence channel. At 2 h post-administration, the mice were sacrificed. The tumor and the liver were excised and embedded in Tissue-Tek OCT compoundTM, frozen in liquid nitrogen, and sectioned into 10 µm thick slices to measure the fluorescence imaging and fluorescence spectra by confocal microscopy.

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ASSOCIATED CONTENT The authors declare no competing financial interest. Supporting Information Details of polymer synthesis; characterization; fluorescence quenching curves; confocal fluorescence images and fluorescence spectra for PEG-PCL-Cy5, PEG-PCLCy5.5, PEG-PS-Cy5.5 and PEG-PS-Cy5 micelles; the whole cell fluorescence spectra for PEG-PCL-FRET micelles with different time points; the blood clearance and micelle degrees for i.v. injected with PEG-PDLLA-FRET.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Xuanrong Sun: 0000-0001-5414-9300 Guowei Wang: 0000-0003-1369-8738 Hao Zhang: 0000-0002-5861-1619 Xin Liu: 0000-0002-2339-3973 Jianbin Tang: 0000-0003-4498-5705 Youqing Shen: 0000-0003-1837-7976 Author Contributions ‡

Xuanrong Sun and Guowei Wang contributed equally to this work.

ACKNOWLEDGEMENTS The authors thank the National Basic Research Program (2014CB931900) and the National Natural Science Foundation (U1501243, 51390481 and 21506192) for financial supports. The authors declare no competing financial interest.

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