Fabrication of Conjugated Amphiphilic Triblock Copolymer for Drug

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Fabrication of Conjugated Amphiphilic Triblock Copolymer for Drug Delivery and Fluorescence Cell Imaging Xuezhi Zhao, Kaicheng Deng, Fangjun Liu, Xiaolong Zhang, Huiru Yang, Jinlei Peng, Zengkui Liu, Liwei Ma, Baoyan Wang, and Hua Wei ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00991 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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ACS Biomaterials Science & Engineering

Fabrication of Conjugated Amphiphilic Triblock Copolymer for Drug Delivery and Fluorescence Cell Imaging

Xuezhi Zhao, Kaicheng Deng, Fangjun Liu, Xiaolong Zhang, Huiru Yang, Jinlei Peng, Zengkui Liu, Liwei Ma, Baoyan Wang, Hua Wei*,

State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, and College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000, China

*Corresponding author E-mail address: [email protected] (H. Wei)

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Abstract：An elegant integration of light-emitting segments into the structure of polymeric delivery systems endows the resulting self-assembled nano-vehicles with the diagnostic ability toward an enhanced therapeutic efficiency. A variety of polyfluorene (PF)-based binary delivery systems have been designed and developed successfully, but PF-based ternary formulations remain rarely explored likely due to the synthetic challenge. To develop a universal synthesis strategy toward linear conjugated amphiphilic triblock copolymer for cancer theranostics, herein we focused on the functionalization of the PF terminus for further chain extension, and prepared well-defined

PF-based

amphiphilic

PF-b-poly(ε-caprolactone)-b-poly(oligo(ethylene methacrylate)

triblock glycol)

copolymers,

monomethyl

ether

(PF-b-PCL-b-POEGMA), by integrated state-of-the-art polymer

chemistry techniques including Suzuki reaction, ring-opening polymerization (ROP), atom transfer radical polymerization (ATRP) and click coupling. The resulting conjugated amphiphilic triblock copolymers can self-assembly into core-shell-corona (CSC) micelles with PF block constructing the inner hydrophobic core for fluorescent tracking, PCL segment forming the hydrophobic middle shell for drug encapsulation, and POEGMA moiety building the hydrophilic outer corona for particulate stabilization. Interestingly, the CSC micelles with hydrophobic PCL middle layer show a greater drug loading capacity as well as a higher fluorescence quantum yield (Φ) relative to the core-shell micelles self-assembled from the control of PF-b-POEGMA diblock copolymers without PCL sequence due to the more hydrophobic spaces and well-separation of PF sequence provided simultaneously by the PCL central block. The efficient cellular uptake of the anticancer drug, doxorubicin (DOX)-loaded CSC micelles together with the in vitro cytotoxicity against the HeLa cells makes the conjugated amphiphilic triblock copolymers developed herein a promising platform for simultaneous cell image and drug delivery, thus offering great potential for cancer theranostics.

Keywords: Polyfluorene; amphiphilic triblock copolymers; controlled drug delivery; cell imaging ACS Paragon Plus Environment

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Introduction Nanoscaled polymeric drug delivery systems such as liposomes, micelles, and dendrimers have drawn much attention from both fundamental research and biomedical/pharmaceutical industries.1,2 Notably, great efforts have been made in recent years to develop the nano-vehicles integrating both imaging modalities and delivery of anti-cancer drug for cancer diagnosis and therapy, also termed as “theranostics”.3-8 Among the various imaging contrast agents, the organic fluorophores have been extensively applied for both in vitro and in vivo bioimaging studies due to their high sensitivity and resolution, tunable optical properties for multiplexed imaging, the capability for long-term tracking9,10 and relatively low toxicity in contrast to the inorganic materials.11-13 A further screening of the organic fluorophores by the imaging properties reveals that the polymeric fluorophores with superiorly optical properties relative to the small molecule fluorophores, such as higher absorption coefficiency, brighter fluorescence and enhanced photostability have attracted considerable interests for bioimaging applications.14-19 Conjugated polymers, due to their high quantum yields and molar absorptivity, tunable properties, easy functionalization, and photostability,20-26 have been extensively explored as the building blocks of the constructed nano-carriers for various advanced applications including in vivo imaging, cell labeling, oxygen sensing, and delivery of therapeutic agents.27-33 Polyfluorene (PF) is known as a high-performance conjugated polymer with excellent chemical and thermal stability.34 Unfortunately, most PF derivatives are only soluble in the organic solvents which



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apparently limits their applications in a water-based biological environment. This problem can be addressed by integrating a hydrophilic polymer chain with the PF block for the preparation of an amphiphilic copolymer. Great efforts have been made in this research field, and a variety of PF-based binary (e.g. diblock copolymers) delivery systems have been designed and developed successfully.35,36 However, PF-based ternary (e.g. triblock copolymers) formulations remain rarely explored likely due to the synthetic challenge. Notably, Wang et al. recently prepared conjugated bottlebrush-like amphiphilic copolymers by decorating the side chain of fluorescent

poly(fluorene-alt-(4,7-bis(hexylthien)-2,1,3-benzothiadiazole))

(PFTB)

backbone with polycaprolactone (PCL) and poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) grafts.3 To develop a universal synthesis strategy towards linear conjugated amphiphilic triblock copolymer for cancer theranostics, herein we focused on the functionalization of the PF terminus for further chain extension, and prepared well-defined PF-based amphiphilic triblock

copolymers,

PF-b-poly(ε-caprolactone)-b-poly(oligo(ethylene

monomethyl ether methacrylate)

glycol)

(PF-b-PCL-b-POEGMA), by a combination of

state-of-the-art synthetic techniques including Suzuki reaction, ring-opening polymerization (ROP), atom transfer radical polymerization (ATRP) and click coupling.

The

resulting

conjugated

amphiphilic

triblock

copolymers

can

self-assembly into core-shell-corona (CSC) micelles. The PF moiety can act as a blue-light emitting fluorophore presenting a good imaging effect in cells. The hydrophobic PCL middle layer endow the CSC micelles with a greater drug loading


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capacity as well as a higher fluorescence quantum yield (Φ) relative to the core-shell micelles self-assembled from the control of PF-b-POEGMA diblock copolymers without PCL sequence. An anticancer drug, doxorubicin (DOX) was loaded in the micelles for anti-cancer drug delivery. The performance of the resulting conjugated amphiphilic triblock copolymers on theranostics for both cancer imaging and therapy was evaluated by fluorescence microscopy and in vitro cytotoxicity study.

Experimental Section Materials ε-Caprolactone (CL) (Sigma-Aldrich) was dried over CaH2 and distilled under reduced pressure prior to use. Oligo (ethylene glycol) monomethyl ether methacrylate (OEGMA, Mn=300 g/mol with 4~5 pendent EO units) from Sigma-Aldrich was purified by passing through a basic alumina column to remove the inhibitor. 2,7-dibromo-9,9-di-n-hexylfluorene,

n-butyllithium,

2-iso-Propoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, tetrakis(triphenylphosphine)-palladium(0), 4-Bromobenzyl alcohol, Sodium carbonate were

purchased

from

J&K

and

used

as

received.

N,N,N',N″,N″-pentamethyldiethylenetriamine (PMDETA) was supplied by Aladdin. Phenylboronic acid, stannous (II) octanoate (Sn(Oct)2), 2-bromoisobutyryl bromide, triethylamine (TEA), copper (I) bromide (CuBr), dimethylacetamide (DMAc), and bipyridine

(bpy)

were

purchased

from

Sigma-Aldrich.

Ethylene

glycol,

tetrahydrofuran (THF), propargyl alcohol were purchased from Tianjin Chemical



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Reagent Factory (China). Sodium azide (NaN3, Sanyou, Shanghai), anisole (Kelong, Chengdu, China) and other reagents were used as received without further purification. 9,9-Dihexyl-2-bromo-7-4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl

fluorene

was

synthesized according to the reported procedures.37

Preparation of Hydroxy-Functionalized Polyfluorene (PF-OH). The general procedure of polymerization was carried out through the Suzuki coupling

reaction.38

9,9-Dihexyl-2-bromo-7-4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl fluorine (1.69 g, 3.15 mmol), sodium carbonate (1.82 g , 17.20 mmol), Pd(PPh3)4 (60.26 mg , 0.052 mmol) and 4-Bromobenzyl alcohol (385.59 mg, 1.89 mmol ) were dissolved in DMAc (25 ml). After three freeze-pump-thaw cycles, the mixture was vigorously stirred at 120 °C for 72 h, and then phenylboronic acid (1.92 g , 15.75 mmol) was added. After another 12 h, the mixture was cooled to room temperature and later filtrated in methanol and desiccated in vacuum.

Synthesis of Bromide-Functionalized Polyfluorene (PF-Br). PF-Br was synthesized by esterification of PF-OH with excess 2-bromoisobutyryl bromide. PF-OH (0.62 g, 0.15 mmol) and TEA (0.15

g, 1.48

mmol)

were dissolved in 16 ml of dry dichloromethane (DCM) and cooled in an ice bath. 2-B romoisobutyryl bromide (0.34 g, 1.5 mmol) was added and the mixture was stirred at 0

°C for 30 min, followed by stirring at room

reaction temperature

for another 72 h. The reaction mixture was later concentrated by vacuum evaporation,


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and further dissolved in THF. The insoluble quaternary ammonium salts were removed by filtration, the filtrate was added dropwise into methanol to precipitate the crude product. The product was purified by re-dissolving/precipitating in THF/methanol for three times, and further dried under vacuum (yield, 92%).

Synthesis of Azide-Functionalized Polyfluorene (PF-N3) PF-Br (0.66 g, 0.15 mmol) and NaN3 (99.23 mg, 1.52 mmol) were dissolved in DMF (10 ml) in a round-bottom flask equipped with a magnetic stirrer, the reaction mixture was stirred at 45 oC for 48 h, the mixture was added dropwise into excess ice-cold methanol to precipitate the crude product. The product was purified by re-dissolving/precipitating in THF/methanol for three times, and further dried under vacuum (yield, 90%).

Synthesis of Amphiphilic Block Copolymers (Alkyne-PCL-b-POEGMA) The synthetic procedures of alkyne-PCL-b-POEGMA was identical to the preparation of 4-arm star-shaped PCL-POEGMA block copolymer reported in our previous study except using propynol as the starting initiator.39 The yield of alkyne-PCL-OH, alkyne-PCL-Br, and alkyne-PCL-b-POEGMA is 86%, 92%, and 83%, respectively.

Preparation of the Conjugated Amphiphilic Triblock Copolymers (PF-b-PCL-b-POEGMA) by Click Coupling



PF-b-PCL-b-POEGMA was prepared by click reaction using CuBr/PMDETA as the catalyst. Typically, PF-N3 (51.32 mg, 0.011 mmol), Alkyne-PCL-b-POEGMA (100.66 mg, 0.005 mmol) and PMDETA (4.66 mg, 0.026 mmol) were dissolved in a 20/80 (v/v %) alcohol/THF mixed solvent (4 ml).

After three freeze-pump-thaw

cycles, CuBr (3.86 mg, 0.026 mmol) was loaded under the nitrogen atmosphere. The reaction mixture was subjected to another three freeze-pump-thaw cycles, which was later sealed and placed in an oil bath thermostated at 45 oC for 48 h. Thereafter, the reaction was exposed to air and diluted with THF followed by precipitation in excess ice-cold n-hexane to harvest the crude product. To remove excess PF-N3, the mixture was dissolved in 2 ml of DMF, placed in a dialysis bag (MWCO, 3.5 kDa) and dialyzed against distilled water for 24 h. Formation of precipitates was observed on the bottom of the dialysis tube due to the phase separation of unreacted insoluble PF from the aqueous phase. After filtration under reduced pressure, the purified PF-b-PCL-b-POEGMA was obtained by freeze-drying (yield, 87%).

Synthesis of Alkyne -Br Alkyne-Br was synthesized by esterification of propynol with 2-bromoisobutyryl bromide similar to the synthesis of PF-Br mentioned earlier. The crude product was purified by column chromatography using 10/1 (v/v) petroleum ether/ethyl acetate as the eluent (Rf = 0.16). The purified product was isolated by evaporation of the solvents and further dried in a vacuum oven (yield, 88%).


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Synthesis of Alkyne-POEGMA Alkyne-POEGMA was prepared by ATRP of OEGMA using Alkyne-Br as an initiator. Typically, Alkyne-Br (52.41 mg, 0.27 mmol), bpy (86.18 mg, 0.54 mmol) and OEGMA (4.14 g, 13.79 mmol) were dissolved in anisole (27.59 ml). After three freeze–pump–thaw cycles, CuBr (39.58 mg, 0.27 mmol) was added under the nitrogen atmosphere. The reaction mixture was sealed after another three freeze–pump–thaw cycles, and placed in an oil bath thermostated at 60 oC. After 8 h, the reaction was exposed to air and diluted with THF. The crude product was collected by precipitation in excess ice-cold n-hexane. the precipitates were dissolved in 2 ml of DMF, placed in a dialysis tube (MWCO, 3.5 kDa) and later subjected to dialysis against distilled water for 24 h to remove the copper catalyst and any unreacted monomer. The purified Alkyne-POEGMA was harvested by freeze-drying (yield, 89%).

Preparation of the Conjugated Amphiphilic diblock Copolymer (PF-b-POEGMA) by Click Coupling PF-b-POEGMA

was

prepared

by

click

coupling

between

PF-N3

and

alkyne-POEGMA. The synthetic procedures were identical to the preparation of PF-b-PCL-b-POEGMA mentioned earlier. The yield of PF-b-POEGMA is 80%.

Characterization of Polymers



1

H NMR spectra were recorded on a JEOL-ECS 400M nuclear magnetic resonance

instrument using CDCl3 as the solvents and TMS as the internal reference. The molecular weight and molecular weight distribution of the polymers were determined by the size exclusion chromatography and multi-angle laser light scattering (SEC-MALLS). SEC using HPLC-grade DMF containing 0.1 wt% LiBr at 60 oC as the eluent at a flow rate of 1 mL/min. Tosoh TSK-GEL R-3000 and R-4000 columns (Tosoh Bioscience) were connected in series to a Agilent 1260 series (Agilent Technologies), an interferometric refractometer (Optilab-rEX, Wyatt Technology) and a MALLS device (DAWN EOS, Wyatt Technology). The MALLS detector was operated at a laser wavelength of 690.0 nm.

Preparation and Characterization of the Micelles Taking PF12-b-PCL44-b-POEGMA45 as an example, PF12-b-PCL44-b-POEGMA45 (1.5 mg) in 1 ml of DMF was dialyzed against distilled water for 24 h to obtain a micelle solution with an concentration of 0.25 mg/ml. The TEM images were recorded on a JNM-2010 instrument operating at an acceleration voltage of 200 keV. The specimens for TEM observation were prepared by placing a drop of micelle solution onto a carbon-coated copper grid. After deposition, excess solution was removed using a strip of filter paper. The sample was further stained using phosphotungstic acid (2% w/w) and dried in air prior to visualization.


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Dynamic light scattering (DLS) was used to determine the average hydrodynamic size of micelles on a Zetasizer (Nano ZS, Malvern, Worcestershire, UK). The detection angle was fixed at 173o. The polymer solution was passed through a Millipore 0.45 µm pore-sized syringe filter prior to measurements. The polymer solutions with a concentration of 0.1 mg/ml were used.

Critical Micelle Concentration (CMC) Determination Fluorescence spectra were acquired on a LS55 luminescence spectrometer (Perkin-Elmer, Waltham, MA, USA). A stock solution of polymer was serially diluted with DI water with the concentrations ranging from 10-5 to 10-2 mg/ml. Excitation was carried at 360 nm, and emission spectra were recorded ranging from 350 to 500 nm. Both excitation and emission bandwidths were 10 nm. From the emission spectra, the intensity (peak height) of the I420 was analyzed as a function of the polymer concentrations. A CMC value was determined from the intersection of the tangent to the curve at the inflection with the tangent through the points at low concentration.

Spectral Feature Evaluation Absorption spectra of polymers were evaluated on a PerkinElmer Lambda 35 UV-vis spectrometer (Perkin-Elmer, Waltham, MA, USA). Phosphate buffer solution (PBS, pH 7.4, 150mM) and THF were used as the solvent, and the concentrations of the solution were 1 mg/mL. Fluorescence spectra were recorded on a LS55 luminescence spectrometer (Perkin-Elmer, Waltham, MA, USA). The concentrations



Page 12 of 42

of the polymer solution were 0.002 mg/ml in PBS 7.4 and 0.0001 mg/ml in THF. Excitation was carried at 360 nm, and emission spectra were recorded ranging from 350 to 600 nm. Both excitation and emission bandwidths were 10 nm. Fluorescence quantum yields (relative values) of samples were calculated according to the following expression:

ΦS =

FS (1 − 10 − AR ) η S2 × × × ΦR FR (1 − 10 − AS ) η R2

(1)

Where the subscripts R and S refer to the reference and the sample, respectively, Φ is the fluorescence quantum yield and ΦR is equal to 0.55, F is the integrated fluorescence intensity under fluorescence emission spectrum, A is the absorbance at the excitation wavelength and ƞ is the refractive index of the solvent.

In Vitro Drug Loading and Drug Release DOX•HCl (1 mg) and TEA (0.26 g) were dissolved in 2 ml of DMSO and stirred overnight in dark at room temperature to obtain free DOX base. Next, the PF12-b-PCL44-b-POEGMA45 (10 mg) in 2 ml of DMSO was added to the above DOX solution. After stirring at room temperature for 1 h, the above mixture was added dropwise into 4 ml of ultra purified water under vigorous stirring. After stirring for another 1 h, the solution was dialyzed against 5 L of distilled water for 24 h, during which the water was refreshed every 8 h. Finally, the drug-loaded micelles were harvested by freeze-drying. To determine the drug loading content (DLC) and entrapment efficiency (EE), the freeze-dried drug-loaded micelles were re-dispersed in PBS (pH 7.4). The concentration of DOX was determined on a Lambda 35 UV-Vis


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spectrometer (Perkin-Elmer) at A485

nm.

The drug loading content (DLC) and the

encapsulation efficiency (EE) was calculated according to the following formula: DLC (%) = Wdrug loaded in particles / Wparticles × 100%

(2)

EE (%) = Wdrug loaded in particles / Wdrug fed for encapsulation × 100%

(3)

In vitro drug release study was investigated in PBS (pH 7.4, 150 mM) and saline sodium citrate (SSC, pH 5.0, 150 mM) at 37 °C. The freeze-dried drug-loaded nanoparticles was re-dispersed in the PBS buffer to prepare a drug-loaded micelle solution with a concentration of 0.5 mg/ml. 1 ml of the solution was transferred to a dialysis bag, and then immersed in a tube containing 25 ml of release medium. The tube was kept in a horizontal laboratory shaker thermostated at a constant temperature of 37 °C and a stirring speed of 120 rpm. At predetermined time intervals, 3 ml of release medium was taken out and replenished with equal volume of fresh medium. The drug concentration was determined by measuring A485 nm using a calibration curve. The amount of DOX released in PBS (pH 7.4) or SSC (pH 5.0) was determined by UV-Vis spectrometer. The experiment was performed in triplicate for each sample.

Cell Imaging HeLa cells were seeded in 6-well plates at a plating density of 5×105 cells per well in 1 ml of complete growth medium and incubated in a 37 °C, 5% CO2 environment for 24 h. Solutions of polymer, DOX-loaded micelle and free DOX were prepared in complete growth medium at concentrations equal to 25% of their respective IC50



Page 14 of 42

values, and were later added to the wells and incubated for 24 h at 37 °C. After incubation, cells were rinsed with PBS and fixed with 4% paraformaldehyde (PFA) solution for 20 min at room temperature. Finally, cells were counterstained with acridine orange (AO). Coverslips were mounted onto glass slides and imaged using a Nikon A1R confocal microscope.

Cell Viability Study The cytotoxicities of various formulations were evaluated in vitro using the MTS assay. The cells were seeded in 96-well plates at a density of 2500 cells per well in 100 µl of complete growth medium and incubated in a 37 °C, 5% CO2 environment for 24 h. Samples were prepared in serial dilutions in water and then diluted 10-fold in OptiMEM medium (Invitrogen). The cells were then rinsed once with PBS and incubated with 40 µl of the sample solutions with different polymer or Dox concentrations at 37 °C for 4 h. Cells were then rinsed with PBS, and the medium was replaced

with

100

µl

of

culture

medium.

At

24

h,

20

µl

of

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra zolium(MTS, Promega) reagent was added to each well. Cells were then incubated at 37 °C, 5% CO2 for 3 h. The absorbance of each well was measured at 490 nm on a Tecan Safire2 plate reader (Männerdorf, Switzerland). Cell viability for each treatment condition was determined by normalizing to the cells only signal.

Results and Discussion


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Synthesis and Characterization of the PF-b-PCL-b-POEGMA Copolymers Well-defined conjugated amphiphilic triblock copolymer, PF-b-PCL-b-POEGMA with two different compositions was prepared in a three-step approach including: (a) synthesis of Polyfluorene (PF-OH) through a Suzuki coupling followed by esterification of PF-OH with excess 2-bromoisobutyryl bromide to prepare PF-Br and azidotion of PF-Br with excess NaN3 to obtain PF-N3 (Scheme 1a), (b) synthesis of alkyne-PCL-OH by Sn(Oct)2-catalyzed bulk ROP of ε-CL using propynol as the initiators

followed

by

esterification

of

alkyne-PCL-OH

with

excess

2-bromoisobutyryl bromide to prepare alkyne-PCL-Br and ATRP of OEGMA using alkyne-PCL-Br as the macroinitiator to obtain alkyne-PCL-b-POEGMA (Scheme 1b), (c)

generation

of

the

PF-b-PCL-b-POEGMA

target by

conjugated click

amphiphilic

coupling

alkyne-PCL-b-POEGMA (Scheme 1c).


triblock

between

copolymer,

PF-N3

and


Scheme 1. Synthesis of (a) PF-N3, (b) alkyne-PCL-b-POEGMA, and (c) PF-b-PCL-b-POEGMA.

The molecular weights (MWs), polydispersity indexs (PDIs) and degree of polymerizations (DPs) of all synthesized polymers were determined by 1H NMR and SEC-MALLS analyses. The PF was prepared through a Suzuki coupling, the typical 1H NMR spectra of PF-OH, PF-Br and PF-N3 are presented in Figure 1. The DP of PF was determined to


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be ~12 by comparing the integrated intensity of peak c assigned to the methylene protons adjacent to the terminal hydroxyl group to that of peak a attributed to the aromatic protons (Figure 1a). Successful synthesis of the PF-OH by Suzuki coupling is further confirmed by the SEC analysis using tetrahydrofuran (THF) as an eluent and polystyrene (PS) as the calibration standards (Figure S1), which reveals a unimodal elution peak with narrow distribution (Mn= 4.23 kDa, PDI= 1.07). Next, the complete esterification was confirmed by a clear shift of the resonance signal of methylene protons adjacent to the terminal hydroxyl groups in PF-OH from 4.77 ppm (Figure 1a) to a position at 5.28 ppm in the lower field (Figure 1b) and the appearance of a new peak d (methyl protons of 2-bromoisobutyryl moieties) at 1.96 ppm (Figure 1b). In addition, the ratio of the integrated intensity of peak d to that of the characteristic peaks of PF repeating units further confirms almost quantitative conversion (Figure 1b). Finally, the successful azidotion of the PF terminuses is characterized by the FT-IR analysis, which reveals clearly the presence of the characteristic peak at approximately 2100 cm−1 attributable to the azide group after conversion (Figure S2). More importantly, the full conversion of the terminal bromine function to an azide terminus was verified by a clear shift of the peak d from 1.96 ppm (Figure 1b) to a position at 1.50 ppm in the higher field (Figure 1c).



Page 18 of 42

Figure 1. 1H NMR spectra of (a) PF-OH, (b) PF-Br and (c) PF-N3 in CDCl3.

We prepared a series of alkyne-functionalized PCL-b-POEGMA with different DPs of PCL and POEGMA. Taking alkyne-PCL44-b-POEGMA45 as an example, the typical

1

H

NMR

spectra

of

alkyne-PCL-OH,

alkyne-PCL-Br

and

alkyne-PCL-POEGMA are presented in Figure 2. The DP of PCL and POEGMA was determined to be ~44 and ~45, respectively, based on the analysis of 1H NMR data reported in our previous study.39


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Figure 2. 1H NMR spectra of (a) alkyne-PCL-OH, (b) alkyne-PCL-Br and (c) alkyne-PCL-b-POEGMA in CDCl3.

Successful synthesis of the well-defined PCL-b-POEGMA diblock copolymers is also confirmed by SEC-MALLS analysis (Figure 3). All synthesized polymers show unimodal SEC elution peaks with narrow distributions, indicating well-controlled ROP and ATRP processes.



Page 20 of 42

Figure 3. SEC elution traces (dRI signals) of (a & b) alkyne-PCL , alkyne-PCL-b-POEGMA, and PF-b-PCL-b-POEGMA and (c) alkyne-POEGMA and PF-b-POEGMA; UV signals of (d) PF12-N3, P0, P1 and P2 using DMF as the eluent.

The prepared Alkyne-PCL-b-POEGMA and the PF-N3 were subsequently coupled by a copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC) “click reaction” between

the

alkyne

and

azido

groups

of

each

polymer

to

generate

PF12-b-PCL33-b-POEGMA45 (P1) and PF12-b-PCL44-b-POEGMA45 (P2) triblock copolymers. The successful synthesis and purification of the target triblock copolymers were confirmed by the appearance of all the characteristic signals attributed to the PF, PCL, and POEGMA block in the 1H NMR spectrum of the


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triblock copolymer (Figure 4). It is important to note that the slight but detectable differences between the dRI-signals of the target triblock copolymer and PCL-b-POEGMA precursor (Figure 3a & b, and S4) is likely due to the small contribution from the short PF block (Figure S3, the dRI signal of PF is partially overlapped with the solvent signal for the SEC-MALLS measurements when using DMF as the eluent). Instead, the signals from UV detector are included (Figure 3d) to show a clear shift of the elution traces toward higher molecular weight for the triblock copolymers relative to that of the PF homopolymer. The results strongly support the occurrence of the click coupling and successful preparation of the target triblock copolymer.

Figure 4. 1H NMR spectra of PF12-b-PCL44-b-POEGMA45 (P2) in CDCl3.



Rationalities for the Polymer Design A diblock copolymer of PF-b-POEGMA can indeed self-assemble into micelles in water and provide a reservoir for drug loading, but actually the hydrophobic core composed of short PF chains can’t provide the resulting micelles with a satisfactory drug loading capcacity. Therefore, we also prepared a long PF block with DP of 18, but this polymer turned out to be insoluble even in DMF, rendering further functionalization impossible. To address simultaneously the low drug loading capacity provided by the short PF sequence and insolubility of the long PF block, the introduction of another hydrophobic block, like PCL is necessary to improve the drug loading capacity without affecting the solubility of PF block. In addition, the incorporation of the hydrophobic PCL central block surrounding the hydrophobic PF block is believed to enhance the fluorescence property of the PF sequence. To provide a direct insight into this point, a control of diblock copolymers, PF12-b-POEGMA45 (P0) without PCL block was prepared. The synthesis of P0 was schematically illustrated in Scheme S1. Successful synthesis of P0 is confirmed by 1H NMR study (Figure S5, S6, & S7) and SEC-MALLS analysis (Figure 3c & d). A detailed comparison study in terms of the drug loading capacity and photophysical properties was made in the following sections.

Size and Morphology of Micelles Size is a critical factor for polymeric micelles due to its significant effect on the in vivo performance of micellar drug carriers. Small size (

Fabrication of Conjugated Amphiphilic Triblock Copolymer for Drug

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