pH-Triggered Polypeptides Nanoparticles for Efficient BODIPY

Subscriber access provided by SUNY DOWNSTATE

Article

pH-triggered Polypeptides Nanoparticles for Efficient BODIPY Imaging-guided Near Infrared Photodynamic Therapy Le Liu, Liyi Fu, Titao Jing, Zheng Ruan, and Lifeng Yan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01320 • Publication Date (Web): 29 Mar 2016 Downloaded from http://pubs.acs.org on April 2, 2016

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 free 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 accessible to all readers and 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.

ACS Applied Materials & Interfaces 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 45

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

pH-triggered Polypeptides Nanoparticles for Efficient BODIPY Imaging-guided Near Infrared Photodynamic Therapy Le Liu, Liyi Fu, Titao Jing, Zheng Ruan, and Lifeng Yan *

CAS Key Laboratory of Soft Matter Chemistry, National Synchrotron Radiation Laboratory, iChEM, and Department of Chemical Physics, University of Science and Technology of China. Hefei, 230026, P.R.China. Fax: +86-551-63603748; Tel: +86-551-63606853; E-mail: [email protected]

1

ACS Paragon Plus Environment


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

Abstract: An efficient pH-responsive multifunctional polypeptide micelle for simultaneous imaging and in vitro photodynamic therapy (PDT) has been prepared. The goal here is to detect and treat cancer cells by near infrared fluorescence (NIRF) imaging and PDT synchronously. A photosensitizer BODIPY-Br2 with efficient singlet oxygen generation was synthesized at first which owns both seductive abilities in fluorescence emission and reactive oxygen species (ROS) generation under light irradiation. Then, amphiphilic copolymer micelles pH-triggered disassembly were synthesized from N-carboxyanhydride (NCA) monomer via a ring-opening polymerization and click reaction for the loading of BODIPY-Br2 by hydrophobic interaction, and the driving force is the protonation of the diisopropylethylamine groups conjugated to the polypeptide sidechains. In vitro tests performed on HepG2 cancer cells conform that the cell suppression rate was improved by more than 40% in the presence of light in the presence of an extremely low energy density (12 J/cm2) with very low concentration of 5.4µM photosensitizer. At the same time, the internalization of the nanoparticles by cells can also be traced by the NIRF imaging, indicating that the NIR nanoparticles presented an imaging guided photodynamic therapy properties. It provides the potential of using polypeptide as a biodegradable carrier for NIR image-guided photodynamic therapy.

Keywords: pH-responsive, N-carboxyanhydride (NCA), polypeptide, photodynamic therapy (PDT), near-infrared fluorescence (NIRF) imaging, singlet oxygen

2


Page 2 of 45

Page 3 of 45

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


Introduction The development of polymeric nanomaterials for both diagnosis and therapy has received increasing attention recently and become an extremely important field in nanomedical research, which was vividly called “see and treat” or theranosis of cancer.1-3 Photodynamic therapy (PDT), in recent years, has attracted a lot of attention for cancer therapy when light was swatch-on in the presence of photosensitizer and oxygen, generating reactive oxygen species (ROS).4 For example, in type II process, when it was irradiated, the photosensitizer could be converted to the TS (triplet state) through intersystem crossing (ISC), and molecular oxygen would immediately absorb the generated energy to form singlet oxygen, which do extremely harm to tumors. Therefore, for a superb photosensitizer, a high singlet oxygen quantum yield would be required which could be achieved by linking heavy atom to improve the ISC, and this effect plays a greater role in BODIPY dyes than other kinds of dyes.5 Besides, BODIPY dyes showed superior photo-physical properties over other existing dyes, such as excellent photo-stability, fluorescence quantum yield and extinction coefficient.6 So BODIPY could be applied in imaging and treating at the same time via heavy atom effect modification. Imaging guided PDT, especially NIR PDT of imaging guided,7 has been widespread concerned concerning the fact that the envisage might be achieved to trace the movement of photosensitizer in vitro or in vivo and monitor the tumor size-changing simultaneously during the cancer therapy.8-14 3



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

However, BODIPY based photosensitizers working in the NIR region is still scarce and there are still challenges to utilize NIR dyes as imaging agent and photosensitizers at the same time, especially in terms of combining with polymeric nano-carriers which are stimuli-responsive.10 In addition, photosensitizers are usually hydrophobic, and nano-carriers should be necessary to deliver them to target tissues or cells.15-16 Wide variety of nanomaterials has been designed for nano-carriers, including liposomes, polymers, QDs, etc, among which polymeric nanostructures have their own unique advantages over others.17 For example, the compositions, sizes, morphologies, even their surface properties could be well designed adapting to the necessity, and because of the enhanced permeability and retention (EPR) effect, these nanoparticles can preferable accumulate in the tumor tissue than in normal one. For better drug delivery, nanomaterials responsive to external stimuli, such as temperature, pH, and enzymes have been well explored, among which pH shows a difference between tumor microenvironment and normal tissues.18 Cancer cells display a lowered extracellular pH that can promote matrix degradation, and many researchers have done a lot of studies on efficiently drug release triggered by acidic environment.19-20 In most cases, the polymeric nanomaterials are core-shell structure comprising of a hydrophilic shell for their stabilization in aqueous environment, such as PEG or poly-oligo (ethylene glycol) methacrylate (POEGMA), and a hydrophobic core which can be used to load anticancer drugs and imaging agents.21 Polypeptides, during 4


Page 4 of 45

Page 5 of 45

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


recent years, has been widely used for their biocompatibility and biodegradability and could be easily prepared using N-carboxy-anhydrides monomer (NCA) monomer via ring-opening polymerization (ROP) is a more effective method of comparing to traditional solid-phase synthesis.22-28 From the above consideration, here we synthesized a pH-responsive copolymer (POEGMA-PAsp) via the ROP of NCA which can be degraded faster at lower pH in cancer cells. In this system, the POEGMA segment acts as a hydrophilic shell for improving the solubility and stabilization, and PAsp segment plays a role of the hydrophobic core of load DOX or BODIPY-Br2 (Scheme 1). Corresponding to our prediction, this core-shell structured micelle could encapsulate DOX or BODIPY-Br2 efficiently via hydrophobic interactions and would degrade to release them at lower pH such as 5.5, as shown in Scheme 1. The MTT assay upon HepG2 cells exposed to light showed an obvious growth inhibition. In addition, a BODIPY-Br2 carrying pH-sensitive micelle which holds great promising for simultaneous imaging and treating has also been developed.

Results and Discussion Synthesis of the BODIPY photosensitizers 4, 4-difluoro-4-bora-3a, a-diaza-s-indacene (BODIPY) has received much attention since its discovery, and has been applied in various fields, such as biological imaging, sensors, light-emitting materials and PDT. BODIPY dyes showed superior photo-physical properties such as high photo-stability, fluorescence quantum yield 5



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

and extinction coefficient over other dyes. In addition, after modifying the BODIPY core using heavy atoms, the S1→T1 transition was increased for higher singlet oxygen generation. On the other hand, BODIPY photosensitizers with NIR absorbance are still being searched to produce a deeper tissues penetration. In this study, as shown in Scheme 2, a BODIPY-Br2 photosensitizer has been synthesized which has strong NIR absorbance and fluorescence emission (λmax = 721nm, λem = 754nm in chloroform, as shown in Figure 1). The 1H-NMR,13C-NMR, 11

B-NMR, FT-IR，LC-MS spectra are shown in supporting information，confirming

the compounds with exact structures and high purity for further use. By introducing bromine atoms, intersystem crossing yield was obviously improved to generate singlet oxygen.6 Singlet oxygen generation capacity was measured using DBPF assay, as shown in Figure 2. Under 808nm irradiation, there is rarely singlet oxygen generated for BODIPY (Fig.S2), however an obvious improvement was obtained for BODIPY-Br2, which are more superior to Rhodamine B (Fig.S2), a kind of reference photosensitizer. The calculated φ∆(1O2) is 0.36 for BODIPY-Br2 in chloroform, indicating efficient singlet oxygen generation. Interestingly, BODIPY-Br4 was also synthesized, but it showed low singlet oxygen generation, might be due to its poor solubility and light absorption capacity (ε= 75000M−1 cm−1 in CHCl3). Besides, the absorbance of BODIPY-Br2 shows no subtraction implying its outstanding photo-stability. Even more strikingly, under 635nm irradiation, BODIPY-Br2 presented an extremely prompt generation of singlet oxygen, and the absorbance of DBPF at 410nm 6


Page 6 of 45

Page 7 of 45

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


decreased swiftly every 10s irradiation. In consideration of this phenomenon, we chose 635nm irradiation to achieve other measurements.

Design, synthesis and characterization of pH-sensitive copolymers ROP of NCA monomers is a common method for preparing polypeptides utilizing the primary amine groups as the initiator. In addition, controlled radical polymerization with lower dispersion index (DI) could be well achieved using reversible addition-fragmentation chain transfer (RAFT) agent as initiator. Therefore, the amphiphilic copolymers (Scheme 3) were prepared by combining the RAFT and ROP polymerizations: 1) Using propargylamine as the initiator, ROP of Asp-NCA monomers to obtain PAA-PAsp; 2) Azido modified RAFT polymerization of OEGMA to get RAFT- POEGMA-APA; 3) Combining the two above segments by click reaction (RAFT-POEGMA-PAsp). Besides, to prepare pH-responsive amphiphilic copolymers that can self-assemble in physiological solution, diisopropylethylamine (DIPEA) was conjugated to PAsp through an amide bond formation, and the final product POEGMA-PAsp was prepared. The 1H-NMR spectra of the copolymer are shown in Fig.3, meanwhile, the disappearance of 2150cm-1 peak of –N3 proves the completion of click reaction (Fig.S3). GPC traces of these three polymers were measured using RI detector to understand the molecular structure of products better, as shown in Fig.4. A shift towards lower elution time was observed as click reaction down, which corresponds to a higher molecule weight. Table 1 list the molecular weights and their distribution 7



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

measured by NMR and GPC respectively, and the obtained Mn differs minor compared to the Mw measured from 1H-NMR. All peaks were single with a PDI around 1.10, indicating an efficient reaction without any dead polymer chains formed during the polymerization. The pKa of RAFT-POEGMA-PAsp was measured via acid-base titration giving the pKa to be 6.4 (Fig. S4), indicating that this amphiphilic copolymer would change from amphiphilic to hydrophilic when the pH was lower than 6.4. Since the pH of the lysosome medium is much lower than this critical value, this pH-responsive copolymer could show its superiority in drug delivering.

Preparation and characterization of pH-responsive micelles Since the copolymers are comprised of a hydrophilic segment (POEGMA) and a hydrophobic segment (PAsp) at neutral pH, and it can self-assemble into micelles to encapsulate anticancer drugs or imaging agents. However, under acidic environment (pKa=6.4, as shown in Fig.S4), these micelles would swell or disassembly due to the protonation of the DIPEA groups conjugated to PAsp. Micelles encapsulating BODIPY-Br2 (Polymer/BODIPY-Br2) to realize imaging PDT were prepared. However, BODIPY-Br2 would forms precipitates when the micelles were disassembled by pH 5.5 for its hydrophobicity, and it would be infeasible to study the release behavior of Polymer/BODIPY-Br2 using traditional dialysis method. Thus, hydrophobic drug DOX was chosen to be the substitute to measure the drug release behavior, and micelles loading doxorubicin hydrochloride 8


Page 8 of 45

Page 9 of 45

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


(polymer/DOX) was prepared. Photosensitizer loading content (PLC) and drug loading content (DLC) were calculated to be 2.02% and 1.04%, while the efficiency turns to be 13.2% and 11.4% respectively. Drug release results under both neutral (pH = 7.4) and acidic (pH = 5.5) conditions were shown in Fig. 5. Under pH 7.4, the drug release was slow in the first 24 h, and in the following 72 h no obvious further release was observed and the total drug release was 27%. While under pH 5.5, a promoted drug release behavior showed, the drug release was fast and sustaining until 96h with totally 60% drug release which was still increasing. The stepped-up drug release resulting from the protonation of the DIPEA indicated the pH-responsive capability of the drug delivery system. The results reveal that the as-prepared polymeric micelles are pH responsive. Size and their distribution of the micelles with or without BODIPY-Br2 were determined by DLS (Fig. 6). DLS showed that the micelles without photosensitizers loaded under pH 5.5 for 2h have a wide distribution with 35nm and 165nm as the peaks, while the size is about 117nm at pH 7.4, indicating the damage of the micelles. Similar result has been found for the micelles with BODIPY-Br2 encapsulated, as shown in Fig.6c and d, at pH 7.4 the size of the micelles is about 106.7nm, a little smaller than the pure polymeric micelles, indicating there exist hydrophobic and electrostatic interaction between polymers and photosensitizers. However, it became larger when the pH was changed to 5.5, indicating an efficient disassembly of the micelles, and during the processes, photosensitizers were released. AFM studies reveal morphologies change at the both pHs, and clearly the micelles were 9



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 10 of 45

disassembled at pH 5.5, indicating the pronation of the relative amino groups of the copolymers. Besides, these micelles had a negative Zeta potential of -8.08mV, allowing the micelles keep stable in blood circulation.

PDT cytotoxicity MTT assay against HepG2 cells was used to evaluate the in vitro PDT cytotoxicity assay of polymer/BODIPY-Br2 or BODIPY-Br2 (Fig. 7). Generally, the experiment was carried out under 635nm irradiation for 10 min with a given fluorescence rate 20 mW/cm2, which was extremely low as far as reported. In addition, the concentration of photosensitizer is only about 5.4 µM for efficient PDT. In comparison with the results of polymer/BODIPY-Br2 or BODIPY-Br2, the suppression rates keep invariant as the concentration of BODIPY-Br2 increased, for its limitation to be transported into cells. However, cells were efficiently killed at a consistent dosage of the Polymer/BODIPY-Br2 when the light irradiation was added. The mortality of the cells with irradiation increased to upto 60% at a concentration of 5.4 µM, 45% higher than that of cells incubated under dark. However, since there were differences between cancer cells and normal cells, such as

endocytosis,

cell

membrane

permeability,

and

other

factors,

the

polymer/BODIPY-Br2 might work in different ways. Considering this, we also carried out the cytotoxicity of polymer/BODIPY-Br2 to normal cells (HL-7702 cells) (Fig S13.). Under irradiation, the cells growth inhibition was increased as the concentration of photosensitizers improved, 23% higher than that of cells incubated 10


Page 11 of 45

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


under dark.

See and treat Depending on its fluorescence property, BODIPY-Br2 could also be used in imaging to study the internalization and localization of the copolymers. HepG2 cells were treated with polymer/BODIPY-Br2 for 24h to be observed through a fluorescence microscope. Figure 8 shows that after cultivated for 24h, the copolymers were well dispersed in HepG2 cells, almost in the cytoplasm. The strong emitted fluorescence indicated the photosensitizer-loaded nanoparticles is satisfactory for bio-imaging. At the same time, FDA and PI were used to monitor the PDT efficiency by imaging of dead (red) and live (green) cells, respectively. The polymeric nanoparticles containing BODIPY-Br2 did not display cytotoxity when incubated without light. However, after exposure to 635nm light, HepG2 cells were effectively killed (Fig. 9). These results indicate that polymer/BODIPY-Br2 cause evident cellular damages with the irradiation of light, even at a low concentration (0.1mg/mL), which is agree with the results of MTT experiments. The above results clearly reveal that the polymer/BODIPY-Br2 nanoparticles could deliver the hydrophobic photosensitizer efficiently inside to cancer cells, in where the nanoparticles were disassembled to release the photosensitizer, which can be traced by NIR fluorescence imaging, then NIR light irradiation was switch on to kill the target cells or tumor by PDT, and the performance can also traced by NIR imaging. 11



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 12 of 45

Conclusions In summary, a pH-sensitive copolymer has been synthesized to encapsulate BODIPY-Br2 photosensitizers for efficient image-guided photodynamic therapy, which is a promising cancer theranostics strategy. Modification of the polypeptides with DIPEA enabled an accelerated disassemble of the polymeric nanoparticles in acidic conditions to release the photosensitizers for efficient PDT. Specifically, the mortality of the cells with irradiation increased 45% higher than the cells incubated in the dark, with an extremely low laser rate and dose of photosensitizer. The strong fluorescence emitted by the BODIPY-Br2 loaded nanoparticles makes it possible for biomedical imaging. All the advantages mentioned above make it a potential nanomaterial for cancer imaging and PDT.

Materials and Methods Materials Chemicals (AR purity), unless indicated, were purchased from Sinoreagent corporation. Tetrahydrofuran (THF) and n-Hexane were refluxed with CaH2 overnight before distillation and immediate use. N, N-Dimethylformamide (DMF) was dried with CaH2 for 36h at room temperature followed by reduced-pressure distillation. 3-chloropropylamine

hydrochloride,

4,4'-Azobis(4-cyanovaleric

acid),

azodiisobutyronitrile (AIBN), sodium azide, β-benzyl-L-aspartate, triphosgene, doxorubicin

(DOX)

hydrochloride,

diisopropylethylamine 12


(DIPEA),

Page 13 of 45

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


propargylamine (PAA), N,N,N’,N’,N’-pentamethyldiethylene triamine (PMDETA), 4-Methoxyphenylboronic

acid,

[1,1’-Bis(diphenylphosphino)-ferrocene]palladium(II)

5-bromo-2-thiophenealdehyde, dichloride

dichloromethane

complex (1:1), ethyl chloroacetate, trifluoroacetic acid (TFA), trifluoroacetic anhydride, borontrifluoride dietherate, were purchased from Aladdin Corporation, China. Oligo (ethylene glycol) methacrylate (OEGMA, Mw=500) was purchased from Sigma Aldrich Corporation, China. Fetal bovine serum (FBS), dulbecco modified eagle medium (DMEM), methyl thiazolyl tetrazolium (MTT), fluorescein diacetate (FDA) and propidium iodide (PI), were obtained from Sangon Corporation, China. Ultrapure water was prepared by a Milli-Q system (18.2 MΩ, Millipore, USA). Dialysis bags (cut off Mw=8000, 2000) were purchased from Bomei biotechnology corporation, China.

Characterization 1

H-NMR,

11

B-NMR and

13

C-NMR studies were carried out on a Bruker NMR

spectrometer (AC 300) using chloroform-d or dimethyl sulfoxide-d6 (DMSO-d6) as solvent. UV-Vis spectra were measured on a Shimadzu UV-2401 PC Ultraviolet. Fluorescence spectra were measured on Shimadzu RF-5301PC with set excitation slit width (10nm) and emission slit width (10 nm). FT-IR spectra were recorded on a Bruker spectrometer (EQUINOX 55) applying potassium bromide (KBr) assay method. Shimadzu Shodex GPC KD-804 column and refractive index detector (RID-10A) equipping Gel Permeation Chromatography (GPC, LC-20AD) were used 13



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 14 of 45

to determine the molecular weights of the samples. The measurement was achieved at 60 oC using DMF as the mobile phase at a decided sample concentration (5mg/mL). The calibration of Mn, Mw and Mw/Mn was computed using monodispersed polystyrene standards. Dynamic light scattering were measured on a Malvern Zetasizer (Nano ZS90) at room temperature. ESI/APCI mass spectra were recorded on a LCQ Advantage MAX Spectrometer, with samples were solving in methanol to obtain the LC spectra. Atomic Force Microscope (AFM) were recorded on a Nanoscope IIIA microscope. The MTT assay absorbance was recorded on Bio-rad iMark microplate reader. The fluorescence microscope measurements were carried out on Olympus U-HGLGPS.

Synthesis of 5-(4-Methoxyphenyl) - thiophene-2-carbaldehyde (1) 6 4-Methoxyphenylboronic

acid

(2.99g,

19.70

mmol)

and

5-bromo-2-thiophenealdehyde (3.78g, 19.70 mmol) were dissolved into toluene (60 mL) and ethanol (20 mL). Then 2M Na2CO3 aqueous solution (20 mL) and [1,1’-Bis(diphenylphosphino)-ferrocene]palladium(II)

dichloride

dichloromethane

complex (1:1) (100mg) was then added. Raise the temperature to 80oC and reacted for 14 h. After cooling, the obtained mixture was then washed with brine and water, and removes the solvent. The raw products were purified using column chromatography (silica gel, 5-30% ethyl acetate/hexane) to gain a light yellow solid (3.22g, 75.2%). 1

H-NMR ( CDCl3, 300MHz) δppm : 3.87 ( s, 3H), 6.96 ( d, J=3.8Hz, 2H), 7.31 ( d,

J=5.4Hz, 1H), 7.63 ( d, J=3.8Hz, 2H) , 7.73 ( d, J=5.4Hz, 1H), 9.87 ( s, 1H) . 14


Page 15 of 45

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


13

C-NMR (CDCl3, 75MHz) δppm : 55.5, 114.6, 123.0, 125.8, 127.8, 137.8, 141.5,

154.5, 160.7, 182.7. MS (m/z, APCI): calcd for C12H10O2S: 218.02; found: 219.05 [M+H]+.

Synthesis of Ethylazidoacetate Sodium azide (2.60g, 0.04mol) was dissolved into anhydrous DMF (20 mL) and stirred for 10min, then ethyl chloroacetate (2.23g, 0.02mol) was dropped into the mixture. Raise the temperature to 40 oC and keep stirring for 5h. After cooling, the solution was then extracted with 20 mL ethyl acetate for 3 times, and the organic layers were washed with water and dried over Na2SO4. A yellow liquid was obtained after the solvents were removed by evaporation.

1

H-NMR (CDCl3, 300MHz) δppm:

1.31 (t, J=7.1Hz, 3H), 3.86 (s, 2H), 4.26 (p, J=7.1Hz, 2H). FTIR (cm-1): 2108.1(-N3).

Synthesis of Ethyl 2-(4-methoxyphenyl)-4H-thieno [3, 2-b] pyrrole-5-carboxylate (2)

Sodium ethoxide (2.30g, 34.88mmol) was dissolved in anhydrous EtOH (80 mL) and stirred for 30 min bubbling with nitrogen. Ethylazidoacetate (4.50g, 34.88mmol) and 1 solution (in 20 mL EtOH, 2.00g, 8.72mmol) were added dropwise slowly in more than 40 min. Then the reaction was kept in an ice-salt bath and stirred for 5 h. A saturated aqueous solution of NH4Cl (100 mL) was added to facilitate yellow precipitation. Using vacuum filtration, the orange solid was collected and washed with water. After allowed to be dried, the solid was dissolved in toluene (20 mL) and 15



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 refluxed at 110 oC for 1.5 h. The solvent was removed by rotary evaporation and finally purified using column chromatography (silica gel, 5-30% ethyl acetate/hexane) to get a yellow solid (1.00 g, 49.5%). 1H-NMR ( CDCl3, 300MHz) δppm: 1.40 ( t, J=7.1Hz, 3H ), 3.85 ( s, 3H ), 4.37 ( m, 2H ), 6.93 ( d, J=8.0Hz, 2H ), 7.07 ( s, 1H ), 7.11 ( s, 1H ), 7.55 ( d, J=8.0Hz, 2H ), 8.98 ( s, 1H ). 13C NMR (CDCl3, 75 MHz) δppm: 14.5, 55.4, 60.6, 105.9, 107.9, 114.3, 123.5, 126.2, 127.1, 127.8, 141.8, 148.2, 159.6, 161.5. MS (m/z, APCI): calcd for C16H15NO3S: 301.08; found: 302.08 [M+H]+.

Synthesis of 2-(4-Methoxyphenyl)-4H-thieno [3, 2-b] pyrrole -5-carboxylic acid (3) Sample 2 (0.30g, 1.09mmol) was firstly dissolved in EtOH (10 mL) and then sodium hydroxide (0.62 g, 15.5 mmol) in water (5 mL) was added dropwise. After refluxing for 2h and cooling, the mixture was acidified with 1M HCl to facilitate a brown solid in an ice bath. The precipitate was collected, washed with water and finally dried by freeze drying (0.26mg, 80%). 1H-NMR ( DMSO-d6 , 300MHz) δppm: 3.79 ( s, 3H ), 6.99 ( d, J=8.8Hz, 2H ), 7.01 ( s, 1H ), 7.24 ( s, 1H ), 7.61 (d, J=8.8Hz, 2H ), 11.95 ( s, 1H ), 12.53( s, 1H ).

13

C NMR (DMSO-d6, 75 MHz) δppm: 55.8,

106.9, 114.5, 121.6, 123.5, 126.1, 127.3, 127.8, 142.2, 145.6, 159.0, 162.1. MS (m/z, APCI): calcd for C14H11NO3S: 273.05; found: 274.05 [M+H]+.

Synthesis of 2, 8-Di (4-methoxyphenyl)-11-trifluoromethyl-dithieno [2, 3-b]-[3, 2-g]-5, 5-difluoro-5-bora- 3a, 4a-dithio-s-indacene (BODIPY) (4) 16


Page 16 of 45

Page 17 of 45

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


Sample 3 (0.20g, 0.70 mmol) was dissolved in TFA (10 mL) bubbling with nitrogen and heated at 40 oC for 10min. Trifluoroacetic anhydride (2 mL) was then added dropwise and the temperature of the reaction was raised to 80 oC for 40 min. The reaction was quenched with saturated aqueous solution of NaHCO3 after cooling to facilitate a blue solid. The solid was collected, washed with water and finally dried by freeze drying. The dried solid was dissolved in toluene (30 mL) and stirred for 5 min. Triethylamine (1.5 mL) and borontrifluoride dietherate (1 mL) were added successively. When the fog disappeared, the reaction temperature was then raised up to 80 oC for 30 min, all under a nitrogen atmosphere. After cooling, the reaction was quenched with saturated aqueous solution of NaHCO3 and washed with water and brine. The solvent was evaporated to obtain a green metallic solid purified by column chromatography 80% chloroform/hexane (80mg, 36.6%). 1H-NMR (CDCl3, 300MHz) δppm: 3.91 (s, 6H ), 7.00 (d, J=8.8Hz, 4H ), 7.35 (s, 2H), 7.71 (d, J=8.8Hz, 4H ). 11

B-NMR(CDCl3, 128MHz) δppm: 0.79. MS (m/z, APCI): calcd for C28H18-

BF5N2O2S2: 584.08; found: 584.09 [M+H]+. ε = 200000M−1 cm−1, ΦF = 0.37 in CHCl3.

Synthesis of 3, 7-Dibromo-2, 8-di (4-methoxyphenyl)-11-trifluoromethyl-dithieno [2, 3-b]-[3, 2-g]-5, 5- difluoro-5-bora-3a, 4a-diaza-s-indacene (BODIPY-Br2) (5) Sample 4 (0.053g, 0.09 mmol) and I2 (0.001g) was dissolved in CH2Cl2 (20 mL). Br2 (0.043g, 0.27 mmol) in CH2Cl2 (5 mL) was added and refluxed at 40 oC for 12 h. After cooling, the reaction mixture was quenched with saturated aqueous solution of 17



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

NaHCO3 and washed with water and brine, dried over anhydrous Na2SO4. The solvents were removed by evaporation to obtain a brown solid (40mg, 59.2%). 1

H-NMR (CDCl3, 300MHz) δppm: 3.90(s, 6H), 7.02 (d, J=14.0Hz, 4H), 7.60 (s, 1H),

7.78 (m, 4H).

11

B-NMR (CDCl3, 128MHz) δppm: 0.51. MS (m/z, APCI): calcd for

C28H18BBr2F5N2O2S2: 741.90; found: 722.90 [M-F]+. ε = 89000M−1 cm−1, ΦF = 0.45 in CHCl3.

Synthesis of S-dodecyl-S’-(α-methyl-α-cyanoacetic acid) trithiocarbonate (RAFT) (6) 1-dodecanethiol (10.12g, 0.05mol) was dissolved into ethanol aqueous solution (10%, 40 mL), followed by adding KOH (3.37g, 0.06mol) and keep stirring for 0.5h at room temperature. Then CS2 (3.0 mL, 0.05mol) was added dropwise over 20min, the mixture was stirred for another 3h. After that, 4-toluene sulfonyl chloride (4.65g, 0.025mol) in DCM (10 mL) was added slowly in an ice-water bath, remove the ice bath and stir for further 5h. The organic layer was collated, and the aqueous layer was then extracted with 10 mL DCM for 3 times. The organic phase mixture was washed by brine and water, dried over Na2SO4. A deep red solid was obtained after the solvents were removed. The dried red solid (9.96g, 0.018mol) and 4, 4'- Azobis (4-cyanovaleric acid) (5.55g, 0.0198mol) were dissolved into acetic ether (80 mL), stirring for 12h at 80 oC. After removing acetic ether, the raw products was purified using column chromatography (silica gel, 0-80% ethyl acetate/hexane) to get a yellow solid (7.18g, 49.5%). 1H-NMR (CDCl3,300MHz) δppm: 0.88 (t, J=6.6Hz, 3H), 1.26(m, 18


Page 18 of 45

Page 19 of 45

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


18H) , 1.70(m, 1H) , 1.77(s, 3H), 2.36 (t, J=7.7Hz, 2H), 2.53 (t, J=7.7Hz, 2H), 3.28 (t, J=7.4Hz, 2H).

Synthesis of 3-azidopropan-1-amine (APA) Sodium azide (6g, 92.3mmol) and 3-chloropropylamine hydrochloride (4g, 30.8mmol) were reacted at 100 oC for 48h in water (40 mL). After cooling, the mixture was then extracted with 10 mL DCM for 3 times, and the combined organic solutions were dried over anhydrous MgSO4. A transparent liquid was obtained after removing the solvent using vacuum evaporation (2.12g, 65.3%). 1H-NMR (CDCl3, 300MHz) δppm: 1.74 (m, 2H), 2.81 (t, J=6.8Hz, 2H), 3.38 (t, J=5.8Hz, 2H).

Synthesis of RAFT-APA (7) RAFT agent 6 (750mg, 1.86mmol) and HOSu (235mg, 2.05mmol) were firstly dissolved in DCM (30 mL), and then DCC (422mg, 2.05mmol) in dry DCM (5 mL) was added in an ice-water bath, keep stirring at room temperature for 36h. After removing the solid by filtration,) 3-azidopropan-1-amine (187mg, 1.86mmol) was dropped and stirred for another 12h. After removing the solvents, the raw product was then purified using column chromatography (silica gel, 0-35% acetic ether /petroleum ether) to get a yellow solid (560mg, 62.2%). 1H-NMR(CDCl3,300MHz) δppm: 0.87 (t, J=6.6Hz, 3H) , 1.65 (m, 2H) , 1.75 (s, 3H), 1.80(m, 4H) , 2.32(s, 4H) , 3.28(m, 2H), 3.37(m, 2H).

19



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

Synthesis of RAFT- POEGMA-APA (8) RAFT-APA 7 (230mg, 0.474mmol), AIBN 13.5mg (0.118mmol, 1/4equiv) and OEGMA (6.8g, 14.2mmol, 30equiv) were dissolved into DMF (5 mL) in a Schlenk tube (argon purged) and keep stirring for 12h. After cooling, purify the mixture by dialysis against ultrapure water. A sticky yellow solid was get using freeze-drying (5.4g, 98.8%).

Synthesis of Asp-NCA L-aspartic acid-4-benzyl ester (1.5g, 13.4mmol) and triphosgene (2.2g, 14.7mmol 1.1equiv) was mixed in dry DMF (40 mL), and keep stirring at 45 oC and bubbling with nitrogen for 2 h. The clear solution was collected and crystallized with dried THF and hexane for 2 times to give a white solid (1.35g, 68%).

Synthesis of PAA-PAsp (9) Propargylamine (21.6mg, 0.392mmol) was added into a Schlenk tube (flame dried and argon purged), add dry DMF (5 mL) to solubilize the initiator. Asp-NCA (937mg, 3.92mmol, 10equiv) in dry DMF (5 mL) were then added into the above initiator solution which was precooled in an ice-water bath with the protection of argon, stirred for 3 days at 0 oC. The obtained solution was then stirred at room temperature for another 12h, purified against ultrapure water by dialysis. A pure white solid was get using freeze-drying (885mg, 92.5%).

20


Page 20 of 45

Page 21 of 45

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


Synthesis of RAFT-POEGMA-PAsp via click reaction (10) Polymer 8 (480mg, 34.7µmol), polymer 9 (93.7mg, 34.7µmol), CuBr (5mg, 34.7µmol), and PMDETA (6mg, 34.7µmol) were dissolved into dry DMF(5 mL) in a Schlenk tube (flame dried and argon purged), keep stirring for 3 days at 30 oC. The obtained mixture was then purified against EDTA aqueous solution and ultrapure water by dialysis. A cream white solid was get using freeze-drying (442mg, 77.2%).

Amino substitution of Polymer 5 (11) Polymer 10 (366mg, 3.05µmol) and DIPEA (87.4mg, 61.0µmol) were dissolved into dry DMF(5 mL) in a Schlenk tube (flame dried and argon purged), keep stirring for 3 days at 45 oC. After cooling, the mixture was then purified against ultrapure water by dialysis. A sticky yellow solid was get using freeze-drying (302mg, 82.2%).

Measurement of pKa The pKa of pH responsive Copolymer 11 was measured via acid-base titration. Polymer 11 (100mg) was dissolved into deionized water (20 mL), the solution’s was adjusted to alkaline using NaOH aqueous solution. Then 1M HCl was discontinuously added and at the same time, the pH of the solution was recorded. Deionized water (20 mL) without polymer was also titrated considering the control group. The pKa were determined by the equation: pKa = 1/2(V0+Vend), in which V0 and Vend mean the volumes of HCl added while the pH beginning and stopping changes gently, respectively . 21



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

Preparation of micelles entrapped with DOX drug (Polymer/Dox) or BODIPY-Br2 photosensitizer (Polymer/BODIPY-Br2) To obtain polymer/DOX, polymer 11 (50mg) and doxorubicin hydrochloride (5.0mg) was dissolved in 1mL DMF, trimethylamine (2.6mg) was added and stirred for 2h. Then deionized waster (10 mL) was dropped and stirred for another 12h, and the solution was then dialyzed against ultrapure water. Similarly, to obtain Polymer/BODIPY-Br2, polymer 11 (30mg) and BODIPY-Br2 (5.0mg) was dissolved in 1mL DMF and stirred for 2h. Then deionized waster (10 mL) was dropped and stirred for another 12h, followed by dialyzing against ultrapure water. Drug loading content (DLC) and photosensitizer loading content (PLC) were calculated by the following equations: DLC (wt %) = (weight of loaded drug/weight of nanogel)×100% PLC (wt %) = (weight of loaded photosensitizer/weight of nanogel)×100% where DLC and PLC refer to the drug loading content and photosensitizer loading content, respectively.

In vitro drug release To evaluate the drug release behavior of the pH-responsive polymer, conditional dialysis method was carried out on polymer/DOX under both neutral (phosphate buffer solution, pH=7.4) and acidic (acetate/ sodium acetate buffer solution, pH=5.5) 22


Page 22 of 45

Page 23 of 45

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


conditions. For both conditions, to buffer solution (40 mL), polymer/DOX solution (700µL, 1mg/mL) in a dialysis bag (cut off Mw=2000) was immersed and conducted on a shaking bath at 37 oC. Samples (2mL) were removed and fresh buffer solutions (2mL) was immediately added at every predetermined time points. Using the fluorospectrophotometer, drug release was quantified (λex= 480nm, λem = 590nm)

Detection of singlet oxygen Singlet oxygen generation of BODIPY, BODIPY-Br2 and Rhodamine B were detected using a lamp at 0.5 mW/cm2. THF (2mL) containing DPBF (90 µM) and dye (5 µM) solutions were stirred in the dark and the absorbance measured at predetermined time points. The reduction of absorbance was considered to quantify the singlet oxygen generation efficiency.

In vitro PDT cytotoxicity assay To evaluate the in vitro PDT cytotoxicity of Polymer/BODIPY-Br2, MTT assay against HepG2 cells was carried out. To a 96-well plate, HepG2 cells were plated with a density of 3000 cells (100 µL) per well. After incubating for 24h, the medium was replaced with fresh DMEM with Polymer/BODIPY-Br2 at various concentrations (0, 0.05, 0.1, 0.15, 0.2 mg/mL) and incubated for another 24h under dark conditions. Then the cells were irradiated or not, after replacing with fresh DMEM. In the irradiation case, a 635nm lamp was used to irradiate the cells for 10 min with a given fluorescence rate 20 mW/cm2, and the spectral output was in the absorption region of 23



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 24 of 45

BODIPY-Br2. Then the cells were incubated for 24h to measure the cell viability using MTT assay, that is, MTT (20 µL, 5 mg/mL) to each well was added and incubated for 4h, and the medium was removed and the crystal was solubilized using DMSO

(150

µL)

per

well.

In

a

similar

way,

the

measurement

of

Polymer/BODIPY-Br2 cytotoxicity for HL-7702 cells was also carried out.

In vitro optical imaging To a 6-well plate, HepG2 cells were plated with a density of 105 cells per well and incubated for 24h. The next day, the medium was replaced with Polymer/BODIPY-Br2 in DMEM (0.1 mg/mL) and incubated for another 24h in darkness. After replacing the medium, the cells could be observed through a fluorescence microscope.

Laser-induced in vitro PDT To a 6-well plate, HepG2 cells were plated with a density of 105 cells per well and incubated for 24h. The next day, the medium was replaced with Polymer/BODIPY-Br2 in DMEM (0.1 mg/mL) and incubated for another 24h in darkness. Then the cells were irradiated or not, after replacing with fresh DMEM. In the irradiation case, a 635nm lamp was used to irradiate the cells for 10 min with a given fluorescence rate 20 mW/cm2, and the spectral output was in the absorption region of BODIPY-Br2, and the cells were incubated for 24h more. The medium was then replaced with fresh DMEM containing FDA (5 µg/mL) and PI (100 µg/mL) and staining for 15min. After replacing the medium, the cells could be observed through a fluorescence microscope. 24


Page 25 of 45

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


Supporting Information 1

H-NMR,

13

C-NMR,

11

B-NMR, FT-IR and LC-MSspectra of BODIPY-Br2

synthesis, absorption spectra of DPBF upon irradiation in the presence of BODIPY and Rhodamine B under 808nm, FT-IR spectrum of RAFT-POEGMA-PAsp and RAFT- POEGMA-APA, and acid-base titration for pKa measurement of RAFT-POEGMA-PAsp.

Acknowledgment

This work is supported by the National Natural Science Foundation of China (No. 51373162).

References 1. Lv, R. C.; Yang, P. P.; He, F.; Gai, S. L.; Yang, G. X.; Dai, Y. L.; Hou, Z. Y.; Lin, J., An Imaging-Guided Platform for Synergistic Photodynamic/Photothermal/ Chemo-Therapy with pH/Temperature-Responsive Drug Release. Biomaterials 2015, 63, 115-127. 2. Lammers, T.; Aime, S.; Hennink, W. E.; Storm, G.; Kiessling, F., Theranostic Nanomedicine. Acc. Chem. Res. 2011, 44 (10), 1029-1038. 3. Ryu, J. H.; Koo, H.; Sun, I. C.; Yuk, S. H.; Choi, K.; Kim, K.; Kwon, I. C., Tumor-Targeting Multi-Functional Nanoparticles for Theragnosis: New Paradigm 25



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 26 of 45

for Cancer Therapy. Adv. Drug Delivery Rev. 2012, 64 (13), 1447-1458. 4. Wang, C.; Cheng, L.; Liu, Y. M.; Wang, X. J.; Ma, X. X.; Deng, Z. Y.; Li, Y. G.; Liu, Z., Imaging-Guided pH-Sensitive Photodynamic Therapy Using Charge Reversible Upconversion Nanoparticles under Near-Infrared Light. Adv. Funct. Mater. 2013, 23 (24), 3077-3086. 5. Kamkaew, A.; Lim, S. H.; Lee, H. B.; Kiew, L. V.; Chung, L. Y.; Burgess, K., BODIPY Dyes in Photodynamic Therapy. Chem. Soc. Rev. 2013, 42 (1), 77-88. 6. Awuah, S. G.; Polreis, J.; Biradar, V.; You, Y., Singlet Oxygen Generation by Novel NIR BODIPY Dyes. Org. Lett. 2011, 13 (15), 3884-3887. 7. Yan, L.; Jing, T.; Fu, L.; Liu, L., Reduction-Responsive Polypeptide Nanogel Encapsulating NIR Photosensitizer for Imaging Guided Photodynamic Therapy. Polym. Chem. 2015.7, 951-957 8. Guan, M.; Dong, H.; Ge, J.; Chen, D.; Sun, L.; Li, S.; Wang, C.; Yan, C.; Wang, P.;

Shu,

C.,

Multifunctional

-Trismethylpyridylporphyrin-Fullerene

Upconversion-Nanoparticles

Nanocomposite:

A

Near-Infrared

Light-Triggered Theranostic Platform for Imaging-Guided Photodynamic Therapy. Npg Asia Mater. 2015, 7, e205. 9. Liu, X.; Que, I.; Kong, X.; Zhang, Y.; Tu, L.; Chang, Y.; Wang, T. T.; Chan, A.; Lowik, C. W. G. M.; Zhang, H., In Vivo 808 nm Image-Guided Photodynamic Therapy Based on an Upconversion Theranostic Nanoplatform. Nanoscale 2015, 7 (36), 14914-14923. 10. Lv, R.; Yang, P.; He, F.; Gai, S.; Yang, G.; Dai, Y.; Hou, Z.; Lin, J., An 26


Page 27 of 45

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


Imaging-Guided Platform for Synergistic Photodynamic/Photothermal/ChemoTherapy with pH/Temperature-Responsive Drug Release. Biomaterials 2015, 63, 115-127. 11. Nair, L. V.; Nazeer, S. S.; Jayasree, R. S.; Ajayaghosh, A., Fluorescence Imaging Assisted Photo dynamic Therapy Using Photosensitizer-Linked Gold Quantum Clusters. Acs Nano 2015, 9 (6), 5825-5832. 12. Song,

X.;

Liang,

C.;

Photosensitizer-Conjugated

Gong,

H.;

Chen,

Q.;

Albumin-Polypyrrole

Wang,

C.;

Liu,

Nanoparticles

Z., for

Imaging-Guided In Vivo Photodynamic/Photothermal Therapy. Small 2015, 11 (32), 3932-3941. 13. Chao, W.; Liang, C.; Yumeng, L.; Xiaojing, W.; Xinxing, M.; Zhaoyi, D.; Yonggang, L.; Zhuang, L., Imaging-Guided pH-Sensitive Photodynamic Therapy Using Charge Reversible Upconversion Nanoparticles under Near-Infrared Light. Adv. Funct. Mater. 2013, 23 (24), 3077-3086. 14. Lin, J.; Wang, S.; Huang, P.; Wang, Z.; Chen, S.; Niu, G.; Li, W.; He, J.; Cui, D.; Lu, G.; Chen, X.; Nie, Z., Photosensitizer-Loaded Gold Vesicles with Strong Plasmonic Coupling Effect for Imaging-Guided Photothermal/Photodynamic Therapy. Acs Nano 2013, 7 (6), 5320-5329. 15. Yuan, Y.; Liu, B., Self-Assembled Nanoparticles Based on PEGylated Conjugated Polyelectrolyte and Drug Molecules for Image-Guided Drug Delivery and Photodynamic Therapy. ACS Appl. Mater. Interfaces 2014, 6 (17), 14903-14910. 16. Ji, S.; Ge, J.; Escudero, D.; Wang, Z.; Zhao, J.; Jacquemin, D. Molecular 27



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

Structure-Intersystem Crossing Relationship of Heavy-Atom-Free BODIPY Triplet Photosensitizers. J. Org. Chem. 2015, 80 (11), 5958-5963. 17. Chen, M.; Yin, M., Design and Development of Fluorescent Nanostructures for Bioimaging. Prog. Polym. Sci. 2014, 39 (2), 365-395. 18. Singh, N. K.; Lee, D. S., In Situ Selling pH- and Temperature-Sensitive Biodegradable Block Copolymer Hydrogels for Drug Delivery. J. Controlled Release 2014, 193, 214-227. 19. Yoshida, T.; Lai, T. C.; Kwon, G. S.; Sako, K., pH- and Ion-Sensitive Polymers for Drug Delivery. Expert Opin. Drug Delivery 2013, 10 (11), 1497-1513. 20. Fu, L.; Sun, C.; Yan, L., Galactose and Near Infrared Fluorescence Probe Conjugated pH-Responsive Copolymer for Imaging of Drug Delivery. J. Controlled Release 2015, 213, E72-E73. 21. Fu, L.; Sun, C.; Yan, L., Galactose Targeted pH-Responsive Copolymer Conjugated with Near Infrared Fluorescence Probe for Imaging of Intelligent Drug Delivery. ACS Appl. Mater. Interfaces 2015, 7 (3), 2104-2115. 22. Xing, T.; Lai, B.; Mao, C. Q.; Yan, L. F., Synthesis of NIR Probe Conjugated Polypeptide for Drug Delivery and Imaging. J. Controlled Release 2013, 172 (1), E50-E50. 23. Xing, T.; Lai, B.; Yan, L. F., Disulfide Cross-Linked Polypeptide Nanogel Conjugated with a Fluorescent Probe as a Potential Image-Guided Drug-Delivery Agent. Macromol. Chem. Phys. 2013, 214 (5), 578-588. 24. Xing, T.; Lai, B.; Ye, X. D.; Yan, L. F., Disulfide Core Cross-Linked PEGylated 28


Page 28 of 45

Page 29 of 45

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


Polypeptide Nanogel Prepared by a One-Step Ring Opening Copolymerization of N-Carboxyanhydrides for Drug Delivery. Macromol. Biosci. 2011, 11 (7), 962-969. 25. Xing, T.; Mao, C. Q.; Lai, B.; Yan, L. F., Synthesis of Disulfide-Cross-Linked Polypeptide Nanogel Conjugated with a Near-Infrared Fluorescence Probe for Direct Imaging of Reduction-Induced Drug Release. ACS Appl. Mater. Interfaces. 2012, 4 (10), 5662-5672. 26. Xing, T.; Yang, X. Z.; Fu, L. Y.; Yan, L. F., Near Infrared Fluorescence Probe and Galactose Conjugated Amphiphilic Copolymer for Bioimaging of HepG2 Cells and Endocytosis. Polym. Chem. 2013, 4 (16), 4442-4449. 27. Xing, T.; Yang, X. Z.; Wang, F.; Lai, B.; Yan, L. F., Synthesis of Polypeptide Conjugated with Near Infrared Fluorescence Probe and Doxorubicin for pH-Responsive and Image-Guided Drug Delivery. J. Mater. Chem. 2012, 22 (41), 22290-22300. 28. Zhao, X.; Chen, Z.; Zhao, H.; Zhang, D.; Tao, L.; Lan, M., Multifunctional Magnetic Nanoparticles for Simultaneous Cancer Near-Infrared Imaging and Targeting Photodynamic Therapy. RSC Adv. 2014, 4 (107), 62153-62159.

29



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 30 of 45

Figures Caption Scheme 1. Structure of polypeptides loading BODIPY-Br2, the micellization and pH triggered drug release followed by NIR PDT. Scheme 2. Synthesis of BODIPY-Br2 photosensitizers. Scheme 3. Synthesis of the pH-responsive amphiphilic copolymer. Figure 1．．Absorption (a) and fluorescence emission (b) spectra of BODIPY and BODIPY-Br2 photosensitizers. Figure 2. Absorption spectra of DPBF upon irradiation in the presence of BODIPY-Br2 under 808 nm (a) or 635 nm (b) for different time, and plots of change in absorbance of DPBF at 410 nm at different irradiation time (λirr=808 nm) in the presence of BODIPY-Br2 and BODIPY using Rhodamine B as the standard in DMSO (c). Figure 3. 1H-NMR spectra of the chemicals (10mg/mL) during the synthesis of RAFT-POEGMA-PAsp: RAFT-POEGMA-APA,

a)

RAFT,

d)PAA-PAsp,

b) f)

RAFT-APA, Amino

c)

substituted

RAFT-POEGMA-PAsp in CDCl3 and e)RAFT-POEGMA-PAsp in DMSO-d6 at room temperature. Figure 4. GPC traces of (1)PAA-PAsp, (2)RAFT-POEGMA, (3)POEGMA - PAsp in DMF measured by GPC with a RI detector. Figure 5. Drug release behavior of Polymer/BODIPY-Br2 under neutral (pH=7.4) and acidic (pH=5.5) conditions. 30


Page 31 of 45

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 6. Size and size distribution of the micelles with or without BODIPY-Br2 under neutral (pH=7.4) and acidic (pH=5.5) conditions determined by DLS: Polymers aqueous solution at pH 7.4 (a) and 5.5 (b), polymers aqueous solution with BODIPY-Br2 at pH 7.4 (c) and 5.5 (d), and typical AFM height images of the polymer/BODIPY-Br2 with pH of 7.4 (e) and 5.5 (f), respectively. Figure 7. The cytotoxicity of BODIPY-Br2 only (a) and Polymer/BODIPY-Br2 (b) to HepG2 cells with (red) or without (black) irradiation. Figure

8.

The

fluorescence

images

of

HepG2

cells

cultured

with

Polymer/BODIPY-Br2 for 24h. Figure 9. Images of HepG2 cells treated with Polymer/BODIPY-Br2 (0.1 mg/mL), with or without illumination with 635 nm laser light for 10min.

Table 1. Molecular weights and polydispersity indexes of the obtained copolymers

31



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

Scheme 1. Structure of polypeptides loading BODIPY-Br2, the micellization and pH triggered drug release followed by NIR PDT.

32


Page 32 of 45

Page 33 of 45

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


Scheme 2. Synthesis of BODIPY-Br2 photosensitizers.

33



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

Scheme 3. Synthesis of the pH-responsive amphiphilic copolymer.

34


Page 34 of 45

Page 35 of 45

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．．Absorption (a) and fluorescence emission (b) spectra of BODIPY (5µM) and BODIPY-Br2 (13µM) photosensitizers in CHCl3 at room temperature.

35



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 2. Absorption spectra of DPBF (90µM) upon irradiation in the presence of BODIPY-Br2 under 808 nm (a) or 635 nm (b) for different time, and plots of change in absorbance of DPBF at 410 nm at different irradiation time (λirr=808 nm) in the presence of BODIPY-Br2 (5µM) and BODIPY (5µM) using Rhodamine B (5µM) as the standard in DMSO (c) at room temperature. 36


Page 36 of 45

Page 37 of 45

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


a)

b)

c)

d)

f)

e)

Figure 3. 1H-NMR spectra of the chemicals (10mg/mL) during the synthesis of RAFT-POEGMA-PAsp: a) RAFT, b) RAFT-APA, c) RAFT-POEGMA-APA, d)PAA-PAsp,

f)

Amino

substituted

RAFT-POEGMA-PAsp

e)RAFT-POEGMA-PAsp in DMSO-d6 at room temperature.

37


in

CDCl3

and


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. GPC traces of (1)PAA-PAsp, (2)RAFT-POEGMA, (3)POEGMA - PAsp in DMF measured by GPC with a RI detector at 60 oC.

38


Page 38 of 45

Page 39 of 45

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. Drug release behavior of Polymer/BODIPY-Br2 under neutral (phosphate buffer solution, pH=7.4) and acidic (acetate/ sodium acetate buffer solution, pH=5.5) conditions at 37oC.

39



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 6. Size and size distribution of the micelles with or without BODIPY-Br2 under neutral (pH=7.4) and acidic (pH=5.5) conditions determined by DLS: Polymers aqueous solution at pH 7.4 (a) and 5.5 (b), polymers aqueous solution with BODIPY-Br2 at pH 7.4 (c) and 5.5 (d), and typical AFM height images of the polymer/BODIPY-Br2 with pH of 7.4 (e) and 5.5 (f), respectively.

40


Page 40 of 45

Page 41 of 45

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 7. The cytotoxicity of BODIPY-Br2 only (a) and Polymer/BODIPY-Br2 (b) to HepG2 cells with (red) or without (black) irradiation.

41



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 8. The fluorescence images of HepG2 cells cultured with Polymer/BODIPY-Br2 for 24h.

42


Page 42 of 45

Page 43 of 45

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 9. Images of HepG2 cells treated with Polymer/BODIPY-Br2 (0.1mg/mL), with or without illumination with 635 nm laser light for 10min.

43



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

Table 1. Molecular weights and polydispersity indexes of the obtained copolymers

a

Sample

MnNMR MnGPC Mw/Mn

PAA-PAsp

1972

2090

RAFT-POEGMA

10085

11025 1.09

RAFT- POEGMA-PAsp 11980

13215 1.10

1.06

Molecular weight determined by1-H NMR integration. b Molecular weight determined by GPC.

44


Page 44 of 45

Page 45 of 45

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


TOC

45


pH-Triggered Polypeptides Nanoparticles for Efficient BODIPY

Recommend Documents