Communication
One-Pot Synthesis of Glycopolymer-Porphyrin Conjugate as Photosensitizer for Targeted Cancer Imaging and Photodynamic Therapya Jiawei Lu, Weidong Zhang, Lin Yuan, Wenjuan Ma, Xiao Li, Wei Lu, Yun Zhao,* Gaojian Chen*
One-pot system combining multi-reactions is used to synthesize novel porphyrin-glycopolymer conjugates. Sodium mercury amalgam is used to catalyze the reactions: 1) reduction of RAFT polymerized poly(2-(methacrylamido) glucopyranose) (PMAG), 2) converting protoporphyrin to protoporphyrinogen, 3) thiol-ene coupling reaction of PMAG and protoporphyrinogen. The product is oxidized in the same pot to generate the final porphyrin-PMAG conjugates. The resulting conjugates are characterized by NMR, GPC, UV-Vis, and fluorescence spectroscopy. Glycoparticles (200 nm) bearing glucose units are formed by dissolving the conjugates in water. Glycoparticles show enhanced binding ability toward Con A, bind K562 cells efficiently and kill these cells under light irradiation in dose and light treatment length dependent manners, illustrating the potential biological applications of the conjugates as photosensitizer for cancer imaging and photodynamic therapy. 1. Introduction
J. Lu, Dr. W. Zhang, X. Li, W. Lu, Prof. G. Chen Center for Soft Condensed Matter Physics and Interdisciplinary Research, Soochow University, Suzhou 215006, P. R. China E-mail: [email protected] J. Lu, W. Lu, X. Li, Prof. L. Yuan Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, P. R. China W. Ma, Prof. Y. Zhao Cyrus Tang Hematology Center, Soochow University, Suzhou 215123, P. R. China E-mail: [email protected] Supporting Information is available online from the Wiley Online Library or from the author.
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Photodynamic therapy (PDT) is developing rapidly for tumor treatment, which utilizes photosensitizer and light irradiation at certain wavelength onto target tumor tissues.[1] Porphyrin compounds are one of the most common photosensitizers used in PDT[2] due to their long triple excited state lifetime,[3] and among which, photofrin has received approval for clinical usage in several countries for the treatment of various cancers. Porphyrin compounds have also been used in photochemical internalization (PCI) to potentiate the biological activity of a large variety of molecules that do not readily penetrate the plasma membrane.[4] However, the non-specific skin phototoxicity, poor water solubility, and inefficient delivery to target tumor tissues of these photosensitizers limit their broad application in cancer treatment currently.
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DOI: 10.1002/mabi.201300451
One-Pot Synthesis of Glycopolymer-Porphyrin Conjugate as Photosensitizer . . . www.mbs-journal.de
Carbohydrates are non-ionic, cell-permeable compounds, and play a pivotal role in biological processes such as cell–cell recognition and cell signaling. The glycomodification of porphyrin could increase its water solubility and enable it for targeted PDT against tumor cells, due to the specific interaction of glycopolymers with lectin type receptors, which are over-expressed in certain malignant cells.[5] Conjugates of photosensitizers such as porphyrins and pathalocyamines with small molecular carbohydrate have been synthesized in order to enhance cellular uptake and PDT efficacy.[6] Whereas individual protein saccharide interactions are typically weak, the multivalent interactions employed in biological systems, known as the ‘‘cluster glycoside effect,’’ can be characterized as high affinity and high specificity. Synthetic polymers with pendent sugar moieties,[7] which are able to interact with lectins as multivalent ligands in a similar manner to natural glycoproteins, will be a good candidate to conjugate with photosensitizer for PDT. Although this type of porphyringlycopolymer conjugate has seldom been reported,[8] a simpler and more efficient reaction to prepare novel porphyrin-glycopolymer conjugates is still in need. Thiol– ene addition or click reaction,[9] either the anti-Markovnikov radical addition[10] or the base/nucleophile catalyzed Michael addition,[11] is the reaction that has attracted the attention of many researchers due to its high efficiency and relatively mild conditions. There are two vinyl groups in protoporphyrin (PpIX), however no report has been found to ‘‘click’’ thiols to the vinyl groups of PpIX directly via the thiol–ene reaction. Researchers reported the addition of thiol-sugar by esterification of PpIX first, converting to isohematoporphyrin, bromation, and reacting with
OH
OH O
HO HO
OH NH
S O
(1)
O
HO HO
OH
OH O
CN
S
NH
(3)
Me
NH N
COOH
NH
CN
n
(2) Me COOH
Me
Me
NH HN NH HN
COOH
Me
OH O
Me Me
S
Me
O
HO HO
Me
Me Me
N HN
(4)
OH
OH O
n
Me
O
HO HO
CN
HS
n
Me
thiol-sugars finally in multi steps.[12] As early as in the 1960s researchers found that the vinyl groups of protoporphyrinogen were reactive enough for the addition of thio-containing compounds and the formation of thioether bonds.[13] Converting PpIX to propoporphyrinogen will make it possible for the modification of PpIX with thiol-teminal polymers. Glucose transporters (GLUTs) are membrane proteins that allow the energy independent transport of glucose across the cell membrane. It is noted that GLUTs, especially GLUT1 or GLUT3, are over-expressed in many cancer cells and possesses high affinity toward glucose. In addition, nanoparticles with glucosefunctionalized coronae have recently been found to have excellent stability in FBS and have minimal interactions with the serum proteins.[14] Bearing these in mind, in the current study, we synthesized a novel porphyrin-glycopolymer conjugate, PpIX-poly(2-(Methacrylamido)glucopyranose), in a simple approach: we synthesized poly(2-(Methacrylamido)glucopyranose) via reversible addition fragmentation chain transfer (RAFT) polymerization firstly, and then conjugated with PpIX via a one-pot reaction combining the multi-step reactions (Scheme 1): 1) reduction of RAFT end group to thiols, 2) reduction of PpIX to Propoporphyrinogen, 3) thiol–ene reaction of Protoporphyrinogen with thiolterminal glycopolymer and 4) the oxidation of Protoporphyrinogen to afford the porphyrin-glycopolymer conjugate. The physic-chemical characteristics of this conjugates were studied, and its binding properties with lectin and the in vitro anti-cancer effect for PDT were assessed as well.
NH CN
S NH N
COOH
n
N HN
Me
COOH
COOH
One-pot conjugation Scheme 1. One-pot reaction to fabricate the Protoporphyrin-glycopolymer conjugate.a) a) Sodium mercury amalgam, sodium acetate buffer; DMSO/acetonitrile.
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2. Results and Discussion 2.1. Synthesis and Characterization of PorphyrinGlycopolymer Conjugate RAFT polymerization was firstly used to synthesize glycopolymers for the subsequent one-pot conjugation reaction: Glycopolymer was synthesized by polymerization of MAG, using 2-cyanoprop-2-yl-a-dithionaphthalate (CPDN) as the RAFT agent and 2,20 -azobis(isobutyronitrile) (AIBN) as the initiator. We obtained glycopolymers poly(2-(methacrylamido) glucopyranose) (PMAG) with various molecular weights by controlling the polymerization time and initial ratios between monomer and RAFT agent. Three glycopolymers (polymer A, C, E) were chosen for further conjugation with PpIX, a summary of their molecular weights and PDIs were shown in Table 1. The chemical structure was confirmed by 1H NMR (Figure S1, Supporting Information). The signals at 7.4–8.1 ppm were attributed to the aromatic protons of the naphthalene units in CPDN, which revealed that the RAFT agents were attached to the polymer. The polymers were characterized by GPC, and present low PDIs. The synthesized glycopolymers PMAGs were then used to prepare the porphyrin-PMAG conjugates. As previously reported, sodium amalgam is able to convert PpIX to Protoporphyrinogen easily.[13a,13b] Thioesters are often cleaved under reducing, for example sodium borohydride is used to reduce RAFT polymers to obtain thiol-terminal molecules.[15] Therefore, the catalytic system (sodium mercury amalgam) is also used to reduce RAFT end groups of polymers and catalyze the coupling reaction. Herein, we tried the multi-step reactions in one-pot using sodium amalgam as catalyst: reduction of PpIX to Propoporphyrinogen, reduction of RAFT end group to thiols, thiol–ene reaction of Protoporphyrinogen with thiol-terminal glycopolymer. In the same pot, the oxidation of Protoporphyrinogen to PpIX was done by adding DMSO to afford the final porphyrinPMAG conjugates. GPC was used to monitor the one-pot conjugation between glycopolymer and PpIX, by taking samples at various times during the reaction. As shown in Figure S2, Supporting Information, after 24 h, the reaction almost complete. To guarantee the full conversion, the reaction
was continued for 48 h. NMR characterization of the porphyrin-PMAG conjugate further confirmed the successful incorporation of porphyrin onto the polymer chain with the observation of several signals between 3.3 and 2.5 ppm (Figure S1, Supporting Information), representing b-pyrrolic methyl groups of the conjugate. Meanwhile, the chemical shifts at 7.4–8.1 ppm disappeared after modification, indicating that almost all end groups in the CPDN units have been reduced with sodium mercury amalgam and then reacted with Protoporphyrinogen. We found that the peaks at 5.2 ppm always existed either before or after modification, indicating that the hemiacetal groups in PMAG were not reduced to hydroxyl group by sodium mercury amalgam, keeping the ring structure of the sugar units intacted. FT-IR (Figure S3, Supporting Information) was another measure to confirm the incorporation of porphyrin onto the polymer, showing stronger absorption band at 1400 cm1 for the conjugate due to the vibrations of the CH2 and CH3 groups of PpIX.[16] The purified final polymers of different molecular weights were compared with starting glycopolymers and their typical GPC traces were shown in Figure 1a. It should be noted that for polymer C, the GPC trace at the lower molecular weight end is not going back to the baseline, because it is connected with the trace of DMF, which has been cut in Figure 1 for better comparison. After modification, the Mn;GPC of polymer B, D, and F increased from 20 000 to 44 900 g mol1, 5800 to 14 900 g mol1, and 10 200 to 14 900 g mol1, respectively, almost doubled that of glycopolymer A, C, and E. The PDIs were slight ascent with shoulder in the low molecular region of GPC traces, indicating that there might be small amount of starting glycopolymers which had not been removed completely via dialysis. All the GPC data were displayed in Table 1. PpIX has an absorption peak in the region of 400–410 and 500–600 nm in DMF. The UV absorption spectra of the porphyrin-PAMG conjugate both in DMF and water were analyzed and compared with the small molecular PpIX. Figure 1b showed an intense and sharp band at 400 nm and multi Q bands from 470 to 600 nm for polymer B in DMF, while there was no absorption at those wavelengths for polymer A, which confirmed the successful incorporation of porphyrin into the polymer. Compared to the UV
Table 1. GPC Characterization of the polymers before and after modification.
Samples Mn;th [g mol1] 1
Mn;GPC [g mol ] PDI a)
342
c)
Aa)
Bb)
Ca)
Db)
Ea)
Fb)
12 800
26 200
6400
13 400
9600
19 800
20 000
44 900
5800
14 900
10 200
18 600
1.13
1.22
1.25
1.30
1.15
1.17
PMAG before modification; b)Porphyrin-PMAG conjugate; c)Determined by GPC.
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One-Pot Synthesis of Glycopolymer-Porphyrin Conjugate as Photosensitizer . . . www.mbs-journal.de
(a) 2.5
(b)
B
A
Polymer B, H2O Protoporphyrin IX, H2O
1.0
F
E
1.2
Polymer B, DMF Protoporphyrin IX, DMF
2.0
0.8
D
Absorbance
dW/dlogM
C 1.5
1.0
0.6
0.4
0.5
0.2
0.0 300
0.0 3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
350
400
450
500
550
600
Wavelength (nm)
LogMw 20
0.20 0.18
18
Absorption intensity at 365 nm
(d)
(c)
16
Intensity (%)
14 12
0.16
(e)
0.14 0.12 0.10 0.08 0.06 0.04 0.02
10
0.00 0.01
0.1
1
-1
8
logC (c, g L )
6 4 2
3.00 μm
0 1
10
100
1000
Size (d, nm)
Figure 1. a) GPC chromatographs of glycopolymer (A,C,E) and porphyrincontaining glycopolymer (B,D,F); b) UVVis spectra of Protoporphyrin IX and polymer B at different solvents; c) SEM image of glycoparticles formed for polymer B in aqueous solution; d) DLS measurement of polymer B in aqueous solution; e) CAC determination by extrapolation of the difference of absorbance at 365 nm for DPH in polymer B solutions.
absorption of PpIX (0.01 mg mL1) in DMF, there was a blue shift for polymer B both in water and DMF, from 377 and 405 nm to 368 and 400 nm, respectively (Figure 1b), which further indicated that porphyrin group was successfully incorporated into the polymer via chemical bonding. Similar results were obtained in aqueous solutions. The UV absorption peaks of the molecules in DMF and water were different, which is due to the differences in polarity in different solvents.[17] Fluorescence spectrum of the porphyrin-PMAG conjugate was further recorded in aqueous solution. Figure S4, Supporting Information showed the fluorescence spectrum of polymer B in aqueous solution (lex ¼ 488 nm). Strong emission peaks (lem ¼ 633 nm) was observed, which could be utilized for diagnosis or imaging applications. 2.2. Formation and Characterization of Nanoparticles in Water As the morphology of amphiphilic polymer chains in selective solvents, especially in aqueous solutions, is not
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only important but also the first step for any of their biorelated potential applications. The porphyrin-PMAG conjugate is like a ABA block copolymer with hydrophobic porphyrin in the middle and hydrophilic glycopolymer at both ends. The aggregation behavior of polymer B in water was characterized by the dynamic light scattering (DLS) technique. From Figure 1d, the mean size of polymer B aggregates was 209 nm. The aggregated nanoparticles of polymer B were further confirmed by SEM. As shown in Figure 1c, it was evident that the self-assembled nanoparticles had a regular spherical shape and are well dispersed. From the SEM image, the average sizes of the particles were 160 nm. The difference in the sizes determined by different methods was mainly attributed to the fact that the size measured by the DLS was the hydrodynamic diameter of micelles in aqueous solution, while the size observed by SEM was diameter of the dried micelles. Similar conclusion was drawn in literature.[18] To further confirm the formation of a self-assembled structure for polymer B in aqueous solutions, critical aggregation concentration (CAC) of polymer B was tested by the
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hydrophobic dye inclusion method (Figure 1e).[19] A value of 0.103 g L1 was obtained. 2.3. Lectin Binding Assay To measure the biofunctionality of the glucose moieties and how the polymeric ligands would react in the biological system, the ligand-lectin binding ability of the glycopolymer micelles was performed by specific binding using Concanavalin A (Con A), a lectin specific for binding glucose and mannose. Bioactivity of the glycoparticles was tested using online DLS measurements by monitoring the size growth with conjugation time (Figure 2a). The particle sizes increased steadily as more lectin was attached to the particles and particles were cross-linked with each other. This experiment was conducted by adding 200 mL of the concentrated lectin stock solution (1 mg mL1) to the polymer solution 600 mL (1 mg mL1). Since Con A has four binding sites, inter-particle cross-linking occurred, and led to an average hydrodynamic diameters of 850 nm after 42 min, which was a significant increase from the original micelle size of around 190 nm (Figure S5, Supporting
(a)
2.4. Cell Adhesion Studies and In Vitro Cytotoxicity To test its high affinity toward certain cells for potential bio-labeling or imaging applications, K562 and normal bone marrow (NBM) cells were chosen as a model. GLUT is a protein that possesses carbohydrate recognition domains (CRD), and it has high affinity towards glucose
I
II
III
IV
V
VI
K562
(b) 1000
Information). Further experiments with only the linear glucose polymer did not result in any visible complexation with Con A (Figure 2a). The result was similar to the reports by Stenzel and co-workers,[20] our understanding was that the PMAG had a weaker binding ability with Con A compared to mannose-containing polymers. However, in the form of nanoparticles with a glucose corona, the binding ability was greatly enhanced due to stronger multivalent effect displayed by the sufficient numbers of glucose groups on the surface of the nanoparticles. This enhanced binding ability guaranteed its further application for cancer imaging and therapy. The interaction of Con A with porphyrin-containing glycoparticles was time dependent similar as others’ reports.[21]
Polymer A Polymer B
800
Size/ nm
600
400
NBM
200
0 0
5
10
15
20
25
30
35
40
Conjugation time/ min
(d)
1.8
1.7
No light Light
100
( e)
1.5
1.4
60
40
1.2 0
100
200
300
Time (s)
400
500
600
Without Polymer B 100
With Polymer B
60
40
20
20
1.3
120
80
80
1.6
Cell viability (%)
Absorbance at 424 nm
120
Polymer B ZnPc
Cell viability (%)
(c)
0
0 0
0.02 0.04 0.06 0.08 0.1 0.2 0.4 0.6 0.8
1
-1
Concentration (mg mL )
0
10
20
30
40
60
Time (min)
Figure 2. a) Dynamic light scattering measurements of the sizes evolution of polymer A and polymer B with Con A versus conjugation times; b) Fluorescent microscopy images of K562 cells and NBM cells mixed with glyco-particles, single-channel fluorescence image (I, II, IV, V), dual-channel fluorescence synchronous acquisition (III, VI), Nuclei were stained with Hoechst 33342 (blue, lex ¼ 405 nm). Aggregations of glyco-particles could be detected as green dots (lex ¼ 488 nm); c) Comparison of the rates of decay of DPBF in DMF using Polymer B and PpIX as the photosensitizers; d) The cell viability of K562 cells treated with porphyrin-PMAG (Polymer B) at various concentrations for 20 min with or without light treatment; e) The cell viability of K562 cells treated with porphyrin-PMAG (Polymer B, 0.02 mg mL1) for various time.
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One-Pot Synthesis of Glycopolymer-Porphyrin Conjugate as Photosensitizer . . . www.mbs-journal.de
and glucosamine. GLUT, especially GLUT1, are overexpressed in many cancer cells including leukemia cells.[22] Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that K562 had a much higher expression of GLUT1, compared to that of NBM cells (Figure S6, Supporting Information). After 4 h incubation of the cells with the conjugate, stronger fluorescence signal were observed on the surface of K562 cells, but relative little on the surface of NBM cells (Figure 2b). The conjugate could aggregate and bind onto K562 cells effectively indicates that the glyco-particles have a high affinity toward GLUT1 proteins expressed in the cell membrane. Singlet oxygen has been implicated as an intermediary species leading to cell death following the excitation of photosensitizers in PDT. Measurements of singlet oxygen yields are thus important in assessing the potential effectiveness of the new synthesized polymeric pre-drug for PDT. The singlet oxygen quantum yield (FD) of Polymer B was measured in DMF by the method of the rate of decay of DPBF in DMF, using ZnPc as the reference (Figure 2c),[23] the value obtained for Polymer B was 0.29, relative to ZnPc whose FD is 0.56, close to the report by Redmond.[24] Next, we tested the efficacy of porphyrin-PMAG to suppress the growth of K562 cells in vitro. The polymer had cytotoxic effect against the growth of K562 in a dose dependent manner; in addition the treatment under light irradiation greatly enhance the cytotoxic effect (Figure 2d), indicating the reactive oxygen species (ROS) formed upon light irradiations kill K562 cells effectively. It should be noted that with increased usage of the porphyrin-PMAG, cell viability decreased even without light treatment, which might be due to the fact that the glycopolymer binds to GLUTs on the surface of the cells and interferes their uptake of glucose, leading to slow cell growth or cell death. When K562 cells were treated with the equal amount of conjugates (0.02 mg mL1), the cytotoxic effect was also light treatment length dependent (Figure 2e).
3. Conclusion In summary, a series of well-controlled glycopolymers were synthesized by RAFT polymerization, followed by employing novel catalytic system (sodium mercury amalgam) to ‘‘activate’’ PpIX, reducing RAFT glycopolymer and carrying out the thiol–ene click reaction in one pot to synthesize the novel porphyrin-glycopolymer conjugate. DLS measurements and SEM observation confirmed the formation of nanoparticles by the self-assembly of conjugates in aqueous solution. The bioactivity of the glyco-particles was examined using lectin Con A (Canavalia ensiformis) and cancer cells (K562) that over-expressed GLUTs (GLUT1). Results revealed that the glucose functionalities in the
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conjugate remained bioactive and showed enhanced affinity toward specific lectins and cells as the conjugate was in the form of nanoparticles. In vitro studies showed that light enhanced the cytotoxic effect of the polymer against K562 cells in dose dependent and light treatment length dependent manners. This provided an efficient and simple approach to synthesize well-defined porphyrincontaining glycopolymers for potential cancer imaging and PDT.
Acknowledgements: The authors thank the National Natural Science Foundation of China (No. 91027040, 21004042, 21374069, 21074083, 21104051), National Key Scientific Project of China (973 Program #2011CB933501), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry for financial support.
Received: October 10, 2013; Revised: October 25, 2013; Published online: November 27, 2013; DOI: 10.1002/mabi.201300451 Keywords: biopolymers; glycopolymers; one-pot; photodynamic therapy; reversible addition fragmentation chain transfer (RAFT)
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One-Pot Synthesis of Glycopolymer-Porphyrin Conjugate as Photosensitizer for Targeted Cancer Imaging and Photodynamic Therapya Jiawei Lu, Weidong Zhang, Lin Yuan, Wenjuan Ma, Xiao Li, Wei Lu, Yun Zhao,* Gaojian Chen*
One-pot system combining multi-reactions is used to synthesize novel porphyrin-glycopolymer conjugates. Sodium mercury amalgam is used to catalyze the reactions: 1) reduction of RAFT polymerized poly(2-(methacrylamido) glucopyranose) (PMAG), 2) converting protoporphyrin to protoporphyrinogen, 3) thiol-ene coupling reaction of PMAG and protoporphyrinogen. The product is oxidized in the same pot to generate the final porphyrin-PMAG conjugates. The resulting conjugates are characterized by NMR, GPC, UV-Vis, and fluorescence spectroscopy. Glycoparticles (200 nm) bearing glucose units are formed by dissolving the conjugates in water. Glycoparticles show enhanced binding ability toward Con A, bind K562 cells efficiently and kill these cells under light irradiation in dose and light treatment length dependent manners, illustrating the potential biological applications of the conjugates as photosensitizer for cancer imaging and photodynamic therapy. 1. Introduction
J. Lu, Dr. W. Zhang, X. Li, W. Lu, Prof. G. Chen Center for Soft Condensed Matter Physics and Interdisciplinary Research, Soochow University, Suzhou 215006, P. R. China E-mail: [email protected] J. Lu, W. Lu, X. Li, Prof. L. Yuan Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, P. R. China W. Ma, Prof. Y. Zhao Cyrus Tang Hematology Center, Soochow University, Suzhou 215123, P. R. China E-mail: [email protected] Supporting Information is available online from the Wiley Online Library or from the author.
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Photodynamic therapy (PDT) is developing rapidly for tumor treatment, which utilizes photosensitizer and light irradiation at certain wavelength onto target tumor tissues.[1] Porphyrin compounds are one of the most common photosensitizers used in PDT[2] due to their long triple excited state lifetime,[3] and among which, photofrin has received approval for clinical usage in several countries for the treatment of various cancers. Porphyrin compounds have also been used in photochemical internalization (PCI) to potentiate the biological activity of a large variety of molecules that do not readily penetrate the plasma membrane.[4] However, the non-specific skin phototoxicity, poor water solubility, and inefficient delivery to target tumor tissues of these photosensitizers limit their broad application in cancer treatment currently.
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DOI: 10.1002/mabi.201300451
One-Pot Synthesis of Glycopolymer-Porphyrin Conjugate as Photosensitizer . . . www.mbs-journal.de
Carbohydrates are non-ionic, cell-permeable compounds, and play a pivotal role in biological processes such as cell–cell recognition and cell signaling. The glycomodification of porphyrin could increase its water solubility and enable it for targeted PDT against tumor cells, due to the specific interaction of glycopolymers with lectin type receptors, which are over-expressed in certain malignant cells.[5] Conjugates of photosensitizers such as porphyrins and pathalocyamines with small molecular carbohydrate have been synthesized in order to enhance cellular uptake and PDT efficacy.[6] Whereas individual protein saccharide interactions are typically weak, the multivalent interactions employed in biological systems, known as the ‘‘cluster glycoside effect,’’ can be characterized as high affinity and high specificity. Synthetic polymers with pendent sugar moieties,[7] which are able to interact with lectins as multivalent ligands in a similar manner to natural glycoproteins, will be a good candidate to conjugate with photosensitizer for PDT. Although this type of porphyringlycopolymer conjugate has seldom been reported,[8] a simpler and more efficient reaction to prepare novel porphyrin-glycopolymer conjugates is still in need. Thiol– ene addition or click reaction,[9] either the anti-Markovnikov radical addition[10] or the base/nucleophile catalyzed Michael addition,[11] is the reaction that has attracted the attention of many researchers due to its high efficiency and relatively mild conditions. There are two vinyl groups in protoporphyrin (PpIX), however no report has been found to ‘‘click’’ thiols to the vinyl groups of PpIX directly via the thiol–ene reaction. Researchers reported the addition of thiol-sugar by esterification of PpIX first, converting to isohematoporphyrin, bromation, and reacting with
OH
OH O
HO HO
OH NH
S O
(1)
O
HO HO
OH
OH O
CN
S
NH
(3)
Me
NH N
COOH
NH
CN
n
(2) Me COOH
Me
Me
NH HN NH HN
COOH
Me
OH O
Me Me
S
Me
O
HO HO
Me
Me Me
N HN
(4)
OH
OH O
n
Me
O
HO HO
CN
HS
n
Me
thiol-sugars finally in multi steps.[12] As early as in the 1960s researchers found that the vinyl groups of protoporphyrinogen were reactive enough for the addition of thio-containing compounds and the formation of thioether bonds.[13] Converting PpIX to propoporphyrinogen will make it possible for the modification of PpIX with thiol-teminal polymers. Glucose transporters (GLUTs) are membrane proteins that allow the energy independent transport of glucose across the cell membrane. It is noted that GLUTs, especially GLUT1 or GLUT3, are over-expressed in many cancer cells and possesses high affinity toward glucose. In addition, nanoparticles with glucosefunctionalized coronae have recently been found to have excellent stability in FBS and have minimal interactions with the serum proteins.[14] Bearing these in mind, in the current study, we synthesized a novel porphyrin-glycopolymer conjugate, PpIX-poly(2-(Methacrylamido)glucopyranose), in a simple approach: we synthesized poly(2-(Methacrylamido)glucopyranose) via reversible addition fragmentation chain transfer (RAFT) polymerization firstly, and then conjugated with PpIX via a one-pot reaction combining the multi-step reactions (Scheme 1): 1) reduction of RAFT end group to thiols, 2) reduction of PpIX to Propoporphyrinogen, 3) thiol–ene reaction of Protoporphyrinogen with thiolterminal glycopolymer and 4) the oxidation of Protoporphyrinogen to afford the porphyrin-glycopolymer conjugate. The physic-chemical characteristics of this conjugates were studied, and its binding properties with lectin and the in vitro anti-cancer effect for PDT were assessed as well.
NH CN
S NH N
COOH
n
N HN
Me
COOH
COOH
One-pot conjugation Scheme 1. One-pot reaction to fabricate the Protoporphyrin-glycopolymer conjugate.a) a) Sodium mercury amalgam, sodium acetate buffer; DMSO/acetonitrile.
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2. Results and Discussion 2.1. Synthesis and Characterization of PorphyrinGlycopolymer Conjugate RAFT polymerization was firstly used to synthesize glycopolymers for the subsequent one-pot conjugation reaction: Glycopolymer was synthesized by polymerization of MAG, using 2-cyanoprop-2-yl-a-dithionaphthalate (CPDN) as the RAFT agent and 2,20 -azobis(isobutyronitrile) (AIBN) as the initiator. We obtained glycopolymers poly(2-(methacrylamido) glucopyranose) (PMAG) with various molecular weights by controlling the polymerization time and initial ratios between monomer and RAFT agent. Three glycopolymers (polymer A, C, E) were chosen for further conjugation with PpIX, a summary of their molecular weights and PDIs were shown in Table 1. The chemical structure was confirmed by 1H NMR (Figure S1, Supporting Information). The signals at 7.4–8.1 ppm were attributed to the aromatic protons of the naphthalene units in CPDN, which revealed that the RAFT agents were attached to the polymer. The polymers were characterized by GPC, and present low PDIs. The synthesized glycopolymers PMAGs were then used to prepare the porphyrin-PMAG conjugates. As previously reported, sodium amalgam is able to convert PpIX to Protoporphyrinogen easily.[13a,13b] Thioesters are often cleaved under reducing, for example sodium borohydride is used to reduce RAFT polymers to obtain thiol-terminal molecules.[15] Therefore, the catalytic system (sodium mercury amalgam) is also used to reduce RAFT end groups of polymers and catalyze the coupling reaction. Herein, we tried the multi-step reactions in one-pot using sodium amalgam as catalyst: reduction of PpIX to Propoporphyrinogen, reduction of RAFT end group to thiols, thiol–ene reaction of Protoporphyrinogen with thiol-terminal glycopolymer. In the same pot, the oxidation of Protoporphyrinogen to PpIX was done by adding DMSO to afford the final porphyrinPMAG conjugates. GPC was used to monitor the one-pot conjugation between glycopolymer and PpIX, by taking samples at various times during the reaction. As shown in Figure S2, Supporting Information, after 24 h, the reaction almost complete. To guarantee the full conversion, the reaction
was continued for 48 h. NMR characterization of the porphyrin-PMAG conjugate further confirmed the successful incorporation of porphyrin onto the polymer chain with the observation of several signals between 3.3 and 2.5 ppm (Figure S1, Supporting Information), representing b-pyrrolic methyl groups of the conjugate. Meanwhile, the chemical shifts at 7.4–8.1 ppm disappeared after modification, indicating that almost all end groups in the CPDN units have been reduced with sodium mercury amalgam and then reacted with Protoporphyrinogen. We found that the peaks at 5.2 ppm always existed either before or after modification, indicating that the hemiacetal groups in PMAG were not reduced to hydroxyl group by sodium mercury amalgam, keeping the ring structure of the sugar units intacted. FT-IR (Figure S3, Supporting Information) was another measure to confirm the incorporation of porphyrin onto the polymer, showing stronger absorption band at 1400 cm1 for the conjugate due to the vibrations of the CH2 and CH3 groups of PpIX.[16] The purified final polymers of different molecular weights were compared with starting glycopolymers and their typical GPC traces were shown in Figure 1a. It should be noted that for polymer C, the GPC trace at the lower molecular weight end is not going back to the baseline, because it is connected with the trace of DMF, which has been cut in Figure 1 for better comparison. After modification, the Mn;GPC of polymer B, D, and F increased from 20 000 to 44 900 g mol1, 5800 to 14 900 g mol1, and 10 200 to 14 900 g mol1, respectively, almost doubled that of glycopolymer A, C, and E. The PDIs were slight ascent with shoulder in the low molecular region of GPC traces, indicating that there might be small amount of starting glycopolymers which had not been removed completely via dialysis. All the GPC data were displayed in Table 1. PpIX has an absorption peak in the region of 400–410 and 500–600 nm in DMF. The UV absorption spectra of the porphyrin-PAMG conjugate both in DMF and water were analyzed and compared with the small molecular PpIX. Figure 1b showed an intense and sharp band at 400 nm and multi Q bands from 470 to 600 nm for polymer B in DMF, while there was no absorption at those wavelengths for polymer A, which confirmed the successful incorporation of porphyrin into the polymer. Compared to the UV
Table 1. GPC Characterization of the polymers before and after modification.
Samples Mn;th [g mol1] 1
Mn;GPC [g mol ] PDI a)
342
c)
Aa)
Bb)
Ca)
Db)
Ea)
Fb)
12 800
26 200
6400
13 400
9600
19 800
20 000
44 900
5800
14 900
10 200
18 600
1.13
1.22
1.25
1.30
1.15
1.17
PMAG before modification; b)Porphyrin-PMAG conjugate; c)Determined by GPC.
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One-Pot Synthesis of Glycopolymer-Porphyrin Conjugate as Photosensitizer . . . www.mbs-journal.de
(a) 2.5
(b)
B
A
Polymer B, H2O Protoporphyrin IX, H2O
1.0
F
E
1.2
Polymer B, DMF Protoporphyrin IX, DMF
2.0
0.8
D
Absorbance
dW/dlogM
C 1.5
1.0
0.6
0.4
0.5
0.2
0.0 300
0.0 3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
350
400
450
500
550
600
Wavelength (nm)
LogMw 20
0.20 0.18
18
Absorption intensity at 365 nm
(d)
(c)
16
Intensity (%)
14 12
0.16
(e)
0.14 0.12 0.10 0.08 0.06 0.04 0.02
10
0.00 0.01
0.1
1
-1
8
logC (c, g L )
6 4 2
3.00 μm
0 1
10
100
1000
Size (d, nm)
Figure 1. a) GPC chromatographs of glycopolymer (A,C,E) and porphyrincontaining glycopolymer (B,D,F); b) UVVis spectra of Protoporphyrin IX and polymer B at different solvents; c) SEM image of glycoparticles formed for polymer B in aqueous solution; d) DLS measurement of polymer B in aqueous solution; e) CAC determination by extrapolation of the difference of absorbance at 365 nm for DPH in polymer B solutions.
absorption of PpIX (0.01 mg mL1) in DMF, there was a blue shift for polymer B both in water and DMF, from 377 and 405 nm to 368 and 400 nm, respectively (Figure 1b), which further indicated that porphyrin group was successfully incorporated into the polymer via chemical bonding. Similar results were obtained in aqueous solutions. The UV absorption peaks of the molecules in DMF and water were different, which is due to the differences in polarity in different solvents.[17] Fluorescence spectrum of the porphyrin-PMAG conjugate was further recorded in aqueous solution. Figure S4, Supporting Information showed the fluorescence spectrum of polymer B in aqueous solution (lex ¼ 488 nm). Strong emission peaks (lem ¼ 633 nm) was observed, which could be utilized for diagnosis or imaging applications. 2.2. Formation and Characterization of Nanoparticles in Water As the morphology of amphiphilic polymer chains in selective solvents, especially in aqueous solutions, is not
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only important but also the first step for any of their biorelated potential applications. The porphyrin-PMAG conjugate is like a ABA block copolymer with hydrophobic porphyrin in the middle and hydrophilic glycopolymer at both ends. The aggregation behavior of polymer B in water was characterized by the dynamic light scattering (DLS) technique. From Figure 1d, the mean size of polymer B aggregates was 209 nm. The aggregated nanoparticles of polymer B were further confirmed by SEM. As shown in Figure 1c, it was evident that the self-assembled nanoparticles had a regular spherical shape and are well dispersed. From the SEM image, the average sizes of the particles were 160 nm. The difference in the sizes determined by different methods was mainly attributed to the fact that the size measured by the DLS was the hydrodynamic diameter of micelles in aqueous solution, while the size observed by SEM was diameter of the dried micelles. Similar conclusion was drawn in literature.[18] To further confirm the formation of a self-assembled structure for polymer B in aqueous solutions, critical aggregation concentration (CAC) of polymer B was tested by the
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hydrophobic dye inclusion method (Figure 1e).[19] A value of 0.103 g L1 was obtained. 2.3. Lectin Binding Assay To measure the biofunctionality of the glucose moieties and how the polymeric ligands would react in the biological system, the ligand-lectin binding ability of the glycopolymer micelles was performed by specific binding using Concanavalin A (Con A), a lectin specific for binding glucose and mannose. Bioactivity of the glycoparticles was tested using online DLS measurements by monitoring the size growth with conjugation time (Figure 2a). The particle sizes increased steadily as more lectin was attached to the particles and particles were cross-linked with each other. This experiment was conducted by adding 200 mL of the concentrated lectin stock solution (1 mg mL1) to the polymer solution 600 mL (1 mg mL1). Since Con A has four binding sites, inter-particle cross-linking occurred, and led to an average hydrodynamic diameters of 850 nm after 42 min, which was a significant increase from the original micelle size of around 190 nm (Figure S5, Supporting
(a)
2.4. Cell Adhesion Studies and In Vitro Cytotoxicity To test its high affinity toward certain cells for potential bio-labeling or imaging applications, K562 and normal bone marrow (NBM) cells were chosen as a model. GLUT is a protein that possesses carbohydrate recognition domains (CRD), and it has high affinity towards glucose
I
II
III
IV
V
VI
K562
(b) 1000
Information). Further experiments with only the linear glucose polymer did not result in any visible complexation with Con A (Figure 2a). The result was similar to the reports by Stenzel and co-workers,[20] our understanding was that the PMAG had a weaker binding ability with Con A compared to mannose-containing polymers. However, in the form of nanoparticles with a glucose corona, the binding ability was greatly enhanced due to stronger multivalent effect displayed by the sufficient numbers of glucose groups on the surface of the nanoparticles. This enhanced binding ability guaranteed its further application for cancer imaging and therapy. The interaction of Con A with porphyrin-containing glycoparticles was time dependent similar as others’ reports.[21]
Polymer A Polymer B
800
Size/ nm
600
400
NBM
200
0 0
5
10
15
20
25
30
35
40
Conjugation time/ min
(d)
1.8
1.7
No light Light
100
( e)
1.5
1.4
60
40
1.2 0
100
200
300
Time (s)
400
500
600
Without Polymer B 100
With Polymer B
60
40
20
20
1.3
120
80
80
1.6
Cell viability (%)
Absorbance at 424 nm
120
Polymer B ZnPc
Cell viability (%)
(c)
0
0 0
0.02 0.04 0.06 0.08 0.1 0.2 0.4 0.6 0.8
1
-1
Concentration (mg mL )
0
10
20
30
40
60
Time (min)
Figure 2. a) Dynamic light scattering measurements of the sizes evolution of polymer A and polymer B with Con A versus conjugation times; b) Fluorescent microscopy images of K562 cells and NBM cells mixed with glyco-particles, single-channel fluorescence image (I, II, IV, V), dual-channel fluorescence synchronous acquisition (III, VI), Nuclei were stained with Hoechst 33342 (blue, lex ¼ 405 nm). Aggregations of glyco-particles could be detected as green dots (lex ¼ 488 nm); c) Comparison of the rates of decay of DPBF in DMF using Polymer B and PpIX as the photosensitizers; d) The cell viability of K562 cells treated with porphyrin-PMAG (Polymer B) at various concentrations for 20 min with or without light treatment; e) The cell viability of K562 cells treated with porphyrin-PMAG (Polymer B, 0.02 mg mL1) for various time.
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One-Pot Synthesis of Glycopolymer-Porphyrin Conjugate as Photosensitizer . . . www.mbs-journal.de
and glucosamine. GLUT, especially GLUT1, are overexpressed in many cancer cells including leukemia cells.[22] Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that K562 had a much higher expression of GLUT1, compared to that of NBM cells (Figure S6, Supporting Information). After 4 h incubation of the cells with the conjugate, stronger fluorescence signal were observed on the surface of K562 cells, but relative little on the surface of NBM cells (Figure 2b). The conjugate could aggregate and bind onto K562 cells effectively indicates that the glyco-particles have a high affinity toward GLUT1 proteins expressed in the cell membrane. Singlet oxygen has been implicated as an intermediary species leading to cell death following the excitation of photosensitizers in PDT. Measurements of singlet oxygen yields are thus important in assessing the potential effectiveness of the new synthesized polymeric pre-drug for PDT. The singlet oxygen quantum yield (FD) of Polymer B was measured in DMF by the method of the rate of decay of DPBF in DMF, using ZnPc as the reference (Figure 2c),[23] the value obtained for Polymer B was 0.29, relative to ZnPc whose FD is 0.56, close to the report by Redmond.[24] Next, we tested the efficacy of porphyrin-PMAG to suppress the growth of K562 cells in vitro. The polymer had cytotoxic effect against the growth of K562 in a dose dependent manner; in addition the treatment under light irradiation greatly enhance the cytotoxic effect (Figure 2d), indicating the reactive oxygen species (ROS) formed upon light irradiations kill K562 cells effectively. It should be noted that with increased usage of the porphyrin-PMAG, cell viability decreased even without light treatment, which might be due to the fact that the glycopolymer binds to GLUTs on the surface of the cells and interferes their uptake of glucose, leading to slow cell growth or cell death. When K562 cells were treated with the equal amount of conjugates (0.02 mg mL1), the cytotoxic effect was also light treatment length dependent (Figure 2e).
3. Conclusion In summary, a series of well-controlled glycopolymers were synthesized by RAFT polymerization, followed by employing novel catalytic system (sodium mercury amalgam) to ‘‘activate’’ PpIX, reducing RAFT glycopolymer and carrying out the thiol–ene click reaction in one pot to synthesize the novel porphyrin-glycopolymer conjugate. DLS measurements and SEM observation confirmed the formation of nanoparticles by the self-assembly of conjugates in aqueous solution. The bioactivity of the glyco-particles was examined using lectin Con A (Canavalia ensiformis) and cancer cells (K562) that over-expressed GLUTs (GLUT1). Results revealed that the glucose functionalities in the
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conjugate remained bioactive and showed enhanced affinity toward specific lectins and cells as the conjugate was in the form of nanoparticles. In vitro studies showed that light enhanced the cytotoxic effect of the polymer against K562 cells in dose dependent and light treatment length dependent manners. This provided an efficient and simple approach to synthesize well-defined porphyrincontaining glycopolymers for potential cancer imaging and PDT.
Acknowledgements: The authors thank the National Natural Science Foundation of China (No. 91027040, 21004042, 21374069, 21074083, 21104051), National Key Scientific Project of China (973 Program #2011CB933501), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry for financial support.
Received: October 10, 2013; Revised: October 25, 2013; Published online: November 27, 2013; DOI: 10.1002/mabi.201300451 Keywords: biopolymers; glycopolymers; one-pot; photodynamic therapy; reversible addition fragmentation chain transfer (RAFT)
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