Michael Addition Polymerization of Trifunctional Amine and Acrylic

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Review

Michael Addition Polymerization of Trifunctional Amine and Acrylic Monomer: A Versatile Platform for Development of Biomaterials Weiren Cheng, De-Cheng Wu, and Ye Liu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01043 • Publication Date (Web): 06 Sep 2016 Downloaded from http://pubs.acs.org on September 7, 2016

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Michael Addition Polymerization of Trifunctional Amine and Acrylic Monomer: A Versatile Platform for Development of Biomaterials Weiren Cheng,† Decheng Wu, *,‡ Ye Liu*,† †Institute of Materials Research and Engineering, A*STAR, 2 Fusionopolis Way Innovis #08-03, Singapore 138634.

‡

Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Polymer

Physics & Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China KEYWORDS. Michael addition polymerization; poly(amino ester)s; poly(amido amine)s; gene delivery; drug delivery; bioimaging

ACS Paragon Plus Environment

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ABSTRACT Michael addition polymerizations of amines and acrylic monomers are versatile approaches to biomaterials for various applications. A combinatorial library of poly(β-amino ester)s and diverse poly(amido amine)s from diamines and diacrylates or bisacrylamides have been reported, respectively. Furthermore novel linear and hyperbranched polymers from Michael addition polymerizations of trifunctional amines and acrylic monomers significantly enrich this category of biomaterials. In this Review, we focus on the biomaterials from Michael addition polymerizations of trifunctional amines and acrylic monomers. Firstly we discuss how the polymerization mechanisms, which are determined by the reactivity sequence of the three types of amines of trifunctional amines, i.e., secondary (2o) amines (original), primary (1o) amines, and 2o amines (formed), are affected by the chemistry of monomers, reaction temperature and solvent. Then we update how to design and synthesize linear and hyperbranched polymers based on the understanding of polymerization mechanisms. Linear polymers containing 2o amines in the backbones can be obtained from polymerizations of diacrylates or bisacrylamides with equimolar trifunctional amine; and several approaches, e.g., 2A2+BB’B”, A3+2BB’B’, A2+BB’B”, to hyperbranched polymers are developed. Further through molecular design of monomers, conjugation of functional species to 2o amines in the backbones of linear polymers and the abundant terminal groups of hyperbranched polymers, the amphiphilicity of polymers can be adjusted, and additional stimuli-, e.g., thermal-, redox-, reactive oxidation species (ROS)- and light-, responses can be integrated with the intrinsic pH-response. Finally we discuss the applications of the polymers for gene/drug delivery and bioimaging through exploring their self-assemblies in various motifs, e.g., micelles, polyplexes particles/nanorings and hydrogels. Redox-responsive hyperbranched polymers can display 300 times higher in vitro gene transfection efficiency and provide a higher in vivo siRNA efficacy than PEI. Also redox-responsive micelle carriers


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can improve the efficacy of anti-cancer drug and the bioimaging contrast. Further molecular design and optimization of this category of polymers together with in vivo studies should provide safe and efficient biomaterials for clinical applications.


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1. INTRODUCTION Polymers are among the most important biomaterials for various applications including drug delivery, diagnosis and tissue engineering. Both synthetic and natural polymers have been explored as biomaterials which must be no toxic, no immunogenic and no carcinogenic, and should finally be excreted. One important category of synthetic polymeric biomaterials contains hydrolysable ester groups such as polylactide (PLA) and polycaprolactones. For specific functions, more functional groups are necessary to be integrated such as to provide stimuli-responsive properties including pH-, redox-, enzyme-, light-responses.1,2 Ring-opening polymerization and condensation polymerizations are indispensable to produce safe and efficient polymeric biomaterials. Among these approaches, Michael addition polymerizations of amines and acrylic monomers are very promising because various types of monomers can be adopted to produce highly functional biocompatible polymers.3-5 Langer’s group initiated the works on linear poly(β-amino ester)s from Michael addition polymerization of diacrylate with diamines containing one primary (1o) amine or two secondary (2o) amines,6 and a combinatorial library of linear poly(β-amino ester)s were prepared.5,6 Ferruti and co-workers have reported a series of diverse linear poly(amido amine)s from polymerizations of bisacrylamides and diamines.4 Furthermore, Michael addition polymerizations of acrylic monomers and trifunctional amines containing one 1o amines and one 2o amines significantly enrich the chemistry, topology and applications of this category of polymers. Reviews on Michael addition polymerizations of diamines and acrylic monomers have been reported, respectively.4,5 In this review, we focus on Michael addition polymerizations of acrylic monomers and trifunctional amines. Michael addition polymerizations of amines and acrylates proceed via stepwise reactions. From diamines and acrylic monomers, the polymerizations produce linear polymers.3-5 However, when trifunctional amines are used instead, the polymerization mechanisms


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become complicated because of the different reactivity of 1o amine, 2o amine (original) and 2o amine (formed) of trifunctional amines. Generally cross-linked polymers are formed in these polymerizations when the reactivity of the monomers is equal when the polymerizations pass the gel point.7 But if the reactivity of monomers are different, polymers with varied chemistry and topologies can be formed via different mechanisms depending on the reactivity profiles of the monomers.7-11 Michael addition polymerizations of trifunctional amines and acrylic monomers have been extensively investigated with a clear understanding of the amino reactivity sequence.12-14 On the basis of this understanding, linear and hyperbranched polymers have been developed. In addition, it is feasible to conjugate functional species to linear polymers via reaction with the 2o amines in the backbones and the abundant terminal groups of hyperbranched polymers. These features facilitate fine-tuning of the chemistry and topology of the polymers and integration of additional stimuli-responsive moieties to produce various nanosized motifs with improved efficacy for gene delivery, drug delivery and bioimaging.

2. POLYMERIZATION MECHANISMS Figure 1 lists the typical trifunctional amines and acrylic monomers adopted for Michael addition polymerizations. The effects of chemistry of monomers and polymerization conditions on the mechanisms are discussed below.

Figure 1.

2.1. EFFECTS OF CHEMISTRY OF MONOMERS


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At that moment when we worked on polymerization of diacrylate BDA and trifunctional amines AEPZ,12 1o amines of trifunctional amines were assumed to be as reactive as 2o amines (formed) and less reactive than 2o (original) amines; and polymerization of BDA and equimolar AEPZ was supposed to follow a A2 + BB’2 approach to yield hyperbranched polymer.15 However, from the

13

C NMR spectrum of the polymer obtained as shown in

Figure 2, we could not ascribe the peaks to the expected hyperbranched polymer, and realized that it is novel linear poly(BDA-AEPZ) with 2o amines in the backbones instead.12

Figure 2.

Further we investigated the polymerization mechanisms of diacrylate BDA with typical trifunctional amines, AEPZ, AMPD, MEDA, EEDA and HEDA, by monitoring the reaction processes using

13

C NMR.13 For AEPZ, the polymerization was found to start with the

reaction of BDA exclusively with equimolar 2o amine (original), forming the intermediate A1B1 without A’1B’1 as described in Figure 3A. Then polymerization of A1B1 yields linear poly(BDA-AEPZ) with the 2o amine (formed) kept intact. The reactivity of the three types of amines of AEPZ is significantly different and follows the sequence of 2o amine (original) > 1o amine >> 2o amine (formed). The same amine reactivity sequence and polymerization mechanism were observed for AMPD.13,16,17 For MEDA, the reactivity sequence is in the same order, but the reactivity difference between the 2o amines (original) and 1o amines is smaller, so both intermediates A2B2 and A’2B’2 are formed simultaneously; but linear poly(BDA-MEDA) with 2o amines in the backbones is still produced as described in Figure 3B.13 In contrast, the amine reactivity sequence of EEDA becomes 1o amines > 2o amines (original) > 2o amines (formed); A3B3 and A’3B’3 intermediates are formed simultaneously;


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and the 2o amines (formed) participate in the reaction forming branched polymers as presented in Figure 3C. Similar profiles were also observed for HEDA.13 For all the trifunctional amines, the reactivity of 1o amines should be similar; however the reactivity of 2o amines depends significantly on the steric hindrance of the second adjacent units which follows this order: cyclic aliphatic rings in AEPZ and AMPD < methyl in MEDA < ethyl in EEDA and hexyl in HEDA < polymer chains. Thus, 2o amines (original) of AEPZ and AMPD are the most reactive followed by 2o amines (original) of MEDA; all these 2o amines (original) are more reactive than the 1o amines and the 2o amines (formed); and the 2o amines (formed) are completely kept out of the reactions. However, the 2o amines (original) in EEDA or HEDA are less reactive than the 1o amines, and 2o amines (formed) participate in the reaction. 13

Figure 3.

In contrast, the effect of acrylic monomers on the polymerization mechanisms is insignificant. The amine reactivity sequence of AEPZ is the same in the polymerizations with various diacrylates, e.g., BDA,12,13 CBDA,14 and A1 monomer;18 bisacrylamides, e.g., BAC, MBA, BAP, HMBA, DDA;16,17,19-21 and divinyl sulfone22.

2.2. EFFECTS OF TEMPERATURE AND SOLVENT Higher reaction temperature results in a smaller reactivity difference among the amines. In polymerization of CBDA with equimolar MEDA, 2o amines (formed) of AEPZ remained inactive at 40 oC or below, but participated in the reactions at 48 oC or above.14 Similarly, in


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polymerization with MBA, 2o amines (formed) remained intact at 50 oC or below;16,19 but joined in the reaction at 60 oC.21 For polymerization with diacylates, chloroform is usually used as a solvent because of the solubility of diacrylates. However solvents can be changed for acrylamide monomers. In protic solvents like methanol and water, an enhanced hydrogen transfer results in a smaller amine reactivity difference. For example, 2o amines (formed) of AEPZ participated in the polymerization with equimolar MBA in methanol or water, but were kept out of the reaction in aprotic dimethylformamide.21 Similarly, 2o amines (formed) of AMPD participated in polymerization

with

equimolar

BAC

in

methanol

but

were

intact

in

aprotic

dimethylsulfoxide.17 On the basis of the clear understanding of the mechanisms of Michael addition polymerizations of trifunctional amines and acrylic monomers, both linear polymers and hyperbranched polymers have been developed.

3. LINEAR POLYMERS 3.1. SYNTHESIS. Linear polymers were obtained from Michael addition polymerizations of bisfunctional acrylic monomers with equimolar trifunctional amines when 2o amine (formed) was kept intact. Linear poly(amino ester)s were produced via the polymerizations of diacrylates and AEPZ, AMPD and MEDA.12-14,18 The first linear poly(amino ester)s developed is poly(BDA-AEPZ) (Figure 2).12 PEG containing diacrylate, PEGDA, was also applied to prepare linear poly(PEGDA-AEPZ).23 Recently, Li group prepared linear poly(amino ester)s via polymerization of AEPZ with equimolar diacrylate A1 (Figure 1).18 Linear poly(amido amine)s were also obtained via polymerizations of AEPZ, AMPD and

MEDA with

bisacrylamides such as MBA, BAC, BAP, HMBA, MDA and DDA.16,17,19-21


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The molecular weights of the typical linear poly(amino ester)s and poly(amido amine)s reported are listed in Table 1. As listed in Table 1, polydispersity indexes (PDIs) of the linear polymers are broad, probably due to the multistage polymerization processes. Tgs of linear poly(amido amine)s are higher than those of poly(amino ester)s due to the stronger interactions between the amide groups. A lower density of amide units and a longer bisacrylamide, i.e., DDA, result in lower Tgs. 16,20

3.2. MODIFICATION Modifications are carried out to tune the amphiphilicity and integrate additional stimuliresponses. 3.2.1. TO TUNE AMPHIPHILICITY. Self-assembly of amphiphilic polymer is important for many applications, and PEGylation is a versatile approach to render hydrophilicity for preparation of amphiphilic polymers.24 As shown in Figure 4A, PEGylation of linear poly(amido amine)s is obtained by grafting PEG via the reaction of 4-nitrophenyl carbonate activated PEG25 with the 2o amines.17 Instead Li group used N-hydroxysuccimide activated PEG.18 PEG were also grafted to linear poly(amido amine)s, poly(DDA-AEPZ) via Michael addition reaction of acryl terminated PEG with the 2o amines which formed micelles in aqueous solution with PEG shells and poly(DDA-AEPZ) cores.20 In another way, PEGDA were applied to tune hydrophilicity of linear poly(amino ester)s as shown in Figure 4B.23 To increase hydrophobicity, cholesterol (CE) is conjugated to poly(BAC-AMPD)-g-PEG via the reaction of cholesteryl chloroformate with 2o amines as shown in Figure 4A, and amphiphilic poly(BAC-AMPD)-g-PEG-g-CE could form micelles with PEG shells and cores composed of CE and poly(BAC-AMPD).17 Using the same approach, CE was conjugated to pH- and thermal-responsive NIPAAm grafted poly(amino ester)s shown in Figure 4B which can form thermal-responsive micelles.23 Hydrophobic linear poly(amido amine)s, poly(BAC-


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AMPD)17 and poly(DDA-AEPZ)20, were also obtained from bisacrylamides containing a long alkyl chain, while hydrophilic poly(MBA-AEPZ) and poly(MBA-AMPD) were obtained from MBA.16

Figure 4.

3.2.2. TO INTEGRATE ADDITIONAL STIMULI RESPONSES. The intrinsic property of amine-containing polymers is pH-responsive, and protonation of amines leads to disassembly of micelles20,23 and faster hydrolysis18. Furthermore, as described in Figure 4B, N-isopropylacrylamide (NIPAAm) was conjugated to 2o amines (formed) of linear poly(amino ester)s to afford thermal response. The lower critical solution temperature (LCST) could be tuned to ca. 36.5oC via adopting suitable PEGDA and NIPAAm grafting degree. Self-assembly of the amphiphilic polymers in aqueous solution at 40 oC and pH 7 produces nanoparticles with diameter of ca. 170 nm which becomes smaller at pH 5 as shown in Figure 4C.23 Redox-responsive linear poly(amido amine)s is also obtained by adopting disulfide-containing bisacrylamides BAC (Figure 4A) which was degraded in the presence of thiol compounds;17,19 and Li group reported reactive oxidation species (ROS)-responsive linear poly(amino ester)s through integration of phenylboronic pinaco ester in diacrylate A1 which could be degraded in the presence of ROS such as H2O2.18

4. HYPERBRANCHED POLYMERS 4.1. SYNTHESIS Hyperbranched polymers were produced by controlling the reaction mode of 2o amines (formed) in the following approaches.


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4.1.1. 2A2 + BB’B” APPROACH. As described in Figure 5A, hyperbranched poly(amino ester)s, poly(BDA2-AEPZ1), is obtained via the polymerization of AEPZ with double molar BDA.26 Firstly, 2o amines (original) of AEPZ reacts with equimolar BDA to form intermediate B’B’’A which reacts with another molar BDA to produce B”A2. Polymerization of B”A2 yields hyperbranched poly(amino ester)s. The abundant terminal acryl groups are tuned to 1o, 2o, and 3o amines via Michael addition reactions with amino compounds, respectively, as shown in Figure 5A. Note that AEPZ was applied to get terminal 1o amines after Michael addition reaction of the 2o amines (original) with the acryl groups. Trifunctional amines were also applied in other organic synthesis through exploring the amino reactivity sequence to prepare functional organic compunds.27 Because it was difficult to differentiate the dendritic, linear terminal units from B”A2, so the degree of branch (DB) of this hyperbranched poly(BDA2-AEPZ1) could not be determined. However the ratio of hydrodynamic radius (Rh) to radius of the gyration radius (Rg) of the polymer was determined to be 1.0 which indicates the hyperbranched topology.26 Recently, with the same approach, hyperbranched poly(amino ester)s was also developed using diacrylate NPDA.28

Figure 5.

Polymerization of AEPZ with double molar MBA, BAC or their mixtures produced hyperbranched poly(amido amine)s followed by reaction of the terminal acryl groups with methyl piperazine (MPZ)29 and AEPZ30,31, respectively. Also hyperbranched poly(amido amine)s were produced from the polymerization of AEPZ with double molar BAC followed by tuning the terminal groups to PNIPAAm with a ratio of Rg/Rh of 0.98,32 PEG33 and hydroxyl/amine/the mixture34.


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4.1.2. A3 + 2BB’B” APPROACH. As described in Figure 5B, hyperbranched poly(amino ester)s containing 1o, 2o and 3o amines as polyethyleimine (PEI), poly(TMPTA1-AEPZ2), is produced from the polymerization of trimethylol-propane triacrylate (TMPTA) with double molar AEPZ via the intermediate A(B’B”)2 formed via the reaction of TMPTA with double molar AEPZ.35,36 It was also difficult to differentiate the dendritic, linear and terminal units from A(B’B”)2, so DB of poly(TMPTA1-AEPZ2) could not be measured. But the ratio of Rh/Rg of the polymer was determined to be 1.1 which reflected the hyperbranched topology.35 Triacrylates of different number of ethyleneoxyl uints were also used.37 In addition, based on the reactivity difference between 1o amines and 2o amines (formed), hyperbranched poly(amido amine)s were obtained from polymerizations of 1,3,5-triacryolhexahydro-1,3,5triazine and n-butylamine (BA) via either A3 + BB’ or A3 + 2BB’ approaches.38 Via A3 + 2BB’ approach, hyperbranched poly(amino ester)s were produced from the polymerization of TMPTA

and

double

molar

diamines,

1-(3-aminopropyl)imidazole

(API),

N,N-

dimethylethylenediamnie (DED) and the mixtures with AEPZ.36 Polymerization of pentaerythritol triacrylate with double molar DED (A3 + 2BB’ approach) produced soluble polymers but which were assumed to be cross-linked polymers by Park group.39

4.1.3. A2 + BB’B” APPROACH. As described in Figure 3C, Michael addition polymerization of BDA with equimolar EEDA or HEDA yielded hyperbranched poly(amino ester)s with a degree of branch (DB) of 33% and 37%, respectively.13 Likewise, hyperbranched poly(amido amine)s was obtained from polymerization of BAC and MBA with equimolar DMDPTA30,40 or APEZ21,41. Table 2 lists the molecular weights and PDIs of the typical hyperbranched poly(amino ester)s and hyperbranched poly(amido amine)s reported. A higher PDI indicates the broad molecular weight distributions probably due to the multi-stages polymerization processes. Tgs


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of poly(amino ester)s are lower than hyperbranched poly(amido amine)s, Tgs of hyperbranched polymers are close to the linear counterparts and increase with polar terminal groups.

4.2. MODIFICATION 4.2.1. TO TUNE AMPHIPHILICITY. As mentioned above, PEGlyation is an important approach to modification of polymers.24 As shown in Figure 6A, amino-terminated PEG was conjugated to acryl-terminated hyperbranched poly(amido amine)s obtained from the polymerization 2BAC+1AEPZ through Michael addition reaction to produce PEGylated hyperbranched poly(BAC2-AEPZ1) which can form micelles with PEG shells and poly(amido amine)s cores.33 In another way, the acryl terminal groups of hyperbrached poly(amido amine)s, poly(BAC2-AMPD1) was changed to 1o amine, then PEGylation was obtained via the reaction with 4-nitrophenyl carbonate activated PEG.42 For hyperbranched poly(1TT-2BA) with amino terminal groups, acryl terminated PEG was used instead to enhance

the

amphiphilicity

of

the

polymers.29

In

addition,

11-

mercaptoundecylphosphorylcholine (HS-PC) was applied to react with the terminal acryl groups of hyperbranched poly(amino ester)s to introduce hydrophilic and biocompatible HSPC shells which could form micelles of a diameter of ca. 100 nm in aqueous solution.28

Figure 6.

4.2.2. TO INTEGRATE ADDITIONAL STIMULI RESPONSES. Thermal-responsive hyperbranched polymer(amido amine)s were developed in two approaches. One approach is to conjugate amino-terminated polyNIPAAm to acryl-terminated hyperbranched poly(amido


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amine)s, poly(BAC2-AEPZ1), to produce thermal-responsive hyperbranched poly(amido amine)s with a profile similar to polyNIPAAm.32 Another way is to prepare hyperbrached poly(amido amine)s, S-HPAA, via Michael addition polymerization of BAC and eqimolar DMDPTA.30,43 LCST of S-HPAA is ca. 35 oC with the thermal-response being caused by hydration and dehydration of N, N-dimethylamine unit. UV-responsive hyperbranched polymers were obtained via integration of O-nitrobenzyl units. Hyperbranched poly(amino ester)s with the UV-responsive O-nitrobenzyl units being integrated in the backbones were obtained from Michael addition polymerization of 2NPDA+1AEPZ.28 As described in Figure 7A, the other approach is to conjugate the terminal acryl groups of hyperbranched poly(amido amine)s obtained from Michael addition polymerization of 2BAC+ 1DMDPTA with 2-((2-nitrobenzyl)thio)ethanol, and thioethanol released under UV-irradiation triggers degradation of disulfide containing polymer backbones indicated by the UV-induced disappearance of polymer nanogels as shown in Figure 7B shows.44

Figure 7.

4.3. PROPERTIES 4.3.1. FLUORESCENT PROPERTIES. Fluorescence technology has been applied in many areas; and fluorescent organic materials typically contain conjugated aromatic units.45 However, similar to fluorescent non-conjugated dendrimers poly(amidoamines) (PAMAM)46 and poly(propyleneimine) (PPI),47 our group observed blue fluorescence from hyperbranched poly(BDA2-AEPZ1) as shown in Figure 8.48 Fluorescence was also observed from hyperbranched poly(amido amine)s from BAC and AEPZ.49-51 Integration of β-cyclodextrin (CD) into hyperbranched poly(amido amine)s from MBA and AEPZ enhanced the


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fluorescence intensity probably due to a higher molecular rigidity.41 Thermal/ultrasonicresponsive semiconductor quantum dot (QD)-hyperbranched poly(MBA2-AEPZ1) hydrogels showed reversible stronger fluorescence than its components.52 However, so far, the mechanisms of the fluorescence from non-conjugated polymers have not been fully understood. Carbamato anions formed via the reaction of amines with carbon dioxide might contribute to this type of fluorescence observed.53

Figure 8.

4.3.2. HYDROGELS FORMATION. Polymer hydrogels have many applications. As shown in Figure 6B, hydrogels with controllable cross-linking degrees from PEGylated hyperbranched poly(amido amine)s, poly(BAC2-AEPZ1)-PEG, can be obtained via intermolecular disulfide exchange reaction which was triggered by pH adjustment.33 Changing pH of aqueous solution of protonated poly(BAC2-AEPZ1)-PEG (Figure 6B(a)) to pH 12 leads to formation of loose hydrogels in 1 h and compacted gel after 24 h (Figure 6B(c)). These processes can be interrupted and reassumed by neutralization and rebasification, respectively (Figure 6B(b*)). Nanogels are ideal drug carriers due to the combined properties of hydrogels and nanoparticles.54 Several approached have been developed to produce nanosized aqueous droplets of disulfide –containing hyperbranched poly(amido amine)s. One approach is via surfactant free approach as shown in Figure 6C. A water-in-oil (W/O) emulsion is generated feasibly from a mixture of water and chloroform solution of disulfide-containing PEGylated hyperbranched poly(amido amine)s, poly(BAC2-AMPD1)-PEG, which function as emulsifier; and then poly(amido amine)s based nanogels are formed via intermolecular disulfide exchange reaction in the water phase.55 Another approach is to produce


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micro/nanogels from aqueous droplets containing hyperbranched poly(BAC2-AEPZ1)-PEG (Figure 6A) in decane or cyclohexane using Span80/tween80 as surfactants; the gel particles obtained were further coated with multi-polymer layers.56 Also cross-linking of thermalinduced self-assembly of disulfide containing hyperbranched poly(amido amine)s poly(BAC2-DMDPTA1) in aqueous solution could produce nanogels.44,57 As shown in Figure 7B, adjusting pH to 9 facilitates the self-assembly of 2-((2-nitrobenzyl)thio)ethanol terminated hyperbranched poly(BAC2-DMDPTA1) in aqueous solution, then heating induces cross-linking of the self-assembly which forms nanogels indicated by being insoluble at pH 7.44 Similar results were observed also from hyperbranched poly(BAC-DMDPTA).30

5. BIOMEDICAL APPLICATIONS 5.1. GENE DELIVERY. Gene therapy is promising in treating genetic or acquired diseases but is hindered from clinical applications due to lack of safe and efficient gene delivery approaches. Although viral vectors are efficient in delivering and transfecting large genetic materials, their intrinsic safety, complexity, undesired side effects and high cost are still daunting hurdles. In comparison, non-viral vectors can be prepared from reproducible and cost-effective materials with well controllable structures without host immunogenicity.58 Polymers are important candidates to prepare non-viral vectors for gene therapy by forming nanosized polyplexes particles via ionic interaction with gene.59 PEI, one of the polymers containing the highest amino content, shows good gene transfection efficiency but also high toxicity probably due to the non-biodegradability. Polymers grafted with low molecular weight PEI, which might have lower cytotoxicity, have been developed for gene delivery.60,61 Other types of polymers including chitosan, polylysine and polyamidoamines have also been investigated.59 In order to improve biocompatibility of polymers, integrations of


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biodegradable groups such as ester groups or disulfide groups into amino polymers such as poly(amino ester)s and disulfide containing poly(anmido amine)s are preferred.5,62-65 In comparison with other types of polymers including linear poly(amino ester)s and poly(amido amine)s from Michael addition polymerization of diamines and acrylic monomers, the low cytoxocity, good biocompatibility, and the feasible tunable chemistry and topology render polymers from Michael addition polymerization of trifunctional amines and acrylic monomers promising for gene delivery. Linear poly(BDA-AEPZ) (Figure 2),12 hyperbranched poly(BDA2-AEPZ1) terminated with 1o, 2o and 3o amines (Figure 5A),66 hyperbranched poly(TMPTA1-AEPZ2) with amine constitution similar to PEI (Figure 5B),35 and hyperbranched poly(amino ester)s from A3 + 2BB’ and A3 + 2BBB’37 all displayed gene transfection efficiency comparable to PEI, indicating the insignificant effects of topology on gene transfection efficiency. But hyperbranched poly(TMPTA1-2DED) showed higher gene transfection than PEI, which is probably due to the presence of more protonated amines at physiological pH to facilitate DNA condensation,36 which is similar to the results observed from linear poly(amino ester)s from diamines.5 From poly(amido amine)s, both DNA polyplexes nanoparticles34 and nano-rings31 were formed. In the absence of L-buthionine sulfoximine (BSO) to retain high cellular redox potential, lower cytotoxicity (Figure 9A) and higher transfection efficiency (Figures 9B and 9C) are observed from redox-responsive hyperbranched poly(2BAC-AEPZ) with different terminal groups without significant effects from serum34 Also the results from linear and other hyperbranched poly(amido amine)s showed that redox-response facilitate safe and efficient gene delivery.19,40 Hyperbranched topology also facilitates a higher gene transfection.

4,21

Higher gene transfection efficiency from redox-responsive polymer and

hyperbranched polymers were also observed for poly(amido amine)s from diamines.4


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Furthermore redox-responsive hyperbranched poly(amido amine)s from DMDPTA rendered transfection efficiency 300 times higher than PEI,40 which might be due to a higher amount of protonated amines at physiological pH similar to poly(amino ester)s

Recently folate-

conjugated hyperbranched poly(BAC-AEPZ) was developed to improve transfection efficiency of MMP-9 siRNA in vitro and in vivo. In comparison to PEI, this system displayed higher in vitro transfection efficiency and greater in vivo efficacy in inhibiting MCF-7 tumor.67 However, the gene transfection efficiencies are still needed to be improved further by overcoming easy clearance by the reticuloendothelial and immune systems and difficult access to the right cell in a complex tissue and the cytoplasm or nucleus in order to reach clinical applications.

Figure 9.

5.2. DRUG DELIVERY. Controlled drug delivery systems can improve the efficacy and reduce the side effects of many types of drugs. One critical type of controlled drug delivery systems is polymer based nanomedicine which can be formulated in forms of micelles, polymersomes, nanogels and nanofibers etc.1 These nanomedicines have been applied to enhance efficacy and reduce side effects of chemotherapy, especially when stimuliresponsive nanocarriers are used to securely encapsulate drug and release it once reaching the target sites and needed. Self-assembly of amphiphilic polymers is one of the most important approaches to nanomedicines which inspire developments of different types of amphiphilic polymers. In addition, surfaces engineering of self-assemblies such as forming polyethylene glycol shells is also important to facilitate targeted delivery.1,24,68,69 It is feasible to tune the amphiphlicity, integrate other functionality and adjust topology of polymers form Michael addition polymerizations of trifunctional amines and acrylic


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monomers to develop stimuli-responsive nanomedicines. Micelles from self-assembly of poly(BAC-AMPD)-g-PEG-g-CE (Figure 4A) was applied for doxorubicin (DOX) delivery.17 Confocal images in Figure 10 indicates that poly(BAC-AMPD)-g-PEG-g-CE micelles can deliver DOX into the cytoplasm and nuclei of HepG2 cells. A higher efficacy as reflected by a lower IC50 than free DOX (1.44 µg/mL to 7.42 µg/mL) was observed. This is probably due to lesser efflux of DOX out of the cells from the redox-induced formation of aggregates of the micelles. Micelles from amphiphilic PEGylated hyperbranched poly(amido amine)s, poly(BAC2-AMPD1)-PEG, was also adopted for DOX delivery with an in vitro efficacy comparable to free DOX42, but better in vivo performances are expected. Also photodegradable hyperbranched poly(amino ester)s for DOX delivery was developed and showed a higher efficacy to kill cancer cells under UV irradiation.28

Figure 10.

5.3. BIOIMAGING. Bioimaging is an important diagnostic tool for various diseases.45,70 Among different imaging techniques, fluorescence imaging can provide cost-effective and real time diagnosis at molecular level; and high contrast images can be obtained using fluorescent probes with stimuli “turn-on” behavior or/and aggregate-induced emission (AIE) characteristic.71 In contrast to most of the works using inert materials including polymers for encapsulation of dyes to formulate imaging probes, as shown in Figure 11, we applies redoxresponsive poly(BAC-AMPD)-g-PEG-g-CE (Figure 4A) to encapsulate redox-responsive AIE dye, TPE-MI, to form redox “turn-on” probes with AIE characteristic. The fluorescence intensity of the probes increases with GSH concentration; and the probes provide images with high contrast due to the much higher redox potentials in intracellular components than in extracellular matrix.72


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Figure 11.

The inherent fluorescent property of hyperbranched poly(amido amine)s were also explored for bioimaging. N-galactosamine or N-glucosamine containing hyperbranched poly(BACAEPZ)50 and β-cyclodextrin containing hyperbranched poly(MBA-AEPZ)41 were developed for bioimaging. In addition, polydisulfide macromolecular magnetic resonance imaging (MRI) contrast agent was developed to provide better imaging quality with lesser side effects.73

6. CONCLUSIONS AND FUTURE PRESPECTIVES The mechanisms of Michael addition polymerizations of trifunctional amines and acrylic monomers are well understood which can guide the design, synthesis and functionalization of novel linear and hyperbranched polymers. Through appropriate monomer design and conjugation of functional species via the reactions with the 2o amines in the backbones of linear polymers and the abundant terminal units of hyperbranched polymers, polymers with tunable amphiphilicity, intrinsic pH-response and additional stimuli-, e.g., redox-, UV-, ROS, thermal-response are obtained. Self-assembly of polymers produces different motifs. Micelles and hydrogels including nanogels are obtained from amphiphilic polymers which can be applied as carriers of active species such as anticancer drugs and imaging probes. Polyplex nanoparticles and nanorings are formed from polymers and DNA. These systems are explored for gene delivery, drug delivery and bioimaging. Redox-responsive hyperbranched polymers can display 300 times higher in vitro gene transfection efficiency, and higher in vivo siRNA efficacy to inhabit cancer cell growth compared to PEI. Redox-induced aggregations of polymer micelles in


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cells render a higher drug efficacy to kill cancer cells, and redox-induced fluorescence from polymer micelle-based probes provides higher contrast bioimaging. This platform can be further enhanced. Firstly methods should be established to accelerate the polymerization rates without losing control of the polymer structures. Currently most of the polymerizations are relatively slow, typically requiring days for high conversions. Hence it is necessary to develop suitable catalysts, but so far very few studies have been performed 74

. Moreover, multistage polymerization routes should be optimized to produce polymers

with low PDIs. One promising approach is to develop controlled Michael addition polymerizations of trifunctional amines and acrylic monomers. Secondly the polymers developed, e.g., pH- and thermal-responsive biodegradable polymers, pH- and ROS- responsive polymers, and pH- and redox-responsive hydrogels and nanogels, can be explored for various applications including drug/gene/protein/cell delivery and tissue engineering. On the other hand, polymers with new functionalities can be developed through molecular design of monomers and developing new polymerization mechanisms and polymer modification approaches. Finally, it is essential to further investigate the clinical performances of this category of polymers. Recently, in vivo data obtained for some polymers suggest that it is promising to develop safe and efficient biomaterials from Michael addition polymerizations of trifunctional amines and acrylic mononers. However, more in vivo evaluations and clinical trials are needed to validate the clinical values of this category of polymers.

■AUTHOR INFORMATION Corresponding Author


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*Email: [email protected]; [email protected]. Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS The financial supports are from A*Star.


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Figure 1. Typical trifunctional amines and acrylic monomers for Michael addition polymerizations.


23

Biomacromolecules

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

55

50

45

Page 24 of 41

40

ppm

Figure 2. 13C NMR spectrum and the structure of linear poly(amino ester)s obtained from Michael addition polymerization of BDA and AEPZ.12 Reproduced from Ref 12 with permission from The Royal Society of Chemistry


24

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A

O

O

NH2

N

+

AEPZ

O O

NH

O

BDA

O

O

1

N

A1'B1'

O

HN

NH

O

O

1

O

N

O

O

O

NH2

N

A1B1 O CH3 N H

NH2

O

O

+

Fast

H2N

CH3

+

Fast

HN

H N

N H

CH3

Fast

L CH2CH3 O

O O O

O

N N H

O

O O

Slow

L'

CH3

O

N N

O

N H

O

A3B3

O

CH2CH3 O

n

CH3 H N CH3

O

O N

D

O O

O

O O

H N N CH3

NH2

A3'B3'

O O

N H

O

n

N

O

CH2CH3 O N

O

Fast

Fast

N H

Poly(BDA-MEDA)

O

O O

L

BDA

EEDA

Slow

A2B2

O

O

NH2

N CH3

O

Slow

O

O O

O O

O

CH3

O

BDA

H N

A2'B2'

O O

N

H N

N H

O

O

MEDA

C

Slow

N

Poly(BDA-AEPZ) O

B

O O

O

HN

O

CH3

H N

O

O N

O

L'

N H

CH3

T

Poly(BDA-EEDA)

Figure 3. Mechanisms of Michael addition polymerization of BDA and equimolar A) AEPZ; B) MEDA; and C) EEDA. Reprinted with permission from ref 13. Copyright 2004 American Chemical Society


25

Biomacromolecules

180

a

100

pH 7

pH 7

150

60 90 40 60

pH 5

20

30

b

0 26

28

30

32

34

36 38 T (oC)

40

42

44

46

Transmittance (%)

80

120 Rh (nm)

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

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0

Figure 4. Modification of polymers to yield A) linear poly(BAC-AMPD)-g-PEG-g-CE; Reprinted with permission from ref 17. Copyright 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim and B) pH- and thermal-responsive poly(amino ester)s. Reprinted with permission from ref 23. Copyright 2008 American Chemical Society C) Temperature dependence of transmittance of 1% (w/v) micelles solution and Rh of micelles in 0.05% (w/v) aqueous solution of CE0.48-g-NIPAAm0.46-g-poly(PEGDA-AEPZ). Reprinted with permission from ref 23. Copyright 2008 American Chemical Society


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Figure 5. Approaches to hyperbranched poly(amino ester)s. Reprinted with permission from ref 26,35. Copyright 2005 American Chemical Society


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A

B

C

Figure 6. A) Preparation of PEGylated hyperbrached poly(BAC2-AEPZ1)-PEG. Reprinted with permission from ref 33. Copyright 2010 American Chemical Society B) Formation and structures of hydrogels: a) 10 wt% poly(BAC2-AEPZ1)-PEG solution, b) loose hydrogel after 1 hour of crosslinking, and c) compact hydrogel after 24 hours of crosslinking. Reprinted with permission from ref 33. Copyright 2010 American Chemical Society C) Formation of nanogels from water/chloroform solution of poly(BAC2-AMPD1)-PEG. Reprinted with permission from ref 55. Copyright 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


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A

B

Figure 7. A) Scheme for hyperbranched poly(amido amine)s which can be UV-triggered self-immolate and thermally induced crosslinking. B) Photographs show the self-assembly of polymer at pH 9 in aqueous solution, thermal-induced formation of nanogels and UVtriggered degradation of nanogels. Reprinted with permission from ref 44. Copyright 2014 American Chemical Society.


29

Biomacromolecules

800

Fluorecence intensity (arbitrary units)

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

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600

400

200

0 250

300

350

400

450

500

550

600

650

Wavelength nm

Figure 8. Emission and excitation spectra of 0.5 mM aqueous solutions of hyperbranched poly(BDA2-AEPZ1)-OH at pH 7 with exposure to air. Reprinted with permission from ref 48. . Copyright 2005 American Chemical Society


30

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A 120 Without BSO

Relative cell viability (%)

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

Biomacromolecules

100 80

With BSO

***

**

*

MCF-7

Hep G2

MCF-7

**

60 40 20 0 Hep G2

1.0 µg DNA

0.5 µg DNA

B


31

Biomacromolecules

C 35

GFP positive cells (%)

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

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30 25

**

Without BSO With BSO

*

20 15 10 5 0 4

10

24

Time (h)

Figure 9. Effects of the presence of 0.2 mM BSO on A) cell viability of Hep G2 and MCF-7 cells transfected with poly(BAC2-AEPZ1)-AEPZ/DNA complexes (all data represent mean ± SD (n = 3, Student’s t-test, *P < 0.05, **P < 0.01, **P < 0.005 )); B) CLSM images of Hep G2 cells transfected with poly(BAC2-AEPZ1)-AEPZ/pEGFP complexes at different transfection time (red: RDM-pDNA, green: GFP, blue: DAPI; and the white bar represents 20 µm); C) GFP expression of poly(BAC2-AEPZ1)-AEPZ/RDM-pEGFP complexes evaluated by flow cytometry analysis (mean and standard deviation (n = 3)). Reprinted with permission from ref 34. Copyright 2013 American Chemical Society


32

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Figure 10. CLSM images of HepG2 cells after incubation with DOX loaded micelles of poly(BAC-AMPD)-g-PEG-g-CE for 48 h (1st row) and 72 h (2nd row) (from left to right, DAPI fluorescence, DOX fluorescence, overlays of DAPI and DOX fluorescence, and a bright field). Reprinted with permission from ref 17. Copyright 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


33

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Figure 11. CLSM imaging of live HepG2 cells after incubation with 0.2 mg/mL of fluorescent nanoparticles for 22 hours without moving the medium containing the nanoparticles and fixation; and [GSH] dependent emission spectra of 1 × PBS buffer solution of 0.2 mg/mL of the fluorescent nanoparticles at pH 7 after reaction with GSH for 15 minutes. Reprinted with permission from ref 72. Copyright 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


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Table 1. Linear polymers from Michael addition polymerizations of trifunctional amines and acrylic monomers. Mw ×(10-3)

Polymer

PDI

Tg (oC)

Ref.

poly(amino ester)s Poly(BDA-AMPD)

19.7

3.48

-8.8

13

Poly(BDA-AEPZ)

23.5

3.11

-21.7

13

Poly(BDA-MEDA)

5.5

2.93

-43.0

13

Poly(CBDA-MEDA)

55.1

1.9

n. r.a

14

Poly(PEGDA-AEPZ)

7.45

1.88

-29.0

23

F (polyA1-AEPZ)

11.7

3.53

n.r.

18

poly(amido amine)s

a

Poly(MBA-AEPZ)

28.1(11.8)

3.4 (1.2)

69.6

16(19)

Poly(MBA-AMPD)

12.7

2.0

71.4

16

Poly(MBA-MEDA)

5.3

1.6

46.6

16

Poly(BAP-AEPZ)

15.6

4.2

n. r.

19

Poly(HMBA-AEPZ)

15.0

4.7

n. r.

19

Poly(BAC-AEPZ)

9.6

2.7

n. r.

19

Poly(BAC-AMPD)

7.2

2.2

n. r.

17

Poly(DDA-AEPZ)

26.0

4.2

14.3

20

Poly(DDA-AMPD)

26.0

5.7

15.5

20

not reported.


35

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Table 2. Hyperbranched polymers from Michael addition polymerizations of trifunctional amines and acrylic monomers. PDI

Tg (oC)

Ref.

38.1

3.70

-14.4

26

29.2

3.41

-28.6

26

poly(BDA2-AEPZ1)-PZ

28.4

3.50

-25.8

26

HPAE-1(Poly(NPDA2-AEPZ2)-PC)

5.8

1.64

n. r.d

28

poly(TMPTA1-AEPZ2)

23.3 (2.7)

1.68 (2.47)

n. r.

35 (37)

2.0

2.42

n. r.

37

P-7E/M (poly(TMPETA1-AEPZ2)

2.3

3.75

n. r.

37


3.2

3.17

n. r.

37

3.3

3.11

-33.9

13

4.3

3.05

-53.4

13

Poly(MBA2-AEPZ1)-MPZa

7.9

3.43

69.9

29

Poly(BAC2-AEPZ1)-PNIPAM

89.0

1.8

n. r.

32

Poly(BAC2-AEPZ1)-PEG

31.9

2.0

n. r.

33

PAA (poly(BAC2-AEPZ1)-AEPZ)

17.7

1.21

n. r.

34

16.7

1.23

n. r.

34

20.6

1.38

n. r.

34

HAPP12 (poly(MBA1BAC2-AEPZ)AEPZ)

38.0

1.7

n. r.

31

HP(Poly((BAC2-DMDPTA1))DMDPTA

7.8

1.9

n. r.

44,57

HP(Poly((CBA2-DMDPTA1))-NBTEc

8.7

2.1

n. r.

44

Polymer

Approach

Mn ×(10-3)

poly(amino ester)s poly(BDA2-AEPZ1)-AEPZ poly(BDA2-AEPZ1)-MPZ


Poly(BDA-EEDA)

2A2 + BB’B”

A3 +2BB’B”

A2 + BB’B”

Poly(BDA-HEDA)

poly(amido amine)s

BAAP (poly(BAC2-AEPZ1)-APDb) BAP1A1 (poly(BAC2-AEPZ1)APD/PEG 350(1:1))

2A2 + BB’B”

a

MPZ: N-methyl piperazine; bAPD: 3-amino-1,2-propanediol; cNBTE: N-(2-aminoethyl)-4(((2-hydroxyethyl)thio)methyl)-3-nitrobenzamide; dnot reported.


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■ REFERENCES (1) Kamaly, N.; Yameen, B.; Wu, J.; Farokhzad, O. C. Chem. Rev, 2016, 116, 2602-2663. (2) Tian, H. Y.; Tang, Z. H.; Zhuang, X. L.; Chen, X. S.; Jing, X. B. Prog. Polym. Sci. 2012, 37, 237-280. (3) Mather, B. D.; Viswanathan, K.; Miller, K. M.; Long, T. E. Prog. Polym. Sci. 2006, 31, 487-531. (4) Ferruti, P. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 2319-2353. (5) Green, J. J.; Langer, R.; Anderson, D. G. Acc. Chem. Res. 2008, 41, 749-759. (6) Lynn, D. M.; Langer, R. J. Am. Chem. Soc. 2000, 122, 10761-10768. (7) Odian, G. In Principles of Polymerization, 4th ed.; John Wiley & Sons: Hoboken, New Jersey, 2004; Chapter 2, p 39. (8) Komber, H.; Voit, B.; Monticelli, O.; Russo, S. Macromolecules 2001, 34, 5487-5493. (9) Durand, D.; Bruneau, C. M. J. Polym. Sci., Part B: Polym. Phys. 1979, 17, 295-303. (10) Liu, Y.; Chung, T. S. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 4563-4569. (11) Durand, D.; Bruneau, C. M. J. Polym. Sci., Part B: Polym. Phys. 1979, 17, 273-294. (12) Liu, Y.; Wu, D. C.; Ma, Y. X.; Tang, G. P.; Wang, S.; He, C. B.; Chung, T. S.; Goh, S. Chem. Commun. 2003, 2630-2631. (13) Wu, D. C.; Liu, Y.; He, C. B.; Chung, T. S.; Goh, S. T. Macromolecules 2004, 37, 6763-6770. (14) Hong, C. Y.; You, Y. Z.; Wu, D. C.; Liu, Y.; Pan, C. Y. J. Am. Chem. Soc. 2007, 129, 5354-5355. (15) Gao, C.; Tang, W.; Yan, D. Y. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 23402349. (16) Wang, D.; Liu, Y.; Hu, Z. C.; Hong, C. Y.; Pan, C. Y. Polymer 2005, 46, 3507-3514. (17) Cheng, W. R.; Kumar, J. N.; Zhang, Y.; Liu, Y. Macromol. Biosci. 2014, 14, 347-358. (18) Song, C. C.; Ji, R.; Du, F. S.; Li, Z. C. Macromolecules 2013, 46, 8416-8425. (19) Lin, C.; Zhong, Z. Y.; Lok, M. C.; Jiang, X. L.; Hennink, W. E.; Feijen, J.; Engbersen, J. F. J. J. Controlled Release 2006, 116, 130-137. (20) Wang, D.; Liu, Y.; Hong, C. Y.; Pan, C. Y. Polymer 2006, 47, 3799-3806. (21) Wang, R. B.; Zhou, L. Z.; Zhou, Y. F.; Li, G. L.; Zhu, X. Y.; Gu, H. C.; Jiang, X. L.; Li, H. Q.; Wu, J. L.; He, L.; Guo, X. Q.; Zhu, B. S.; Yan, D. Y. Biomacromolecules 2010, 11, 489-495.


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Michael Addition Polymerization of Trifunctional Amine and Acrylic

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