Facile Synthesis and Properties of Multifunctionalized Polyesters by

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Facile Synthesis and Properties of Multifunctionalized Polyesters by Passerini Reaction as Thermosensitive, biocompatible and Triggerable Drug Release Carriers Man Zhao, Na Liu, Ronghui Zhao, Pengfei Zhang, Shengnan Li, Ying Yue, and Kui-Lin Deng ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00095 • Publication Date (Web): 12 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019

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ACS Applied Bio Materials

Facile Synthesis and Properties of Multifunctionalized Polyesters by Passerini Reaction as Thermosensitive, biocompatible and Triggerable Drug Release Carriers Man Zhao, Na Liu, Rong-Hui Zhao, Peng-Fei Zhang, Sheng-Nan Li, Ying Yue, Kui-Lin Deng* College of Chemistry & Environmental Science, Affiliated Hospital, Hebei University, Baoding 071002, China,

Email: [email protected]

ABSTRACT We developed a facile synthesis for a series of multifunctionalized polyesters by Passerini three-component polymerization (Passerini-3CP) in “one-pot” method at room temperature using serial dicarboxylic acids, dialdehyde and tert-butyl isocyanide as monomers. First, the effects of monomer feed ratio, monomer concentration and different dicarboxylic acids involved in the polymerization were systematically investigated. The in-situ FTIR and GPC measurements have suggested a step-growth mechanism for Passerini-3CP. Second, five succinic acid-end-capped polyethylene glycols (S-PEG) with different molecular weight 400, 800, 1000, 2000 and 4000 g/mol were prepared and selected as dicarboxylic acids for the subsequent Passerini-3CP to fabricate the thermosensitive and biocompatible polyesters. Among five resulting polyesters, four polyesters from S-PEG-400, S-PEG-800, S-PEG-1000 and S-PEG-2000 show reversible response to the external temperature, and the lower critical solution temperature (LCST) in water is in the range of 28.5-84.2oC. Through the copolymerization of S-PEG-400 and S-PEG-800, the LCSTs for functional polyesters can be conveniently controlled to be 38.7, 42.3 and 58.0℃, respectively. After 24-72 hours of incubation in polyesters solution, the viability rate of HeLa cells reached up to 80-107%, showing its excellent biocompatibility. The cleavable polyesters were also prepared by integrating S-S bonds onto their backbones in Passerini-3CP of 3,3'-dithiodipropionic acid as one comonomer for the biomedical applications. With the aid of the hydrophobicity of doxorubicin (DOX) and thermosensitivity of polyesters, the doxorubicin-loaded carriers with the size of 200-400 nm and core-shell structure were easily obtained by dialysis below LCST and subsequent heating to 1

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LCST. The effective release of DOX from the carriers can be triggered by the characteristic reaction of L-glutathione (GSH) with S-S bonds in the functionalized polyester backbones. Keywords: Passerini Polymerization, Thermosensitive Biocompatible Cleavable Polyester, Triggered Drug Release INTRODUCTION Multi-component reactions (MCRs) involve more than two reactants to form one single final product in a one-pot method, and are featured by highly atom-efficient and straightforward practical procedures.1,2 The best known and well-established isocyanide-based multi-component reactions include Passerini three-component reaction (Passerini-3CR), Ugi four-component reaction (Ugi-4CR) and Groebke three-component reaction (Groebke-3CR) and so on.3-5 For example, Passerini-3CR first reported in 1921, combines an oxo-component (aldehyde or ketone), a carboxylic

acid,

and

α-acyloxycarboxamides.6-8

an

isocyanide,

leading

to

the

formation

of

combinatorial

It is worth mentioning that that no by-product is formed from reactant

to final product during the whole Passerini-3CR process. Furthermore, Passerini-3CR can be smoothly performed at room temperature and in air via “one-pot” method. Early, MCRs were mainly applied in combinatorial and medicinal chemistry because of their easy access to diversity and complex structure of compounds in the total synthesis of natural products.1, 2, 9

Until the last few years, MCRs have gained an increasing focus in polymer chemistry. The

combination of multicomponents provides a facile tool to regulate the structures and properties of the resulting polymers and easy introduction of desired groups is also enabled in the polymerization.1,10-20 In fact, as long as two reactants in the MCR systems are bifunctional compounds such as dicarboxylic acids (or dialdehydes, diisocyanides and diamines), or one reactant contains two different functional groups such as amino acids (or carboxyl aldehydes), MCRs theoretically evolve into a linear polymerization. For instance, the combination of a dicarboxylic acid/dialdehyde, a dicarboxylic acid/diisocyanide, or a dialdehyde/diisocyanide lead to the 2


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generation of alpha-amide substituted polyesters, alternating poly(ester-amide)s and polyamides with ester side chains, respectively.1 Recently, Meier et al. systematically reported the advances about Passerini and Ugi reaction in polymer science,1 and a lot of subsequent outstanding researches.18-21 Since 2012, Zi-Chen Li has prepared a variety of novel functional polymers by Passerini-3CP.10-14 Most remarkable of all, the unique and high-efficient mechanism of the modular nature of multi-component polymerization allows the synthesis of polymers with the desired novel properties and structure by the simple combinations of specific monomers. For example, Passerini-3CP of photo-responsive 2-nitrobenzaldehyde, adipic acid and 1,6-diisocyanohexane,

led to poly(ester-amide)s with

controllable decomposition behaviors.22 After 20 min UV-irradiation (at 365 nm) to poly(ester-amide) solution, the complete degradation of polymer backbone was observed by the cleavage of the 2-nitrosobenzyl moiety. Zi-Chen Li14 reported the novel H2O2-cleavable poly(ester-amide)s by the in-situ formation of benzyl ester bond in the polymer backbone from phenylboronic acid ester via Passerini-3CP. Song and coworkers23 introduced a photoswitchable azobenzene-substituted carboxylic acid into the polymer by Passerini reaction. Interestingly, the deformation of the particles from polymer and agglomeration to larger aggregates could be triggered via the isomerization of the azobenzene moieties in the exposure at UV light (at 365 nm). Meanwhile, some other polymers with unique structure or property were also prepared by Passerini-3CP such as some dendrimers with both ABC and ABB branching structures,24 functional poly(ester amide)s,25 divergent dendrimer,26 versatile polyesters from AB-type monomer,

27

and

tunable polymers.28 In addition, Youhua Tao15-16 also demonstrated that Ugi reaction of natural amino acids leads to a biocompatible polypeptoids with antibacterial activity, and a highly water-soluble alternating polypeptoids. Lei Tao29-30 designed and prepared well-defined poly(1,4-dihydropyridine)s

and

multifunctional

protein

conjugation

via

multicomponent

polymerization. Inspired by the recent developments in the MCRs field11-20 and our previous investigations about 3



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the thermosensitive polymers31-34, the aim of this study was to apply the efficient Passerini-3CP for the

design

and

preparation

of

multifunctionalized

polyesters

with

thermosensitivity,

biocompatibility and cleavability for the potential biomedical applications. Using biscarboxylated polyethylene glycol with different molecular weight as diacids in the Passerini-3CP system, the hydrophilicity and hydrophobicity of polyesters were facilely regulated, leading to the thermosensitivity of polyesters. Furthermore, the majority of polyethylene glycol blocks in the polyester backbone directly endowed the resulting polyesters with excellent biocompatibility. Passerini-3CP of 3, 3'-dithiodipropionic acid monomer also led to the facile synthesis of the cleavable polyesters. Additionally, the release of doxorubicin was successfully triggered by the characteristic reaction of L-glutathione (GSH) with S-S bond in polyester backbones. In brief, Passerini-3CP offers a universal methodology toward facile and efficient preparation of functionalized polyesters or polyamides for the potential applications. EXPERIMENTAL Preparation of Polyesters from Five Common Aliphatic Diacids by Passerini-3CP Firstly, the five common aliphatic diacids selected in this study including malonic acid, butanedioic acid, glutaric acid, adipic acid and sbacic acid were used as one component in the Passerini-3CP system to evaluate the effect of various conditions on the polymerization. In the representative preparation of poly(sebacic acid-glutaraldehyde-tert-butyl isocyanide) (PSGBI), 0.8255 g (4 mmol) sebacic acid and 0.4005 g (4 mmol) re-distilled glutaraldehyde were added into a 25 mL flask containing 4 mL dichloromethane. The mixture was stirred for about 2 h, so that sebacic acid fully pre-reacted with glutaraldehyde. And then, 0.6787 g (8 mmol) tert-butyl isocyanide was dropwise added into the flask, and the flask was sealed quickly and stirred for 48 hours at room temperature. After completion, the solution is poured into a beaker containing cold ethyl ether in order to precipitate and filter the crude PSGBI. The purified polyester PSGBI was obtained with a yield of 72.9% after the crude PSGBI was re-dissolved in CH2Cl2 and precipitated 4


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in cold ethyl ether. Similarly, poly(malonic acid-glutaraldehyde-tert-butyl isocyanide) (PMGBI), poly( butanedioic acid-glutaraldehyde-tert-butyl

isocyanide)

(PBGBI),

poly(glutaric

acid-glutaraldehyde-tert-butyl isocyanide) (PGGBI), and poly(adipic acid-glutaraldehyde-tert-butyl isocyanide) (PAGBI) were prepared by Passerini-3CP according to the above procedure. The synthetic route is shown in Scheme 1. Preparation of Thermosensitive and Biocompatible Polyesters from Five S-PEG by Passerini-3CP Five biscarboxylated polyethylene glycols described in Supporting Information including S-PEG-400, S-PEG-800, S-PEG-1000, S-PEG-2000 and S-PEG-4000 were used as diacid component in the Passerini-3CP to prepare the polyester with thermosensitivity and biocompatibility. In the representative preparation of poly(S-PEG-800-glutaraldehyde-tert-butyl isocyanide) (PPGBI-800), 2.100 g, (2.1 mmol) S-PEG-800 and 0.215 g, (2.1 mmol) anhydrous glutaraldehyde were dissolved in 2.1 mL anhydrous CH2Cl2. After stirring for 2 hours at room temperature, 0.364 g, (4.2 mmol) tert-butyl isocyanide was added and stirred for 48 hours. After completion, the solution was dripped into cold ethyl ether to precipitate target polyester PPGBI-800. To purify the crude PPGBI-800, PPGBI-800 aqueous solution is dialyzed in deionized water for two days with a 1000 Da dialysis membrane, and the water is changed every few hours. Finally, the PSGBI-800 solution is then lyophilized for 8 hours. The white solid powder, purified PPGBI-800 was obtained with a yield of 83.4%. Similarly, poly(S-PEG-400-glutaraldehyde-tert-butyl isocyanide) (PPGBI-400), poly(S-PEG-1000-glutaraldehyde-tert-butyl isocyanide) (PPGBI-1000), poly(S-PEG-2000-glutaraldehyde-tert-butyl

isocyanide)

(PPGBI-2000),

and

poly(S-PEG-4000-glutaraldehyde-tert-butyl isocyanide) (PPGBI-4000) were prepared according to the above procedure. The synthetic routes are also shown in Sheme 1. Preparation of Cleavable Polyesters from 3,3'-dithiodipropionic Acid by Passerini-3CP For the synthesis of cleavable polyesters, we introduced a new monomer, 3,3'-dithiodipropionic 5



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acid into the polyester backbone. In the synthesis of poly (3,3'-dithiodipropionic acidS-PEG-800-glutaraldehyde-tert-butyl isocyanide) (PSPGBI-800-65, the mole feed ratio of 3,3'-dithiodipropionic acid is 65%), 0.5363 g (2.6 mmol) 3,3'-dithiodipropionic acid, 1.4000 g (1.4 mmol) S-PEG-800 and 0.4005 g (4 mmol) glutaraldehyde were added into a 25 mL flask to dissolve in 4 mL dichloromethane. The mixture was stirred for about 2 h, so that two kinds of diacids fully pre-reacted with glutaraldehyde. And then, 0.6787 g (8 mmol) tert-butyl isocyanide was added to the flask drop by drop, and the flask was sealed quickly and stirred for 48 hours at room temperature. After the reaction is completed, the solution is poured into a beaker containing cold ethyl ether for precipitation and the crude PSPGBI-800-65 is filtered. The purified polyester PSPGBI-800-65 was obtained with a yield of 76.7% after the crude PSPGBI-800-65 was re-dissolved in methanol and re-precipitated in cold ethyl ether. In addition, the changing feed ratio of 3,3'-dithiodipropionic acid to S-PEG-800 led to the production of PSPGBI-800-60 and PSPGBI-800-55 with different LCSTs. Preparation of DOX-loaded Cleavable Polyester Nanocarrier The fabrication of DOX-loaded cleavable polyester nanocarriers was performed with the aid of thermo-sensitivity of PSPGBI-800-65 and hydrophobicity of DOX. Briefly, DOX hydrochloride (0.006 g), TEA (0.023 g) and PSPGBI-800-65 (0.030 g) were dissolved in DMSO (8 mL) at room temperature. DOX hydrochloride first reacted with TEA to produce hydrophobic DOX. After dissolution, the dialysis bag containing DOX, TEA, thermosensitive PSPGBI-800-65 and DMSO was dialyzed against water for 6 h at room temperature. In this process, DOX precipitated due to its high hydrophobicity. After complete precipitation, the dialysis system was heated slowly to 45 oC (higher than LCST of PSPGBI-800-65). At this moment, PSPGBI-800-65 chains converged on the surface of DOX particles in the heating run. The final DOX-PSPGBI-800-65 nanocarriers were obtained after lyophilizing over 24 h. Drug loading capacity (DLC) and drug loading efficiency (DLE) were calculated according to the following formula: 6


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DLC (%) = (weight of loaded DOX / weight of DOX-PSPGBI-800-65 carriers) × 100% DLE (%) = (weight of loaded DOX / weight of input DOX ) × 100% The weight of loaded drug was determined with the UV-Vis absorption spectra at 480 nm and calculated by using standard absorbance technique. Results and Discussion Strategy for Molecular Design and Synthesis of the Multifunctional Polyesters In Passerini-3CP, all the chemical elements from the three monomers are bonded together onto the macromolecular chains without any by-products. At the same time, due to the existence of highly reactive isocyanide monomer, the polymerization can be smoothly completed in “one-pot” method at room temperature in the air. Based on the recent investigations11-20 and our previous studies,31-34 the strategies for the synthesis of multifunctional polyesters in this investigation are listed as shown in Scheme 1:

Scheme 1. Synthesis of multifunctionalized polyesters by Passerini-3CP 7



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First, the polyesters containing amide side groups were prepared by Passerini-3CP of five common aliphatic diacids with glutaraldehyde and tert-butyl isocyanide as monomers. The optimum conditions of Passerini-3CP were synthetically evaluated by adjusting the monomer ratio, concentration, solvent, temperature, time and structure of dicarboxylic acids. Second, the thermosensitive polymers with biocompatibility have great application values and exploitation prospects in the biomedicine fields.34-35 Five polyethylene glycols with different Mn, which have been approved by the FDA for practical use in medicine,36 were used as dicarboxylic acid components in Passerini-3CP system. In this way, the hydrophilicity and hydrophobicity of polyesters can be effectively adjusted by introducing different polyethylene glycols, so as to achieve their relative balance, leading to excellent thermosensitivities for polyesters. Polyethylene glycol blocks, which is an overwhelming majority in the synthesized polyester chains, can endow the polyesters with good biocompatibility. And also, the triggerable release of anticancer drugs has attracted much attention in the field of medicine, and the preparation of cleavable polymeric materials is one of the key links.37-38 In this investigation, 3,3'-dithiodipropionic acid was selected as one of the components of Passerini-3CP to prepare the cleavable polyesters containing S-S bond on the backbones. Finally, the DOX-loaded nanocarriers with core-shell structure were prepared by a two-step successive procedure based on the thermosensitivity of polyester and the hydrophobicity of DOX. The effective triggered-release of DOX in a simulated environment was also successfully achieved from the nanocarriers in the presence of L-glutathione. Structural Characterization of Multifunctionalized Polyesters The chemical structure of the multifunctional polyesters was characterized by FTIR, 1H NMR, 13C NMR and GPC measurements described in the previous paper34. Figure 1 is the FTIR spectra of representative PSGBI, PPGBI-800 and PSPGBI-800-65 respectively. In the FTIR of PPGBI-800 (B), the stretching and bending vibration peaks of N-H from the amide groups in PSGBI chain appeared at 3419 cm-1 and 1536 cm-1. The absorption peak at 1677 cm-1 can be attributed to the 8


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stretching vibration peak of C=O on the amide bond. The absorption peak of C=O on the ester group in PPGBI-800 appeared at 1736 cm-1. Due to the high relative content of PEG block in PPGBI-800 chains, the strongest stretching vibration peak of C-O-C was observed at 1107 cm-1. For PSPGBI-800-65 (C), the more characteristic absorption of S-S bond on its backbone was found at 652-571 cm-1. Other absorption peaks, such as the stretching vibration absorption of C=O from the ester and amide groups, appeared at 1735 cm-1 and 1670 cm-1, respectively. The absorption peak at 3392 cm-1 corresponds to the stretching vibration of N-H from the amides. The FTIR of other functional polyesters and S-PEG can be seen in Supporting Information (from Figure S1 to Figure S10).

Figure 1. FTIR spectra of three representative multifunctional polyesters (A: PSGBI, B: PPGBI-800 and C: PSPGBI-800-65) For the 1H NMR of PPGBI-800 showed in Figure 2(B), the proton resonance peaks at 1.00-1.40 ppm correspond to H atoms on methyl from tert-butyl isocyanide and methylene from 9



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glutaraldehyde. The chemical shifts at δ = 1.40-1.75 ppm can be attributed to H atom on the other methylenes from glutaraldehyde. In addition, the characteristic proton resonance peak of NH on the amide group was observed at δ = 6.0-6.2 ppm. The proton resonance peak of H atoms on the repeating unit-OCH2CH2- appeared at 3.3-3.7 ppm and the peak intensity was the highest due to the relatively high content of PEG block. Two methynes and two methylene show their peaks at 4.9 ppm and 4.2 ppm, respectively. In Figure S22 and Figure S23 listed in Supporting Information, the 2D NMR measurements (COSY and HSQC) further proved the correctness of structure of PPGBI-800.

Figure 2. 1H NMR spectra of PSGBI (A, in DMSO-d6), PPGBI-800 (B, in CDCl3) and PSPGBI-800 (C, in CDCl3) 10


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Similarly, in the 1H NMR of PSPGBI-800 (C), two -CH2 groups close to S-S bond on the 3,3'-dithiodipropionic acid unit was observed at 2.85 and 2.95 ppm, respectively. The highest peak at δ = 3.5-3.7 ppm indicates the existence of PEG blocks. The percentage of 3,3'-dithiodipropionic acid unit in PSPGBI-800 chain can be calculated according to the integral area of the two above-mentioned peaks. When the feed ratio of 3,3'-dithiodipropionic acid was 55%, 60% and 65%, the structure unit ratio in the copolyester was measured as 60.2 % for PSPGBI-800-55, 63.5% for PSPGBI-800-60 and 72.4% for PSPGBI-800-65, respectively. The 1H NMR spectra of other multifunctional polyesters such as PBGBI, PAGBI, PPGBI-1000 and so on, including

13C

NMR

spectrum of PSGBI can be observed in Supporting Information (from Figure S11 to Figure S21). Optimization of Passerini-3CP Conditions In the step-growth polymerization, the purity of monomers, the ratio of functional groups of monomers and other external factors such as time, temperature and solvent may have important effects on the polymerization. As showed in Supporting Information (Table SI-1), among water, methanol, tetrahydrofuran, toluene, chloroform and dichloromethane as polymerization solvents, PSGBI prepared in dichloromethane showed the highest Mn and followed by in toluene and chloroform. Increasing reaction temperature can not effectively promote the Passerni-3CP, so it is reasonable to carry out the Passerni-3CP at room temperature (at 25 oC). As the monomer concentration is 0.5 M, both the Mn and the yield of PSGBI are relatively small. The Mn of PSGBI is similar for the monomer concentration of 1.0 M, 1.5 M and 2.0 M, which agrees with the reported results.6 In addition, the number of methylenes (-CH2) between two carboxyls shows a significant influence on the Passerni-3CP. For sebacic acid, Passerni-3CP successfully occurs to produce PSGBI with higher Mn and yield. But, Passerni-3CPs of malonic acid, succinic acid and glutaric acid were failed because of no detection of three polyesters (PMGBI, PBGBI and PGGBI). When the polymerization time is less than 48 hours, the Mn of PSGBI increases with 11



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increasing polymerization time, reflecting the characteristics of step-growth polymerization. Continuous prolongation of polymerization time has no obvious effect on the Mn of PSGBI, but increases the yield of PSGBI. Usually, 48 hours were chosen as the reaction time of Passerni-3CP in this study. When the feed ratios of sebacic acid, glutaraldehyde and tert-butyl isocyanide were 1:1:1.8, 1:1:2.0, 1:1:2.2 and 1:1:2.4, the Mn of PSGBI were 2.06, 2.99, 2.08 and 2.08 × 104 g/mol, respectively. That is, the equivalent feed ratio of monomers leads to the highest the Mn of PSGBI. Obviously, this is completely consistent with the basic principles of step-growth polymerization, and is also accorded with the results described by Zi-Chen Li.6,10 In this study, the optimization conditions for Passerini-3CP should be performed at room temperature, with the equivalent feed ratio of monomers and in dichloromethane for 48 h. Mechanism of Passerini-3CP Detected by In-situ FTIR and GPC

Figure 3. Tracking of characteristic absorption from polyester by In-situ FTIR (A: at 1739, 1723; B:1677 and 1519 cm-1) In-situ infrared spectroscopy is one of the most important methods for tracking chemical reactions.39 In this study, the in-situ infrared spectroscopy was used to determine the change of the absorption peaks in the range of 1000-1900 cm-1 during Passerini-3CP. As shown in Figure 3 (A), the stretching vibration absorption of C=O group from carboxylic acid and aldehyde components appeared at 1723 cm-1. With the prolongation of polymerization time, the peak intensity at 1723 12


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cm-1 gradually decreased until it finally disappeared. At the same time, the absorption peak of C=O (at 1739 cm-1) from the formed ester group appeared and strengthened continuously. Similarly, the absorption peaks (at 1677 cm-1 and 1519 cm-1 in Figure 3 (B)) which represent the formation of amide groups simultaneously formed during Passerini-3CP, gradually becomes much sharper and stronger.

Figure 4. GPC curves of PSGBI with different polymerization time Figure 4 is a set of GPC curves of PSGBI with different polymerization times. It can be seen that with the increase of reaction time, the eluent time of PSGBI samples becomes shorter, that is, its molecular weight (Mn) for PSGBIis gradually increasing. For example, when the polymerization time was 12, 24, 36 and 48 hours, the Mn of PSGBI was 0.86, 1.27, 1.47 and 2.16 × 104 g/mol, respectively. Based on the above results, Passerini-3CP in this study showed a step-growth characteristic, which was also consistent with the results reported by Zi-Chen Li. 6,10,40 Regulation of Thermosensitivities for Polyesters Generally, thermosensitive polymers contain hydrophilic and hydrophobic groups, and the hydrophilic and hydrophobic interactions reach or approach a relative balance.35 According to the design principle of thermosensitive polymer,31-32 five polyesters from aliphatic diacids inevitably show no temperature-sensitive characteristics because of their much higher hydrophobicity. In this study, we selected five more hydrophilic biscarboxylated polyethylene glycols with different Mn as 13



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monomers in the Passerin-3CP. In this way, the hydrophilicity and hydrophobicity of synthesized polyesters can be effectively adjusted by polyethylene glycols, thus endowing polyesters with excellent thermosensitivity.

Figure 5. Thermosensitivity of a series of PPGBIs in the solution In Figure 5 plotted by the reported UV measurement34, PPGBI-400, PPGBI-800, PPGBI-1000 in water exhibited good thermosensitivity, and their lowest critical solution temperatures (LCST) were 28.5, 69.2 and 72.8 oC, respectively. PPGBI-2000 is highly soluble in water, showing temperature sensitivity only in the normal saline (0.9%), and its LCST is 84.2 oC. However, the hydrophilicity of PPGBI-4000 is the highest, and no thermosensitivity is observed in both pure water and normal saline. Actually, Figure 5 also reveals that with an increase of Mn of S-PEG, from 400 to 2000 g/mol, the LCST of PPGBI-400, PPGBI-800, PPGBI-1000, PPGBI-2000 was increased accordingly. The increase of Mn of S-PEG means a significant decrease of content of hydrophobic amide-ester moiety on PPGBI chains. In addition, the copolymerization is one of the effective means to adjust the response-temperature of thermosensitive polymers.41-42 In this study, the effect of copolymerization on thermosensitivity of polyesters was studied by the combination of S-PEG-400 and S-PEG-800. When the feed ratios of S-PEG-400 to S-PEG-800 are 75:25, 50:50, 25:75, the LCST of copolyesters (CoP-75-25, CoP-50-50 and CoP-25-75) can be easily adjusted to 38.7, 42.3 and 58.0 oC. With the increase in the number of hydrophilic (-OCH2CH2-) groups in the main 14


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chains, the hydrophilicity of copolyesters increases gradually, and the LCST value increases continuously.

Figure 6. Reversible response to temperature of PPGBI-400 (A) and thermosensitivity of three copolyesters with S-S Bonds (B) Taking PPGBI-400 as an example, the reversible curves of the transmittance of PPGBI-400 with temperature were measured in Figure 6 (A). During the heating process, the transmittance of PPGBI-400 aqueous solution decreases rapidly from 100% to 0.4%, while in the cooling process, the transmittance of PPGBI-400 solution rises rapidly from 0.4% to 100%, which can be reversed many times. Macroscopically, we can clearly observe the reciprocating process from clarification to turbidity of PPGBI-400 solution, and again from turbidity to clarification. The thermosensitive PPGBI-400 exhibits a "hysteresis" phenomenon during the heating-cooling process, and its value is 3~4 oC. This small "hysteresis" phenomenon is related to the relatively high content of PEG block in the PPGBI-400 chains. The longer -(CH2CH2O)y- chain effectively reduces the H-bonding among macromolecular chains according to the reported descriptions.43-44 Figure 6(B) presented the thermosensitivity of copolyesters synthesized by Passerini-3CP of S-PEG-800 and 3,3'-dithiodipropionic acid (mol feed ratio of 55%, 60% and 65%). LCSTs for PSPBGI-800-55, PSPBGI-800-60 and PSPBGI-800-65 were measured as 48.5, 40.4 and 33.4 oC, respectively. More 3,3'-dithiodipropionic acid units represents more hydrophobicity of copolyesters. 15



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In this study, PPPBI-800 with Mn of 2.17, 2.63 and 3.32 × 104 g/mol were prepared by changing reaction time. The LCST of PPPBI-800s decreases with the increase of their Mn as shown in the Supporting Information (Figure S26). This is consistent with the reported results of thermosensitive polymers such as poly(N-vinyl isobutyramide45 and poly(carbonate ether).46 The main reason is that with the increase of Mn, the stronger the H-bonding formed among polyester chains via amides, the much lower the external temperature is enough for polyester to complete aggregation and separation from the solution in the heating process. The contact angle can reflect the hydrophobicity and hydrophobicity of polymers.47 In this study, the contact angle of synthesized polyesters was measured by static suspension drop method with contact angle tester. As shown in Figure 7, the contact angles of PPGBI-400, PPGBI-800, PPGBI-1000, PPGBI-2000 and PPGBI-4000 with water droplets at room temperature are 59o, 46o, 43o, 31o and 23o, respectively. For the three copolyesters (mol feed ratio of S-PEG-400 75%, 50% and 25%), the contact angles similarly decrease with the increasing more hydrophilic S-PEG-800 in Passerin-3CP. In summary, the hydrophilicity of polyesters becomes stronger and stronger as the number of -(CH2CH2O)y- increases, leading to a decrease of the contact angles. This result is also consistent with the thermosensitivity principle of different polyesters presented in Figure 5.

Figure 7. Contact angle of multifunctional polyesters to water at room temperature 16


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Biocompatibility Evaluation of Multifunctional Polyesters Because the thermosensitive polymers have great potential in the biomedical field, good biocompatibility is one of the inevitable requirements for thermosensitive polymers.32-35 Polyethylene glycol used in this investigation is currently one of the few medical polymers approved by FDA.36 The described MTT cell counting method34 was used to evaluate the cytotoxicity of polyesters as shown in Figure 8. After 24, 48 and 72 h culture of HeLa cells in the concentration of 0.5, 5, 25, 50 and 100 μg/mL of PPGBI-800 solution, the survival rate of HeLa cells was still as high as 85%, or even more than 107%, respectively. The results showed that PPGBI-800 had no obvious cytotoxicity to HeLa cells, i.e. indicating excellent biocompatibility. The existence of polyethylene glycol and its higher content theoretically endow the multifunctional polyesters with expected excellent biocompatibility.

Figure 8. Viability of HeLa cells of multifunctional polyesters (A: PPGBI-800; B: PPSGBI-800-65) Similarly, we further evaluated the cytotoxicity of cleavable PPSGBI-800-65 using as drug-loaded carriers. As shown in Figure 8(B), after 24, 48 and 72 hours of culture, the survival rate of HeLa cells is still as high as 80~98%, suggesting excellent biocompatibility of PPSGBI-800-65. For both PPGBI-800 and PPSGBI-800-65, the increasing tendency of HeLa cell viability is also observed after 72 h. This may be due to the adaptation for cells to the environment of 17



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multifunctional polyesters. Fabrication of Cleavable DOX-loaded Nanocarriers with Core-shell Structure

Figure 9. Illustration of formation for DOX-loaded nanocarrier (A), the morphology observed by SEM (B) and size of DOX-loaded nano-particles tested by laser particle size analyzer (C) In recent years, the controllable drug release system has become important research focuses in the biomedicine.48-50 In this study, the synthesis of thermosensitive, biocompatible and cleavable polyesters provides a possibility for the construction of triggered-release carrier systems. DOX, as a model anticancer drug, is one of the most commonly used chemotherapeutic drugs and a popular research tool due to its inherent fluorescence. PPSGBI-800-65 with LCST of 33.4 oC was consciously selected in order to avoid the re-destruction of DOX-loaded carriers due to its themosensitivity on the release temperature (normally at body temperature). Hereby, as shown in Figure 9 (A), we fabricate a DOX nanocarrier by the thermo-sensitivity of PPSGBI-800-65 and the hydrophobicity of DOX. Prior to the loading of DOX into the carriers, DOX·HCl first reacted with TEA in DMSO to detach the HCl and render the drug hydrophobicity. In the dialysis process with water below LCST, DOX slowly formed some nanocarriers from DMSO solution due to its high hydrophobicity. And then, PPSGBI-800-65 chains collapsed and aggregated in the subsequent 18


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heating to LCST due to its thermo-sensitivity. In the meantime, the precipitated PPSGBI-800-65 chains aggregated on the DOX-particle surface, leading to the formation of DOX-loaded nanocarrier with core-shell structure. The morphology and size of DOX-loaded carriers from PPSGBI-800-65 were tested by SEM and laser particle size analyzer measurements described in the previous paper34 shown in Figure 9(B). It was found that DOX-loaded carriers are nearly spherical in shape, and the average diameter is about 300 nm. The glossy and overall uniform surface of the nonocarrier was also observed. In Figure 9(C), the measurement by laser particle size analyzer presents a hydrodynamic diameter of 276 nm and a polydispersity index value of 0.184. This result is also in line with the observation by SEM measurement. Also, the well-known Tyndall effect was clearly observed when a beam of light from laser pointer passed through the DOX-loaded nanocarrier system (as showed in Figure S27). This further proved the size dimension of nanometers of DOX-loaded carrier. In addition, the theoretical DLC was set as 20 wt% in DOX-loaded carriers from PPSGBI-800-65. The calculated DLC and DLE was 13.2% and 70.8%, respectively by determining by the UV measurements. Triggered Release of DOX from Nanocarriers As well as known, the intracellular tumor microenvironment shows obvious divergence with normal cells, especially for the concentration of reductive GSH. Generally,The concentration of GSH in tumor cells is approximately 2~10 mM, much higher than that in the extracellular fluids (approximately 2~20 μM).51-53 To examine the effect of the drug-carrier shell and release environment, we performed DOX release experiments at 37 oC with DOX-loaded carrier from PPSGBI-800-65 and PPGBI-400 in the presence or absence of GSH. As shown in Figure 10, no obvious different DOX release behaviors were found in the presence or absence of GSH when the DOX-loaded carrier was constructed from PPGBI-400. After 17 h, the accumulative release of DOX was 48% in the absence of GSH, 51% in the presence of GSH, respectively. For DOX-loaded carriers from PPSGBI-800-65, in the presence of GSH (10 mM), much faster DOX release rate 19



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from DOX-loaded carriers was observed, that is, 87% of the loaded DOX was released over 17 h. However, for the release system in the absence of GSH, only 42% of loaded DOX was released after the same time. The reason can be ascribed to the cleavage of S-S bonds in PPSGBI-800-65 backbone, which causes destruction and detachment of the drug-carrier shell, and accelerating the release of loaded DOX. The above results suggest that the thermo-sensitive cleavable PPSGBI-800-65 may achieve a triggered-release drug delivery system in the presence of reducing agents (GSH) near the cancer cells. At the same time, PPSGBI synthesized from PEG blocks render excellent biocompatibility for the drug delivery systems.

Figure 10. Triggered-release of DOX from nanocarriers CONCLUSIONS We have described a facile strategy for the synthesis of a group of multi-functionalized polyesters with thermosensitivity, biocompatability and cleavability via Passerini-3CP. The Passerini-3CP undergo a step-growth mechanism, and multifunctional polyesters with more than 1 × 104 g/mol can be easily prepared at room temperature in “one-pot” method. Of most importantly, a set of synthesized polyesters from carboxylized PEG shows excellent thermosensitivity, their tunable LCST is in the range of 28.5-84.2oC, covering the room temperature and body temperature. The 20


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functionalized polyesters from both carboxylized PEG and 3,3'-dithiodipropionic acid exhibit no cytotoxicity, indicating the outstanding biocompatibility. The DOX-loaded carriers were prepared with the aid of the hydrophobicity of DOX and thermosensitivity of the functional polyesters. Finally, the effective release of DOX from nanocarriers can be triggered by the characteristic reaction of GSH with S-S bond on the polyester backbone. In conclusion, Passerini-3CP provides a robust and efficient technique for the preparation of a multifunctional polyester or polyamide by the simple combination of different acids, aldehydes (ketones) and isocyanides. ASSOCIATED CONTENT Supporting Information Materials, Measurements of contact angle for thermosensitive polyesters, In Vitro release of DOX in the absence or presence of GSH, Synthesis of biscarboxylated polyethylene glycol (S-PEG) with different Mn; Scheme S1 Synthesis of Five S-PEGs, Table S1 Effect of the experimental conditions on Passerini-3CP, Table S2 LCSTs of polyesters from several S-PEG; FTIR spectra of PEG-400, S-PEG-400, PMGBI, PBGBI, PGGBI, PAGBI, PPGBI-400, PPGBI-1000, PPGBI-2000, PPGBI-4000 and PSPGBI-800-55; 1H NMR spectra of S-PEG-400, PPGBI-400, PPGBI-1000, PPGBI-2000, PPGBI-4000, PMGBI, PBGBI, PPGBI, PAGBI and PSPGBI-800-55;

13C

NMR

spectrum of PSGBI; 2D NMR spectrum (COSY) of PPGBI-800; 2D NMR spectrum (HSQC) of PPGBI-800; 1H NMR spectra of PPGBI-800 in D2O at 35 oC and 55 oC; Thermosensitive PPPBI-800 with different Mn; Tyndall effect of DOX-loaded nano-carrier system exposed to a beam of light. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] Phone: +86-312-5971137. ORCID 21



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Kuilin Deng: 0000-0001-5813-4544 Author Contributions Man Zhao, Na Liu are contributed equally to this work. Notes The authors declare no competing financial interest.

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Facile Synthesis and Properties of Multifunctionalized Polyesters by

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