Fabrication of Hyperbranched Block-Statistical Copolymer-Based

Subscriber access provided by READING UNIV

Article

Fabrication of Hyperbranched Block-statistical Copolymerbased Prodrug with Dual Sensitivities for Controlled Release Luping Zheng, Yunfei Wang, Xianshuo Zhang, Liwei Ma, Baoyan Wang, Xiangling Ji, and Hua Wei Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00699 • Publication Date (Web): 07 Dec 2017 Downloaded from http://pubs.acs.org on December 8, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 50 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

Bioconjugate Chemistry

Fabrication of Hyperbranched Block-Statistical Copolymer-Based Prodrug with Dual Sensitivities for Controlled Release

Luping Zheng,a Yunfei Wang,a Xianshuo Zhang,a Liwei Ma,a Baoyan Wang,a Xiangling Ji,b Hua Wei*,a

a

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

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China

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

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 50

ABSTRACT: Dendrimer with hyperbranched structure and multivalent surface is regarded as one of the most promising candidates close to the ideal drug delivery systems, but the clinical translation and scale-up production of dendrimer has been hampered significantly by the synthetic difficulties. Therefore there is considerable scope for the development of novel hyperbranched polymer that can not only address the drawbacks of dendrimer but maintain its advantages. The reversible addition–fragmentation

chain

transfer

self-condensing

vinyl

polymerization

(RAFT-SCVP) technique has enabled facile preparation of segmented hyperbranched polymer (SHP) by using chain transfer monomer (CTM)-based double-head agent during the past decade. Meanwhile, the design and development of block-statistical copolymers has been proved in our recent studies to be a simple yet effective way to address the extracellular stability vs intracellular high delivery efficacy dilemma. To integrate the advantages of both hyperbranched and block-statistical structures, we herein reported the fabrication of hyperbranched block-statistical copolymer-based prodrug with pH and reduction dual sensitivities using RAFT-SCVP and post-polymerization click coupling. The external homo oligo(ethylene glycol methyl ether methacrylate) (OEGMA) block provides sufficient extracellularly colloidal stability for the nanocarriers by steric hindrance, and the interior OEGMA units incorporated by the statistical copolymerization promote intracellular drug release by facilitating

the

permeation

of

GSH

and

H+

for

the

cleavage

of

the

reduction-responsive disulfide bond and pH-liable carbonate link as well as weakening the hydrophobic encapsulation of drug molecules. The delivery efficacy of


Page 3 of 50 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


the target hyperbranched block-statistical copolymer-based prodrug was evaluated in terms of in vitro drug release and cytotoxicity studies, which confirms both acidic pH and reduction-triggered drug release for inhibiting proliferation of HeLa cells. Interestingly, the simultaneous application of both acidic pH and GSH triggers promoted significantly the cleavage and release of CPT compared to the exertion of single trigger. This study thus developed a facile approach towards hyperbranched polymer-based prodrugs with high therapeutic efficacy for anti-cancer drug delivery.



Page 4 of 50

Introduction Dendrimer with hyperbranched structure for unimolecular nanoparticles formation and multivalent surface with various functionalities is regarded as one of the most promising candidates close to the ideal drug delivery systems, 1-4

but the clinical translation and scale-up production of dendrimer has suffered

significantly from the synthetic difficulties and generally low yields, especially for the preparation of dendrimer with relatively high generations due to the severe steric hindrance as well as the compromised stability for the dendrimer-drug conjugates, compared to the parent dendrimer, resulting from the aggregation caused by the hydrophobic interactions of drug molecules conjugated to the periphery of a dendrimer. 5-8 Therefore, there is considerable scope for the development of novel hyperbranched polymer that can not only address the drawbacks of dendrimer but maintain its advantages. To this end, segmented hyperbranched polymer (SHP)9–14 with long linear chains between branched points has drawn great attention in recent years. Compared

to

classical

dendrimer

or

highly

compact

conventional

hyperbranched polymer synthesized via AB2 or A2+B3 condensation polymerization,15–19 the minimized steric hindrance between sparsely-branched backbones of SHP offers unique functionalities unavailable in more densely branched structures. In the last decade, self-condensing vinyl polymerization (SCVP)20–28 has emerged as a robust strategy towards hyperbranched vinyl polymers with versatile functionalities.29–32 Compared to the iterative multi-step


Page 5 of 50 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


syntheses required for the preparation of a dendrimer, a SHP can be efficiently prepared via one-pot SCVP33 based on controlled radical polymerization (CRP) techniques, such as atom transfer radical polymerization (ATRP)34–39 and reversible addition–fragmentation chaintransfer (RAFT) polymerization14,40–43. SCVP-generated segmented hyperbranched polymers (SHPs) thus integrate the structural advantages of branched polymers and relatively easy synthesis procedures adopted for the preparation of linear polymers, therefore were chosen as the parent polymer for post polymerization conjugation of anti-cancer drug in this work. Meanwhile, the design and development of a block-statistical copolymer with a statistical sequence has been proved in our recent studies to be a simple yet effective way to achieve intracellular high delivery efficacy.44,45 The statistical polymer block containing copolymerization-incorporated hydrophilic units served as the cargo-loading domain in these designs. Interestingly, these statistically incorporated hydrophilic moieties were able to promote the intracellular release of cargoes towards enhanced therapeutic efficacy by facilitating excellent permeation of the external media, and by weakening the electrostatic/hydrophobic interactions between the polymer chain and the cargoes.45 The key innovation of this paper lies in the fabrication of hyperbranched block-statistical copolymer-based prodrug with pH and reduction dual sensitivities for double triggers-enhanced anti-cancer drug delivery by integrating the facile



preparation strategy of RAFT-SCVP towards hyperbranched polymer and the structural virtues of block-statistical copolymer. RAFT-SCVP of glycidyl methacrylate (GMA) and oligo(ethylene glycol methyl ether methacrylate) (OEGMA) monomers was firstly performed to afford a hyperbranched core of h-P(GMA-st-OEGMA) statistical copolymer using reducible chain transfer monomer (CTM). The resulting hyperbranched core was used as a multimacro-CTA to further perform polymerization of OEGMA to produce a hyperbranched block-statistical copolymer, h-P(GMA-st-OEGMA)-b-P(OEGMA) with controlled dimension (Scheme 1). Finally, the anticancer drug, camptothecin (CPT) was attached to the pendent azide group in the side chain of GMA units by click coupling to prepare the target hyperbranched block-statistical copolymer-based prodrug, h-P((GMA-CPT)-st-OEGMA)-b-P(OEGMA). The delivery efficacy of the target hyperbranched block-statistical copolymer-based prodrug was evaluated in terms of in vitro drug release and cytotoxicity studies.


Page 6 of 50

Page 7 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. Synthesis of h-P(GMA-st-OEGMA)-b-P(OEGMA).

Results and discussion Discussion regarding the advantages and disadvantages of using self-assembled micelle structures versus non-assembling polymer-drug conjugates for tumor delivery In the case of the classical micelles self-assembled from amphiphilic copolymers, the core-shell micellar structure can increase the water solubility and bioavailability of the lipophilic drug by core encapsulation, prolong its circulation time in the bloodstream by shell stabilization, and enable passive tumor targeting by the enhanced permeability and retention (EPR) effect. But they suffer primarily from the structural stability and integrity of the supramolecular nanostructures due to the inevitable dissociation of the micelle aggregates into individual polymer chains under large volume dilution by gastric, blood, or other body fluids, salinity fluctuations, and interactions with cells and biomolecules available in the blood after intravenous administration. Such destabilization may lead to premature release of the loaded drugs and inferior tumor targeting ability, further resulting in severe side effects and compromised therapeutic efficacy in vivo. Regarding the non-assembling polymer-drug conjugates, the anticancer drug, such as DOX and cisplatin, is conjugated covalently to the polymer backbone usually via stimulus-responsive or degradable links (e.g., pH- or reduction-responsive bond) preferentially cleavable in neoplastic tissues (mildly acidic pH and reductive



environment) and stable in blood plasma (physiological pH and mildly oxidizing extracellular milieu). The polymer-drug conjugates therefore exhibit better protection for drug molecules using covalent links than the micelles using hydrophobic interactions for drug encapsulation. Such enhanced stability for drug molecules completely eliminates inevitable leakage and premature release of loaded drugs occurring from conventional micelles upon intravenous administration owing to the diffusion and dynamic instability, thus permits precise tailoring of drug pharmacokinetics in vivo, and improves the in vivo drug efficacy as well as reduces the drug-associated side effects. However, the preparation of polymer-drug conjugates generally requires sophisticated chemistry design including multi-step synthesis, and drug modification, which apparently hampers their scale-up production and clinical translations. To fabricate a formulation with both enhanced stability and superior protection over drug molecules, herein we explored the facile preparation of hyperbranched block-statistical copolymer-based prodrug with dual sensitivities for controlled drug release by RAFT-SCVP and post-polymerization click coupling.

Synthesis of h-P(GMA-st-OEGMA) RAFT-SCVP enables precisely controlled synthesis of the target branched copolymers with variable degrees of branching (DBs), molecular weight (MW), and CTA functionality (F) under optimized conditions.39–41 Herein, RAFT copolymerization of GMA and OEGMA using ACP as a CTM was performed


Page 8 of 50

Page 9 of 50 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


to generate hyperbranched h-P(GMA-st-OEGMA) with a controlled number of dithiobenzoate moieties, reactive epoxy groups, pendent hydrophilic OEGMA units and cleavable disulfide linkage in each branching point. As the copolymerization ([ACP]0: [GMA]0 :[OEGMA]0: [AIBN]0=1:40:20:0.33, [M]0= 0.6M) was performed in DMAc at 70 °C for 10 h, h-P(GMA-st-OEGMA) was obtained in 80% monomer conversion (run 3 of Table 1). In its 1H NMR spectrum (Figure 1a), the appearance of all the characteristic signals of each moiety confirms the successful synthesis of target hyperbranched statistical copolymers, including aromatic and remaining vinyl protons originating from ACP at δ7.93, 7.53, 7.37 ppm (PhH), and 6.43, 6.12, 5.86 ppm (CH2=CH), the protons adjacent to the disulfide bridge at δ 4.26 (COOCH2CH2S) and 2.95 ppm (COOCH2CH2S), and the characteristic signals of GMA units at 3.19 (CHO of epoxy), 2.81 and 2.60 (CH2O of epoxy) and OEGMA units at 3.35 (OCH3). The number-average molecular weight and polydispersity were determined by SEC-MALLS to be 27.7 kDa and 1.41, respectively. The unimodal and nearly symmetrical elution peak demonstrates well controlled RAFT-SVCP process (Figure 2). The number-average CTA functionality per hyperbranched P(GMA-st-OEGMA) was optimized and calculated to be 4.72 for polymer in run 3 based on the following formula, ,



in which MWACP, MWGMA, and MWOEGMA were molecular weights of ACP, GMA, and OEGMA by comparing the integral ratio at 3.19 and 7.93 ppm, the molar ratio of GMA and OEGMA to dithiobenzoate was determined to be 19 (R=2I3.19/I7.93) and 9 (R=2I3.35/3I7.93), and the resulting hyperbranched statistical copolymer was thus denoted h-P(GMA19-st-OEGMA9). Based on the FACP, each hyperbranched h-P(GMA-st-OEGMA) consists of ~89 GMA units and ~42 OEGMA units, by assuming that no CTA functionality was lost during RAFT-SCVP. Owing to the high reactivity of the epoxy groups of GMA units, the resulting statistical copolymer afforded a versatile platform for various postpolymerization modifications. Based on a previous mechanism proposed by Zhao et al.,30 the RAFT-SCVP used in this study with a relatively high molar feed of monomer and CTM (60:1) and a medium monomer conversion could be roughly divided into two stages. The initial stage was primarily dominated by the CTM-mediated RAFT generation of a large amount of polymerizable linear P(GMA-st-OEGMA) copolymer (i.e., macro-CTA). Meanwhile, a small amount of star and branched copolymers were produced as well due to the simultaneous participation of the acrylate group in CTM in the RAFT copolymerization. In the following stage, the acrylate group in the remaining CTM and resultant macro-CTA further participated in the RAFT process and formed the branching units; RAFT copolymerization was conducted until all the polymerizable acrylate groups were completely consumed, leading to the gradual increase of the degree of branching. A relatively broad distribution with a PDI of 1.41 was thus


Page 10 of 50

Page 11 of 50 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


recorded in the SEC elution trace of the resultant polymers. The results agree well with the typical PDIs (1.55-2.43) of the RAFT-SCVP-synthesized polymers reported in the literature,30 and is believed to be the characteristics of a RAFT-SCVP process.

Table 1. Summary for RAFT-SCVP copolymerization of ACP with OEGMA and GMA under various conditions. Mnb Run

a

x:y:1:z

Conc (M)

Time (h)

PDI

RBc

RBc

(x)

(y)

b

(kDa)

FACP

1

20:10:1:0.2

0.8

10

16.4

1.44

13

6.5

3.85

2

40:20:1:0.2

0.6

10

24.0

1.26

26

12

3.10

3

40:20:1:0.33

0.6

10

27.7

1.41

19

9

4.72

a

Polymerization

conditions,

[GMA]0:[OEGMA]0:[ACP]0:[AIBN]0=x:y:1:z,

[GMA]0+[OEGMA]0= 0.8 mol/l for run 1, and 0.6 for runs 2 & 3, in DMAc at 70oC.

b

Number-average molecular weight (Mn) and polydispersity (PDI)

determined SEC-MALLS. cDetermined by 1H NMR analysis where RB means each branched repeat unit. FACP means number-average CTA functionality per branched copolymer.



Page 12 of 50

Figure 1. 1H NMR spectra of (a) h-P(GMA-st-OEGMA), and (b) HP1, h-P(GMA19-co-OEGMA9)-b-(OEGMA)9 in CDCl3.

RAFT

synthesis

of

h-P(GMA-st-OEGMA)-b-P(OEGMA)

using

h-P(GMA-st-OEGMA) as a multimacro-CTA Since the statistical copolymer alone can’t provide sufficiently colloidal stability


Page 13 of 50 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


for nanoparticle formation, h-P(GMA19-st-OEGMA9) with the highest FACP value was next used as a multimacro-CTA to polymerize OEGMA for the production of target hyperbranched block-statistical copolymer with a hydrophilic POEGMA stabilizing outer corona. Successful chain extension with OEGMA was confirmed by the clear shift of SEC elution traces towards higher MW with polymerization time (Figure 2). Given the composition of h-P(GMA19-st-OEGMA9) multimacro-CTA, the degree of polymerization (DP) of OEGMA homo-block was determined by comparing the characteristic signals of GMA units and OEGMA units as aforementioned (Figure 1b). The MWs, PDIs, and DPs of all the polymers synthesized were summarized in Table 2. Note that the small shoulders recorded at low–MW side for HP2-4 (Figure 2) probably indicate the presence of some dead cores without reactive CTA groups to which no OEG monomer can be grafted. The formation of these dead cores is feasibly attributed to the inevitable occurrence of side reactions, such as termination, irreversible transfer, and branch-branch coupling in the preceding RAFT-SCVP process. Similar phenomena were also reported in the literature.30 The HP1, h-P(OEGMA9-st-GMA19)-b-OEGMA9 with the most symmetrical elution peak and narrowest PDI was therefore chosen as a platform for further drug conjugation and subsequent biological evaluation.



Figure 2. SEC elution traces of h-P(GMA19-st-OEGMA9) macro-CTA, and h-P(GMA19-st-OEGMA9)-b-P(OEGMA) prepared at different polymerization time (HP1-4) (Eluent: DMF)


Page 14 of 50

Page 15 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Average size of HP1-4 in PBS (pH 7.4) determined by DLS at a polymer concentration of 0.5 mg/ml .

To investigate the ability for nanoparticle formation, DLS was further used to determine

directly

the

average

hydrodynamic

size

of

h-P(GMA19-st-OEGMA9)-b-POEGMA prepared at different polymerization time. Due to the hyperbranched structure and amphiphilicity of the polymer constructs, HP1-4 can form nanoparticles in an aqueous phase with mean diameters ranging from 36.5 to 49.6 nm (Figure 3). The size increases clearly with polymerization time for the polymer panel, and so does the size distribution, which agrees well with the SEC results. To clarify the relationship between the hyperbranched molecular structure of HP1 and its self-assembled nanoparticles in an aqueous solution, poly(methyl methacrylate) (PMMA) calibration standards with four different MWs were subjected to SEC-MALLS analyses firstly. The results summarized in Table S1 demonstrate that the MW determined by SEC-MALLS matches well with the data provided by the supplier, which confirms the reliability of SEC-MALLS analyses used in this study. The PMMA calibration standards with a Mn of 28.0 kDa and 98.8 kDa show similar root-mean-square z-average radius of gyration, values of ~15 nm, it is therefore reasonable to postulate that the hyperbranched polymer HP1 with a Mn of 40,4 kDa that is within this MW range should display a less than 15 nm due to the more compact structure of branched polymer over linear formulation. The size of HP1 in



DMF determined by DLS is 22.6 nm in diameter (Figure S1), i.e., 11.3 nm in radius, which is in excellent agreement with the estimated aforementioned. The overall results strongly support the formation of unimolecular nanoparticles in DMF for HP1. Next, the self-assembled morphology of HP1 in DMF and an aqueous solution was visualized by TEM observation, respectively to provide direct evidence regarding the nanoparticulate formation. Interestingly, both images show the presence of spherical nanoparticles with quite similar mean diameter around 20-30 nm in a dried status (Figure S2), which confirms the ability for HP1 to self-assemble into identical unimolecular nanoparticles in both organic and aqueous phases due to its hyperbranched architecture with enhanced stability. Such phenomena are similar to the cross-linked structure with enhanced stability reported in the literature.46,47 Finally, the CAC value of HP1 was investigated using pyrene as a fluorescence probe, and was determined to be ~0.021 mg/ml (Figure S3(a)). That’s to say, the aggregation of unimolecular micelles into micelle aggregates takes place above this critical concentration. To further provide an insight into this point, the size of HP1 at various concentrations in an aqueous phase was examined by DLS. As expected, the average size determined below the CAC is significantly smaller than that acquired above (Figure S3(b)), which indicates the association of the unimolecular micelles into larger micelle aggregates occurs in the range varying from below to above the critical concentration. Given that a polymer concentration of 0.5 mg/mL higher than the CAC was adopted in Figure 3, the size present should be attributed to the micellar aggregates formed by the hyperbranched polymer HP1-HP4.


Page 16 of 50

Page 17 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table

Summary

2.

for

RAFT-synthesized

h-P(GMA19-st-OEGMA9)-b-POEGMA under various polymerization time using h-P(GMA19-st-OEGMA9) as a multimacro-CTA. Time Run

M nb

Mnc PDIb

Sample (h)

Branch Composition

(kDa)

(kDa) h-P(OEGMA9-st-GMA19)

1

HP1

2

58.0

1.30

40.4 -b-OEGMA9 h-P(OEGMA9-st-GMA19)

2

HP2

4

78.1

1.48


3

HP3

6

85.5

1.77


4

HP4

8

102.8

1.64

73.0 -b-OEGMA32

a

Polymerization

conditions,

[OEGMA]0

:[macro-CTA]0:[AIBN]0=

200:1:0.33, [OEGMA]0= 0.5 mol/l in DMAc at 70oC. bNumber-average molecular weight (Mn) and polydispersity (PDI) determined SEMALLS. c

Determined by 1H NMR analysis.



Scheme 2. Synthesis of hyperbranched block-statistical copolymer-based prodrug, h-P((GMA-CPT)-st-OEGMA)-b-P(OEGMA) by click coupling.

Drug conjugation and CAC determination Inspired by the modification strategy of CPT reported by Cheng’s group,48 herein CPT was conjugated to HP1 by click coupling through a dually responsive link containing both pH-liable carbonate group and reduction-cleavable disulfide bond, which was highlighted using green and blue colors, respectively in Scheme 2. Given the high coupling efficacy of Cu(I)-catalyzed azide−alkyne cycloaddition and expensive and limited amount of CPT drug available, the conjugation reaction was carried out at a 1:1 molar feed ratio of CPT and HP1 in our experiment. However, the conjugation efficacy turned out to be much lower than expected. Although the reaction time was further increased to 3 days, the conjugation efficacy remains relatively low around ~ 20%, likely due to the steric hindrance


Page 18 of 50

Page 19 of 50 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


OEGMA chains, which prevents the accessibility of azide groups to free CPT molecules. The conjugation efficacy could be improved by adopting a higher molar feed ratio of CPT and azide groups. Such optimization is currently underway. The DLC was determined to be 14.3 %. The CAC of HP1-CPT was determined using fluorescence spectrometer using the fluorescent properties of CPT conjugated (Figure 4a). The fluorescence intensity of CPT at 423 nm keeps relatively constant at low concentrations, but increases dramatically at a certain concentration, indicating the formation of aggregates. Such concentration was determined to be 4.79 mg/l for HP1-CPT. The size of nanoparticles is a crucial factor affecting their properties and performance in vivo. The ideal scale (< 200 nm) of nanoparticles is expected to restrict their uptake by the mononuclear phagocyte system and allow for the passive targeting of cancerous or inflamed tissues through the enhanced permeation and retention (EPR) effect.3,49 The average size of HP1-CPT nanoparticle prodrugs was determined by DLS at various concentrations ranging from 10-1 to 10-5 mg/ml to investigate the stability against dilution (Figure 4b). The HP1-CPT nanoparticle prodrugs show average size of ~200 nm at the higher concentrations of 10-1 and 10-2 mg/ml, which is significantly larger than that (~150 nm) at low concentrations of 10-3.5, 10-4 and 10-5 mg/ml. The results indicate the formation of aggregates at higher concentrations, and agree well with the CAC data.



Figure 4. (a) Plots of I423 in the fluorescence spectra as a function of logarithm of HP1-CPT concentration, (b) average size of nanoparticle prodrugs self-assembled from HP1-CPT at various polymer concentrations determined by DLS (p*< 0.005).

Stability and reduction and acidic pH-triggered disassembly of nanoparticle prodrugs Due to the presence of disulfide bonds and carbonate links in the structure, HP1-CPT nanoparticle prodrugs are expected to show dual sensitivities. To investigate the reduction and acidic pH-triggered disassembly behaviors, the size change of nanoparticle prodrugs incubated for different periods of 6 and 24 h under various conditions was monitored by DLS (Figure 5).


Page 20 of 50

Page 21 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Size variation of HP1-CPT nanoparticle prodrugs incubated under various conditions for (a) 6 h, and (b) 24 h determined by DLS.

The nanoparticles are stable at pH 7.4 (typical extracellular pH) without any change of the unimodal size distribution (dark line), but incubation at pH 5.0 (intracellular endosomal/lysosomal pH, red line) or in the presence of 10 mM GSH at pH 7.4 (mimicking the intracellular reducing environment, blue line) results in bi-modal distributions with a small population centered at ~0.5-1 nm and a large population located at ~100-120 nm. The small population observed is feasibly attributed to the free CPT molecules, which were attributed to the cleavage of the disulfide bond by GSH or hydrolysis of canbonate group by acidic pH, and subsequent release from the nanoparticle prodrugs. A survey of literature47,48,50,51 indicates that the thiol-disulfide reshuffling is a spontaneous and a fast process, i.e., the CPT prodrug cleaved by the intracellular GSH can undergo spontaneously efficient reshuffling and decomposition to sequentially release the thiirane and carbon dioxide, affording CPT parent drug (Scheme 3), which is the same formula produced



by the acidic pH-triggered hydrolysis (Scheme 4). We therefore conclude the CPT prodrug prepared herein can generate the identical CPT parent molecules with biological activity by either the intracellular reducing GSH or an acidic pH.

Scheme 3. The cleavage mechanism of CPT parent drug from the polymer-drug conjugate triggered by the intracellular reducing GSH.

Scheme 4. The cleavage mechanism of CPT parent drug from the polymer-drug conjugate triggered by an acidic pH.

More importantly, compared to the insignificant difference bettween the size distributions at 6 and 24 h applied with only single trigger, simutaneous appllication of both acidic pH and reduction triggers, i.e., exerction of pH 5.0 and 10 mM GSH, promoted significantly the cleavege and release of free CPT from the nanoparticle prodrugs, which is reflected by the remarkably increased intensity for the small population centered at ~0.5-1 nm, and greatly decreased average size for the large


Page 22 of 50

Page 23 of 50 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


population located at several hundred nanometers after incubation for 24h. To identify the small population recorded at ~0.5-1 nm, the size of free CPT in PBS (pH=7.4) at a concentration of 0.5 mg/ml was further determinted by DLS (Figure S4). It is worth pointing out that a minimal amount of methanol was added to promote the dissolution of CPT in PBS considering its hydrophobicity. The results reveal an average size of 0.65 nm with narrow distribution, in consistent with the small population recorded at ~0.5-1 nm (Figure 5), which confirms such small population is assigned to the free CPT molecules as expected. The much more complete release of free CPT parent drug by two triggers leads to the remarkably increased intensity for the small population. Meanwhile, the HP1-CPT polymeric prodrugs become more hydrophilic due to the cleavage and subsequent release of CPT from the conjugates, which accounts for the greatly decreased average size for the large population located at several hundred nanometers. To provide direct evidence regarding the degradation of HP1-CPT polymeric prodrug under reductive and acidic environment, the polymer-drug conjugates incubated at pH 5.0 and in the presence of 10 mM GSH for different periods of time were subjected to SEC-MALLS analyses. The results (Figure 6) show that, compared to the starting uni-modal elution peak of HP1-CPT, a clear change of the elution traces towards bi-modal distributions was recorded after incubation of HP1-CPT in the reductive and acidic environment, which demonstrates the degradation of the parent hyperbranched HP1-CPT conjugates into linear polymer due to the GSH-triggered cleavage of the disulfide link for dissociation of the branched structure



and acidic pH-induced break of the carbonate bond for drug release. More importantly, a longer incubation time resulted in significantly reduced intensity for the elution peak of HP1-CPT centered at 13~14 min, but dramatically enhanced signal located at 19~20 min attributed to the linear polymer, indicating clearly the occurrence of more complete degradation with loner incubation time. The results agree well with the size variation of HP1-CPT micelle prodrugs in the reductive and acidic environment monitored by DLS.

Figure 6. SEC elution traces of HP1-CPT incubated at pH 5.0 and in the presence of 10 mM GSH for different periods of time. The sample solution was withdrawn at pretermined time intervals, and subjected to freeze-drying prior to SEC-MALLS analyses.

In vitro drug release study To further validate the GSH and acidic pH-triggered drug release, in vitro drug


Page 24 of 50

Page 25 of 50 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


release profiles of HP1-CPT nanoparticle prodrugs were studied at four different conditions chosen for the size variation study (Figure 7). The nanoparticle prodrugs were highly stable under physiological conditions with less than 5% of the CPT released over 4 d at pH 7.4. In contrast, incubation of nanoparticle prodrugs at pH 5.0 or with 10 mM GSH resulted in ~25% CPT release from the polymer after 96 h. Concurrent application of pH 5.0 and 10 mM GSH further promoted cumulative drug release to 45% within 96 h. The overall results are in excellent agreement with the size change study, which confirms the dual sensitivities, i.e., both acidic pH and reduction-triggered cleavage and release of CPT molecules from the nanoparticle prodrugs. It has been repeatedly highlighted that the linear polymer-CPT conjugates showed significantly prolonged plasma half-life and enhanced distribution to tumor tissue when compared to CPT alone due to the so-called EPR effect of the nanoparticulate polymer-drug conjugates.52,53 Very recently, Liu et al. fabricated self-reporting theranostic nanocarriers of CPT based on hyperbranched polyprodrug amphiphiles (hPAs) for synergistic imaging/chemotherapy and enhanced tumor uptake.54 In vivo blood circulation of the resulting hyperbranched unimolecular micelles of hPAs examined upon administration into healthy rats showed a half-lives (t1/2) of ~10 h. Clinical studies have suggested that circulation durations of spherical nanoparticles are generally extended by 3-fold in humans compared to those in rats. Thus, hPAs exhibited long circulation times as expected, which is quite typical for the branched and hyperbranched star polymers due to the almost neutral surface of the micellar



coronas and the hyperbranched chain topology with excellent chain flexibility and deformability.55 In this study, the hyperbranched block-statistical copolymer-based dual-responsive nanocarriers of CPT showed accelerated drug release at ~45% under the exertion of concurrent triggers of pH 5.0 and 10 mM GSH in 24 h (Figure 7). Such drug release duration is clearly shorter than the plasma half-life of CPT-polymer conjugates reported in the literature mentioned above, therefore the hyperbranched unimolecular micellar prodrug of CPT developed herein is believed to achieve long circulation and preferential accumulation in tumor tissue required for in vivo applications.

Figure 7. In vitro drug release profiles of HP1-CPT nanoparticle prodrugs under various conditions.


Page 26 of 50

Page 27 of 50 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


In vitro cytotoxicity study Finally, the cytotoxicity of HP1-CPT prodrugs to HeLa cells was assessed by MTS

cell

viability

assay

(Figure

h-P((GMA-N3)19-co-OEGMA9)-b-P(OEGMA)9

8). (HP1-N3)

The polymer

parent was

non-toxic to HeLa cells (with cell viability above 80%) up to a concentration of 44.0 mg/ml (data not shown). The half maximal inhibitory concentration (IC50) of free CPT, and HP1-CPT prodrugs is 42.42 (27.81, 64.68) µg/ml, 365.1 (293.9, 453.5) µg/ml. The increased IC50 (concentration for 50% cell killing) of prodrugs compared to free CPT is likely due to slower internalization mechanism (endocytosis vs direct membrane permeation) and the release kinetics of free drug from the polymer.



Page 28 of 50

Figure 8. In vitro cytotoxicity of free CPT, and HP1-CPT nanoparticle prodrugs in HeLa cells. Cell viability was determined by MTS assay and expressed as % viability compared to control untreated cells.

Conclusions In summary, we fabricated hyperbranched block-statistical copolymer-based prodrug with pH and reduction dual sensitivities using RAFT-SVCP and post-polymerization click coupling. The resulting HP1-CPT prodrugs were capable of forming unimolecular nanoparticles with enhanced stability. Most importantly, due to the presence of acidic pH-liable carbonate group and reduction-cleavable disulfide bridge in the structure, the simultaneous application of both acidic pH and GSH triggers promoted significantly the cleavage and release of CPT compared to the exertion of single trigger. This study thus developed a facile approach towards hyperbranched polymer-based prodrugs with high therapeutic efficacy for anti-cancer drug delivery.

Experimental Section Materials All solvents, monomers, and other chemicals were

purchased from

Sigma-Aldrich unless otherwise stated. GMA (99%) and OEGMA (Mn= 300 g/mol with 4-5 pendent EO units) were passed through a basic alumina column to remove the inhibitor prior to use. 2,2′-Azobis(isobutyronitrile) (AIBN, 99%) was recrystallized twice from ethanol. 4-Cyanopentanoic acid dithiobenzoate


Page 29 of 50 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


(CPADB)49, 2-((2-hydroxyethyl)disulfanyl)ethyl4-cyano-4-(phenylcarbonothioylthio)-penta noate,55

2-((2-(acryloyl

oxy)ethyl)disulfanyl)ethyl

4-cyano-4-(phenylcarbonothioylthio) pentanoate (ACP)42 were synthesized according to the reported procedures. N, N′-Dicyclohexylcarbodiimide (DCC, 98%), glutathione (GSH), N, N, N, N", N"-pentamethyldiethylenetriamine (PMDETA) were purchased from Aladdin. 4-Dimethylamino pyridine (DMAP, 99%) and CPT were purchased from J&K. Dichloromethane (DCM), tetrahydrofuran (THF), anisole and N, N-dimethylformamide (DMF) were purchased from Tianjin Chemical Re agent Factory (China) and further purified according to the standard protocols. PMMA calibration standards with four different MWs were brought from Waters (USA), and used as received.

Synthesis of hyperbranched statistical copolymer, h-P(GMA-st-OEGMA) by RAFT-SCVP To a Schlenk tube were added ACP (150.6 mg, 0.321 mmol), GMA (1.75 ml, 12.84 mmol), OEGMA (1.84 ml, 6.42 mmol) and AIBN (17.39 mg, 0.11 mmol). Thoroughly dried N, N-Dimethyl acetamide (DMAc) was added to the above mixture until the total volume was 2.31 mL. The solution was deoxygenated by three freeze-pump-thaw cycles, and then immersed in an oil bath preheated at 70oC. After reaction for 10 h, the polymerization was quenched by freezing in the liquid nitrogen. After thawing, the polymer



Page 30 of 50

solution was diluted with THF and the product was precipitated in ice-cold anhydrous ethyl ether three times to yield pink powder (Yield: 80%). The number-average molecular weight and polydispersity were determined to be Mn =27.7kDa and PDI= 1.41, respectively. Based on the absolute molecular weight and NMR analysis, the number-average CTA functionality (FCTA) was determined to be 4.72, and each dithiobenzoate moiety corresponded to 19 GMA units and 9 OEGMA units.

RAFT

synthesis

of

hyperbranched

h-P(GMA-st-OEGMA)-b-P(OEGMA)

using

block-statistical

copolymer,

h-P(GMA-st-OEGMA)

as

a

multimacro-CTA To a Schlenk tube were added h-P(GMA-st-OEGMA) (1.37 g, 49.50 µmol), OEGMA (2.83 ml, 9.89 mmol), AIBN (2.71 mg, 16.5 µmol), and DMAc (12.7 ml) (Molar feed ratio of [macro-CTA]: [OEGMA]: [AIBN]= 1:200:0.33). The solution was split in equal volumes into several glass vials. Thereafter, each solution was deoxygenated by three freeze-pump-thaw cycles, and then immersed in an oil bath preheated at 70°C to start the polymerization. The polymerization was quenched by immersing the vials in the liquid nitrogen at predetermined time intervals of 2, 4, 6, and 8 h. After thawing, the polymer solution was diluted with THF and precipitated in ice-cold n-hexane three times to yield pink sticky solid. The product was separated by filtration and further


Page 31 of 50 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


purified three times by redissolving/reprecipitating with THF/n-hexane, and finally dried in vacuum.

Synthesis of functional CPT for post-polymerization conjugation (Scheme 5) Synthesis of 2-((2-hydroxyethyl) disulfanyl) ethyl pent-4-ynoate Compound A was synthesized by the esterification reaction between pentinoic acid and hydroxyethyl disulfide in the presence of DCC and DMAP. Briefly, hydroxyethyl disulfide (6.17 g, 40mmol), DCC (4.95 g, 24 mmol) and DMAP (244 mg, 2.0 mmol) was dissolved in 100 ml of anhydrous DCM. A solution of pentinoic acid (1.96 g, 20.0 mmol) in anhydrous DCM was added dropwise to the above mixture at 0oC under N2 flow with stirring for a period of 30 min. Then the reaction mixture was further stirred at room temperature for 4h. Thereafter, a white byproduct DCU was filtrated out. The filtrate was concentrated, and further purified by column chromatography with eluents of ethyl acetate/hexane (1/4, v/v) to obtain a colorless liquid (2.70 g, yield: 63%). 1

H NMR (400 MHz, CDCl3, ppm, Figure S5): δ 4.35 (t, J = 6.8 Hz, 2H), 3.84

(q, J = 5.6 Hz, 2H), 2.90 (t, J = 6.8 Hz, 2H), 2.84 (t, J = 5.6 Hz, 1H), 2.58 (s, 1H), 2.57 – 2.53 (m, 2H),2.50 – 2.45 (m, 2H), 1.98 (t, J = 2.8 Hz, 1H).



Page 32 of 50

Scheme 5. Synthesis of functional CPT for postpolymerization click coupling.

Synthesis

of

compound

B,

((S)-2-((2-((((4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizi no[1,2-b]quinolin-4-yl)oxy)carbonyl)oxy)ethyl)disulfanyl)ethyl pent-4-ynoate) CPT (0.390 g, 1.12 mmol) was dissolved in 20 ml of DCM, and was added slowly to a phosgene-toluene solution (1.34 g，4.5 mmol) at 0oC. After stirring at room temperature overnight, the solvent and excessive phosgene was removed under vacuum. The resulting chloroformate was redissolved in 10 ml of DCM, which was added to a 50 ml of DCM/THF mixed solution (1/4, v/v) containing compound A (262.5 mg, 1.12 mmol), DCC (1.31 g, 5.60 mmol), DMAP (0.28 g, 2.30 mmol), and Et3N (226.67m g, 2.24 mmol). After stirring for 2h, the mixture was concentrated under vacuum, extracted with 100 ml of ethyl acetate (EtOAc), washed excessively with water (100 ml × 6). The organic layer was dried over sodium sulfate and the solvent was removed by


Page 33 of 50 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


rotary evaporation, affording a light yellow powder as the product (0.23 g, yield: 74%).1H NMR (400 MHz, CDCl3, ppm, Figure S6):δ 8.42 (s, 1H), 8.24 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.88 – 7.83 (m, 1H), 7.69 (t, J = 7.2 Hz, 1H), 7.34 (s, 1H), 5.71 (d, J = 17.2 Hz, 1H), 5.40 (d, J = 17.2 Hz, 1H), 5.31 (s, 2H), 4.43 – 4.34 (m, 2H), 4.31 (t, J = 6.8 Hz, 2H), 2.94 (t, J = 6.8 Hz, 2H), 2.90 (t, J = 6.4 Hz, 2H), 2.56 – 2.45 (m, 4H), 2.29 (dq, J = 14.8, 7.2 Hz, 1H), 2.16 (dq, J = 14.8, 7.2 Hz, 1H), 1.97 (t, J = 2.4 Hz, 1H), 1.02 (t, J = 7.6 Hz, 3H).

Postpolymerization modification of h-P(GMA-st-OEGMA)-b-P(OEGMA) by ring-opening of GMA units using sodium azide h-P(GMA-st-OEGMA)-b-P(OEGMA) (1.23 g, containing ~1.83 mmol of epoxy groups) was dissolved in 8 ml of DMF. Sodium azide (356 mg, 5.48 mmol) and ammonium chloride (293 mg, 5.48 mmol) were added to the above solution, and the mixture was stirred at 50oC overnight. After removal of most DMF under reduced pressure, the product was dissolved in a 4 ml of mixed solvents of H2O and DMF (1/1, v/v), then the product was purified by extensive dialysis (MWCO, 3.5 kDa) against distill water. The final product was harvested by freeze-drying (Yield: 64%). IR (KBr, wavenumber, cm-1) (Figure S7): 3404 (OH), 2104 (C-N=N=N), 1729 (C=O). The fully opening of the epoxy ring in the side chain of GMA units by sodium azide is confirmed by the complete disappearance of the characteristic signals attributed to the epoxy rings of GMA units (2.64, 2.84, 3.19 ppm) and appearance of the new



Page 34 of 50

resonance signals at 3.56 (d, 1H) and 4.06 (c, 2H) ppm in the 1H NMR spectrum of h-P((GMA-N3)-st-OEGMA)-b-P(OEGMA) (Figure S8) after azidotion. Therefore the amount of azide groups introduced to HP1 is equivalent to that of the GMA units.

Synthesis

of

hyperbranched

block-statistical

copolymer-based

prodrug,

h-P(GMA-CPT)-st-OEGMA)-b-P(OEGMA) by click coupling In a typical run, h-P((GMA-N3)-st-OEGMA)-b-P(OEGMA) (38.81 mg, containing ~0.06mmol of azide), B (35 mg, 0.06 mmol), PMEDTA (10 mg, 0.06 mmol), 8 ml of DMF were introduced into a 10 ml Schlenk flask. The solution was deoxygenated by three freeze-pump-thaw cycles, and then CuBr was added under the protection of N2 flow. After another three freeze-pump-thaw cycles, the flask was immersed in an oil bath thermostated at 45oC for three days. Then the reaction was quenched by immersing the flask in the liquid nitrogen. After thawing, the product was diluted with 2 ml of THF and precipitated in ice-cold n-hexane three times to remove any unreacted compound B. Thereafter, extensive dialysis (MWCO, 3.5 kDa) against distilled water was applied to remove the copper catalyst. The final product was harvested by freeze-drying (Yield: 79%). The conjugation efficacy of CPT, i.e., the amount of azide groups further converted for CPT conjugation by click coupling was determined by comparing the UV absorbance of polymer with and without CPT at 369 nm followed by quantification using the following standard curve of CPT in DMF, AHP1-CPT –AHP1 = 35.8006 ×C (µg/ml)－0.00144, (Figure S9, R2 = 0.99)


Page 35 of 50 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


where AHP1-CPT and AHP1 are the UV absorbance of CPT-conjugated polymer, HP1-CPT and parent CPT-free polymer in DMF (0.05 mg/ml) at 369 nm, respectively, and C is the concentration of CPT (µg/ml). The drug loading content (DLC) was calculated using the following equation,

DLC ሺ100%ሻ =

‫݃݅݁ݓ‬ℎ‫ݎ݁݉ݕ݈݋݌ ݊݅ ܶܲܥ ݂݋ ݐ‬ × 100 % ‫݃݅݁ݓ‬ℎ‫ݎ݁݉ݕ݈݋݌ ݂݋ ݐ‬

Polymer characterization 1

H NMR spectra were recorded on a JNM-ECS spectrometer at 400 MHz. The size

exclusion chromatography and multi-angle laser light scattering (SEC-MALLS) analyses were carried out to determine the molecular weight and molecular weight distribution of the polymers. SEC using HPLC-grade DMF containing 0.1 wt% LiBr at 60 °C as the eluent at a flow rate of 1 ml min-1 Tosoh TSK-GEL R-3000 and R-4000 columns (Tosoh Bioscience) were connected in series to an Agilent 1260 series (Agilent Technologies), an interferometric refractometer (Optilab-rEX, Wyatt Technology) and a MALLS device (DAWN EOS, Wyatt Technology). The MALLS detector was operated at a laser wavelength of 690.0 nm. The FT-IR spectroscopic measurements were conducted on a NEXUS 670 FT-IR spectrometer (Nicolet, WI, USA) and solid samples were pressed into potassium bromide (KBr) pellet prior to the measurements. The TEM images were recorded on a JNM-2010 instrument operating at an acceleration voltage of 200 keV. To prepare specimens for TEM



Page 36 of 50

observation, a drop of micelle solution was deposited onto a carbon-coated copper grid. After deposition, excess solution was removed using a strip of filter paper. The sample was further stained using phosphotungstic acid (2% w/w) and dried in air prior to visualization. Fluorescence spectra were recorded on a LS55 luminescence spectrometer (Perkin-Elmer, Waltham, MA, USA). Pyrene was used as a hydrophobic fluorescent probe. Aliquots of pyrene solutions (2 × 10−5 M in acetone, 1 ml) were added to containers, and the acetone was allowed to evaporate. Ten milliliter aqueous polymer solutions at different concentrations were then added to the containers containing the pyrene residue. It should be noted that all the aqueous sample solutions contained excess pyrene residue at the same concentration of 2 × 10−6 M. The solutions were kept at room temperature for 24 h to reach the solubilization equilibrium of pyrene in the aqueous phase. Excitation was carried at 339 nm, and emission spectra were recorded ranging from 350 to 600 nm. Both excitation and emission bandwidths were 10 nm. From the pyrene emission spectra, the intensities (peak height) of the third and the first band were analyzed as a function of the polymer concentrations. A critical aggregation concentration (CAC) value was determined from the intersection of the tangent to the curve at the inflection with the horizontal tangent through the points at low concentration.

Preparation

of

h-P((GMA-CPT)-st-OEGMA)-b-P(OEGMA)

prodrugs and determination of the CAC


nanoparticle

Page 37 of 50 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


Classical dialysis method was applied for the fabrication of nanoparticle prodrug.

Briefly,

h-P((GMA-CPT)-st-OEGMA)-b-P(OEGMA)

(10

mg)

dissolved in 2 ml of DMF was added dropwise into 10 ml of deionized water. After addition, the solution was transferred to a dialysis tube (MWCO: 3.5 kDa), and subjected to dialysis against deionized water for 24 h. The nanoparticle prodrug was harvested by freeze-drying (Yield: 79%). The CAC of nanoparticle prodrug was quantified by monitoring the typical fluorescence intensity of CPT in the polymer prodrug. The polymer prodrug solutions were prepared with different concentrations varying from 1.0×10−5 to 0.5 mg/ml. At the fixed concentration, the absorbance at 423 nm increased with the polymer solution concentration. The CAC was determined by the inflection point .

Stability and reduction and acidic pH-triggered disassembly of Nanoparticle prodrugs The stability, and reduction and acidic pH-triggered disassembly of nanoparticle prodrugs under various conditions were investigated by dynamic light scattering (DLS) on a Zeta sizer (Nano ZS, Malvern, Worcestershire, UK) at a fixed detection angle of 173°.

In vitro drug release study The CPT release profiles were investigated at 37 °C in four different release media of phosphate buffer (PBS, pH7.4, 150 mM), phosphate buffer (pH 5.0,



Page 38 of 50

150 mM), and phosphate buffer (pH 7.4, 150 mM) with GSH (10 mM), phosphate buffer (pH 5.0, 150 mM) with GSH (10mM). A typical procedure was as follows, certain amount of the freeze-dried nanoparticle prodrugs were re-dispersed in PBS (pH7.4, 150 mM) to prepare a solution of 1.0 mg/ml. An aliquot solution of 1.0 ml was transferred to a dialysis bag (MWCO, 3.5 kDa), and then immersed in a Falcon tube containing 25 ml of the release medium thermostated at 37 °C with constant shake (120 rpm). At predetermined time intervals, 3 ml of the release medium was taken out and replenished with equal volume of fresh medium. The drug concentration was determined by measuring the absorbance of CPT at 369 nm based on a previously established calibration curve in PBS (Figure S10).

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

At

20

h,


20

µl

of

Page 39 of 50 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


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

ASSOCIATED CONTENT Supporting information SEC-MALLS analyses (Mn, PDI, and ) of PMMA calibration standards with four different molecular weights, TEM observation of the self-assembled morphology of HP1 in DMF and an aqueous solution, determination of CAC value of HP1 using pyrene as a fluorescence probe, and examination of the size of HP1 at various concentrations in an aqueous phase by DLS, DLS measurements of HP1 in DMF and CPT in PBS, additional 1H NMR data for CPT modification, FT-IR and 1H NMR spectra of HP1-N3, and standard curves of CPT in DMF and PBS are available in Table S1, and Figure S1-10.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]



ORCID Hua Wei: 0000-0002-5139-9387

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors acknowledge the financial support from the National Natural Science Foundation of China (51473072 and 21504035), the Thousand Young Talent Program, and the Fundamental Research Funds for the Central Universities (lzujbky-2016-ct05), and Open Research Fund of State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences.

ABBREVIATIONS RAFT-SCVP, reversible addition–fragmentation chain transfer self-condensing vinyl polymerization; SHP, segmented hyperbranched polymer; CTM, chain transfer monomer; OEGMA, oligo(ethylene glycol methyl ether methacrylate); GSH, glutathione; CPT, camptothecin; CRP, controlled radical polymerization; ATRP, atom transfer radical polymerization; RAFT, reversible addition–fragmentation chain transfer; SCVP, self-condensing vinyl polymerization; GMA, glycidylmethacrylate.

References


Page 40 of 50

Page 41 of 50 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


(1) Hartlieb, M., Floyd, T., Cook, A. B., Cano, C. S., Catrouillet, S., Burns, J. A., and Perrier, S. (2017) Well-defined hyperstar copolymers based on a thiol–yne hyperbranched core and a poly(2-oxazoline) shell for biomedical applications. Polym. Chem. 8, 2041–2054. (2) Fan, X. S., Zhang, W. W., Hu, Z. G., and Li, Z. B. (2017) Facile synthesis of RGD-conjugated unimolecular micelles based on a polyester dendrimer for targeting drug delivery, J. Mater. Chem. B. 5, 1062–1072. (3) Zhao, S. J., Yang, H. R., Zuo, C., Sun, L., Ma, L. W., and Wei, H. (2016) pH-sensitive drug release of star-shaped micelles with OEG brush corona. RSC Adv. 6, 111217–111225. (4) Qiu, L., Liu, Q., Hong, C. Y., and Pan, C. Y. (2016) Unimolecular micelles of camptothecin-bonded hyperbranched star copolymers via β-thiopropionate linkage: synthesis and drug delivery. J. Mater. Chem. B, 4, 141–151. (5) Zolotarskaya, O. Y., Xu, L. Y., Valerie, K., and Yang, H. (2015) Click synthesis of a

polyamidoamine

dendrimer-based

camptothecin

prodrug.

RSC

Adv.

5,

58600–58608. (6) Bugno, J., Hsu, H. J., and Hong, S. (2015) Recent advances in targeted drug delivery approaches using dendritic polymers. Biomater. Sci. 3, 1025–1034. (7) Svenson, S. (2015) The dendrimer paradox – high medical expectations but poor clinical translation. Chem. Soc. Rev. 44, 4131–4144. (8) Zhang, C. Y., Pan, D. Y., Luo, K., Li, N., Guo, C. H., Zheng, X. L., and Gu, Z. W. (2014) Dendrimer–doxorubicin conjugate as enzyme-sensitive and polymeric



Page 42 of 50

nanoscale drug delivery vehicle for ovarian cancer therapy. Polym. Chem. 5, 5227–5235. (9) Muthukrishnan, S., Erhard, D. P., Mori, H., and Müller, A. H. E. (2006) Synthesis and

characterization

of

surface-grafted

hyperbranched

glycomethacrylates.

Macromolecules 39, 2743–2750. (10) Han, J., Li, S., Tang, A., and Gao, C. (2012) Water-soluble and clickable segmented hyperbranched polymers for multifunctionalization and novel architecture construction. Macromolecules 45, 4966–4977. (11) Konkolewicz, D., Poon, C. K., Gray-Weals, A., and Perrier, S. (2011) Hyperbranched alternating block copolymers using thiol–yne chemistry: materials with tuneable properties. Chem. Commun. 47, 239–241. (12) Clarke, N., de Luca, E., Dodds, J. M., Kimani, S. M., and Hutchings, L. R. (2008) Hypermacs-long chain hyperbranched polymers: A dramatically improved synthesis and qualitative rheological analysis. Eur. Polym. J. 44, 665–676. (13) Pfaff, A., and Müller, A. H. E. (2011) Hyperbranched glycopolymer grafted microspheres. Macromolecules 44, 1266–1272. (14) Li, S., Han, J., and Gao, C. (2013) High-density and hetero-functional group engineering of segmented hyperbranched polymers via click chemistry. Polym. Chem. 4, 1774–1787. (15) Caldéron, M., Quadir, M. A., Sharma, S. K., and Haag, R. (2010) Dendritic polyglycerols for biomedical applications. Adv. Mater. 22, 190–218. (16)

Jikei, M., and Kakimoto, M. (2001) Hyperbranched polymers: a promising new


Page 43 of 50 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


class of materials. Prog. Polym. Sci. 26, 1233–1285. (17) Gao, C., and Yan, D. (2004) Hyperbranched polymers: from synthesis to applications. Prog. Polym. Sci. 29, 183–275. (18) Paleos, C. M., Tsiourvas, D., and Sideratou, Z. (2007) Molecular engineering of dendritic polymers and their application as drug and gene delivery systems. Mol. Pharm. 4, 169–188. (19) Yu, X., Liu, Z., Janzen, J., Chafeeva, I., Horte, S., Chen, W., Kainthan, R. K., Kizhakkedathu, J. N., and Brooks, D. E. (2012) Polyvalent choline phosphate as a universal biomembrane adhesive. Nat. Mater. 11, 468–476. (20) Vogt, A. P., and Sumerlin, B. S. (2008) Tuning the temperature response of branched poly(N-isopropylacrylamide) prepared by RAFT polymerization. Macromolecules 41, 7368–7373. (21) Cheng, C., Wooley, K. L., and Khoshdel, E. (2005) Hyperbranched fluorocopolymers

by

atom

transfer

radical

self-condensing

vinyl

copolymerization. J. Polym. Sci., Part A: Polym. Chem. 43, 4754–4770. (22) Muthukrishnan, S., Mori, H., and Müller, A. H. E. (2005) Synthesis and characterization

of

methacrylate-type

hyperbranched

glycopolymers

via

self-condensing atom transfer radical copolymerization. Macromolecules 38, 3108–3119. (23) Min, K., and Gao, H. (2012) New method to access hyperbranched polymers with uniform structure via one-pot polymerization of inimer in microemulsion. J. Am. Chem. Soc. 134, 15680–15683.



Page 44 of 50

(24) Tsarevsky, N. V., Huang, J., and Matyjaszewski, K. (2009) Synthesis of hyperbranched degradable polymers by atom transfer radical (co)polymerization of inimers with ester or disulfide groups. J. Polym. Sci., Part A: Polym. Chem. 47, 6839–6851. (25) Hawker, C. J., Frechet, J. M. J., Grubbs, R. B., and Dao, J. (1995) Preparation of hyperbranched and star polymers by a "living", self-condensing free radical polymerization. J. Am. Chem. Soc. 117, 10763–10764. (26) Ambade, A. V., and Kumar, A. (2000) Controlling the degree of branching in vinyl polymerization. Prog, Polym. Sci. 25, 1141–1170. (27) Baudry, R., and Sherrington, D. C. (2006) Synthesis of highly branched poly(methyl methacrylate)s using the “strathclyde methodology” in aqueous emulsion. Macromolecules 39, 1455–1460. (28) Baudry, R., and Sherrington, D. C. (2006) Facile synthesis of branched poly(vinyl alcohol)s. Macromolecules 39, 5230–5237. (29) Jesberger, M., Barner, L., Stenzel, M. H., Malmström, E., Davis, T. P., and Barner-Kowollik,

C.

(2003)

Hyperbranched

polymers

as

scaffolds

for

multifunctional reversible addition–fragmentation chain-transfer agents: a route to polystyrene-core-polyesters

and

polystyrene-block-poly(butyl

acrylate)-core-polyesters. J. Polym. Sci., Part A: Polym. Chem. 41, 3847–3861. (30) Zhang, C. B., Zhou, Y., Liu, Q., Li, S. X., Perrier, S., and Zhao, Y. L. (2011) Facile synthesis of hyperbranched and star-shaped polymers by RAFT


Page 45 of 50 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


polymerization based on a polymerizable trithiocarbonate. Macromolecules 44, 2034–2049. (31) Zhang, M. J., Liu, H. H., Shao, W., Ye, C. N., and Zhao, Y. L. (2012) Versatile synthesis of multiarm and miktoarm star polymers with a branched core by combination of menschutkin reaction and controlled polymerization. Macromolecules 45, 9312–9325. (32) Han, J, Li, S. P., Tang, A. J., and Gao, C. (2012) Water-soluble and clickable segmented hyperbranched polymers for multifunctionalization and novel architecture construction. Macromolecules 45, 4966–4977. (33) Fréchet, J. M. J., Henmi, M., Gitsov, I., Aoshima, S., Leduc, M. R., and Grubbs, R. B. (1995) Self-condensing vinyl polymerization: an approach to dendritic materials. Science 269, 1080–1083. (34) Matyjaszewski, K. (2012) Atom transfer radical polymerization (ATRP): current status and future perspectives. Macromolecules 45, 4015–4039. (35) Rikkou-Kalourkoti, M., Matyjaszewski, K., and Patrickios, C. S. (2012) Synthesis, characterization and thermolysis of hyperbranched homo- and amphiphilic co-polymers prepared using an inimer bearing a thermolyzable acylal group. Macromolecules 45, 1313–1320. (36) Matyjaszewski, K., and Tsarevsky, N. V. (2014) Macromolecular engineering by atom transfer radical polymerization. J. Am. Chem. Soc. 136, 6513–6533.



(37) Pugh, C., Singh, A., Samuel, R., and Ramos, K. M. B. (2010) Synthesis of hyperbranched polyacrylates by a chloroinimer approach. Macromolecules 43, 5222–5232. (38) Muthukrishnan, S., Jutz, G., André, X., Mori, H., and Müller, A. H. E. (2005) Synthesis of hyperbranched glycopolymers via self-condensing atom transfer radical copolymerization of a sugar-carrying Acrylate. Macromolecules 38, 9–18. (39) Powell, K. T., Cheng, C., and Wooley, K. L. (2007) Complex amphiphilic hyperbranched fluoropolymers by atom transfer radical self-condensing vinyl (co)polymerization. Macromolecules 40, 4509–4515. (40) Chiefari, J., Chong, Y. K., Ercole, F., Krstina, J., Jeffery, J., Le, T. P. T., Mayadunne, R. T. A., Meijs, G. F., Moad, C. L., and Moad, G., et al. (1998) Living free-radical polymerization by reversible addition−fragmentation chain transfer: the RAFT process. Macromolecules 31, 5559–5562. (41) Wang, Z. M., He, J. P., Tao, Y. F., Yang, L., Jiang, H. J., and Yang, Y. L. (2003) Controlled chain branching by RAFT-based radical polymerization. Macromolecules 36, 7446–7452. (42) Zhang, M. J., Liu, H. H., Shao, W., Miao, K., and Zhao, Y. L. (2013) Synthesis and properties of multicleavable amphiphilic dendritic comblike and toothbrushlike copolymers comprising alternating PEG and PCL grafts. Macromolecules 46, 1325–1336.


Page 46 of 50

Page 47 of 50 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


(43) Li, S. P., and Gao, C. (2013) Dendritic molecular brushes: synthesis via sequential RAFT polymerization and cage effect for fluorophores. Polym. Chem. 4, 4450–4460. (44) Wei, H., Volpatti, L. R., Sellers, D. L., Maris, D. O., Andrews, I. W., Hemphill, A. S., Chan, L. W., Chu, D. S. H., Horner, P. J., and Pun, S. H. (2013) Dual responsive, stabilized nanoparticles for efficient in vivo plasmid delivery. Angew. Chem. Int. Ed. 52, 5377–5381. (45) Zhou, X. F., Chang, C., Zhou, Y., Sun, L., Xiang, H., Zhao, S. J., Ma, L. W., Zheng, G. H., Liu, M. Z., and Wei, H. (2017) A comparison study to investigate the effect of the drug-loading site on its delivery efficacy using double hydrophilic block copolymer-based prodrugs. J. Mater. Chem. B 5, 4443–4454. (46) Wei, H., Wu, D. Q., Li, Q., Chang, C., Zhou, J. P., Zhang, X. Z., and Zhuo, R. X. (2008) Preparation of shell cross-linked thermoresponsive micelles as well as hollow spheres based on P(NIPAAm-co-HMAAm-co-MPMA)-b-PCL. J. Phys. Chem. C 112, 15329–15334. (47) Vien T. H, Sandra B, Paul L. D. S, and Martina H. S. (2012) Acid degradable cross-linked micelles for the delivery of cisplatin: a comparison with nondegradable cross-Linker. Chem. Mater. 24, 3197-3211. (48) Cai, K., Song, Z., Yin, Q., Zhang, Y., Uckun, F. M., Jiang, C., and Cheng, J. (2015) Dimeric drug polymeric nanoparticles with exceptionally high drug loading and quantitative loading efficiency. J. Am. Chem. Soc. 137, 3458-3461.



Page 48 of 50

(49) Mitsukami, Y., Donovan, M. S., Lowe, A. B., and McCormick, C. L. (2001) Water-soluble polymers. 81. direct synthesis of hydrophilic styrenic-based homopolymers and block copolymers in aqueous solution via RAFT. Macromolecules 34, 2248–2256. (50) Andrew G. C., Ou, Y. C., Zhang, P. C., and Cui, H. G. (2014) Linker-determined drug release mechanism

of free camptothecin from

self-assembling drug amphiphiles Chem. Commun., 50, 6039-6042. (51) Hu, X. L, Tian, J., Liu, T., Zhang, G. Y., and Liu, S. Y. (2013) Photo-triggered release of caged camptothecin prodrugs from dually responsive shell cross-linked micelles. Macromolecules, 46, 6243−6256. (52) Schluep, T., Cheng, J. J., Khin, K. T., and Davis, M. E. (2006) Pharmacokinetics and biodistribution of the camptothecin-polymer conjugate IT-101 in rats and tumor-bearing mice. Cancer Chemother. Pharmacol. 57, 654-662. (53) Choi, K. Y., Yoon, H. Y., Kim, J. H., Bae, S. M., Park, R. W., Kang, Y. M., Kim, I. S., Kwon, I. C., Choi, K., Jeong, S. Y. et al. (2011) Smart nanocarrier based on PEGylated hyaluronic acid for cancer therapy. ACS Nano 5, 8591-8599. (54) Hu, X. L., Liu, G. H., Li, Y., Wang, X. R., and Liu, S. Y. (2015) Cell-penetrating

hyperbranched

polyprodrug

amphiphiles

for

synergistic

reductive milieu-triggered drug release and enhanced magnetic resonance signals. J. Am. Chem. Soc. 137, 362-368.


Page 49 of 50 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) Tao, L., Liu, J. Q., Tan, B. H., and Davis, T. P. (2009) RAFT Ssynthesis and DNA binding of biodegradable, hyperbranched poly(2-(dimethylamino)ethyl methacrylate). Macromolecules 42, 4960–4962.




Page 50 of 50

Fabrication of Hyperbranched Block-Statistical Copolymer-Based

Recommend Documents