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Delivery of Amonafide from Fructose-coated Nanodiamonds by Oxime Ligation for Treatment of Human Breast Cancer Jiacheng Zhao, Mingxia Lu, Haiwang Lai, Hongxu Lu, Jacques Lalevée, Christopher Barner-Kowollik, Martina H. Stenzel, and Pu Xiao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01592 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 11, 2018

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Biomacromolecules 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.

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Biomacromolecules

1

Delivery of Amonafide from Fructose-coated

2

Nanodiamonds by Oxime Ligation for Treatment of

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Human Breast Cancer

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Jiacheng Zhao,a Mingxia Lu,a Haiwang Lai,a Hongxu Lu,a Jacques Lalevée,b Christopher

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Barner-Kowollik,c,d Martina H. Stenzel,*,a and Pu Xiao*,a, b

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a

7

South Wales, Sydney, Australia; bInstitut de Science des Matériaux de Mulhouse IS2M, UMR

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CNRS 7361, ENSCMu-UHA, 15, rue Jean Starcky, 68057 Mulhouse Cedex, France; cSchool of

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Chemistry, Physics and Mechanical Engineering, Queensland University of Technology (QUT),

Centre for Advanced Macromolecular Design, School of Chemistry, The University of New

10

2 George Street, Brisbane, QLD 4000, Australia; dMacromolecular Architectures, Institut für

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Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstr.

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18, 76128 Karlsruhe, Germany.

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KEYWORDS: amonafide, glycopolymer, nanodiamonds, drug delivery, oxime ligation

14

ABSTRACT: Introducing a strategy towards polymer/nanodiamond hybrids with high polymer

15

grafting density and accessible polymer structural characterisation is of critical importance for

16

nanodiamonds surface modification and bioagent attachment for their biomedical application.

17

Here, we report a glycopolymer/nanodiamond hybrid drug delivery system, which was prepared

18

by grafting amonafide-conjugated glycopolymers onto the surface of nanodiamonds via oxime

ACS Paragon Plus Environment

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

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ligation.

2

vinylbenzaldehyde-co-methyl methacrylate), featuring pendant aldehyde groups, is prepared via

3

RAFT polymerization. The anticancer drug amonafide is conjugated to the polymer chains by via

4

imine chemistry, resulting in acid degradable imine linkages. The obtained amonafide-

5

conjugated glycopolymers are subsequently grafted onto the surface of aminooxy-functionalised

6

nanodiamonds via oxime ligation. The molecular weight of the conjugated polymers is

7

characterised by Size Exclusion Chromatography (SEC), while the successful conjugation and

8

corresponding grafting density is characterised by Nuclear Magnetic Resonance (NMR), Fourier

9

transform infrared spectroscopy (FTIR), and Thermogravimetric Aanalysis (TGA). Our results

10

indicate that the mass percentage of amonafide in the polymer chains is around 17% and the

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surface density of polymer chains is 0.24 molecules/nm2. The prepared drug delivery system has

12

a hydrodynamic size around 380 nm with low PDI (0.3) and can effectively deliver amonafide

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into breast cancer cell and significantly inhibit the cancer cell viability. In 2D cell culture

14

models, the IC50 values of ND-Polymer-AMF delivery system (7.19 µM for MCF-7; 4.92 µM for

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MDA-MB-231) are lower than those of free amonafide (11.23 µM for MCF-7; 13.98 µM for

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MDA-MB-231). An inhibited cell viability of nanodiamonds/polymer delivery system is also

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observed in 3D spheroids models, suggesting that polymer-diamonds hybrid materials can be

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promising platforms for breast cancer therapy.

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INTRODUCTION:

Poly(1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose)-b-poly(3-

20

Nanodiamonds featuring good biocompatibility,1-2 non-bleaching fluorescence,3-4 and a

21

functional surface5, have been widely exploited for biomedical applications.6-10 However, the

22

tendency of unmodified nanodiamonds to agglomerate in solution hampers their application in

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drug delivery.11 Surface functionalization of nanodiamonds with synthetic polymers is a


2

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promising strategy to enhance their colloidal stability based on the shield effect of the polymeric

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steric barrier, reducing the surface interaction between the nanoparticles.12 Surface modification

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of nanodiamonds with hydrophilic polyoxyethylene was reported to significantly enhance their

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dispersibility.13 Polymers are normally attached to nanodiamond surface via either physical

5

adsorption14-15 or covalent conjugation.16-19 To anchor synthesized polymers on surfaces via

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covalent bonds, two strategies, referred to as “grafting-from” and “grafting-onto”, are commonly

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followed.20-22 It has been reported that poly(oligo(ethylene glycol) methyl ether methacrylate)-

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coated nanodiamonds, prepared via the “grafting-from” approach, exhibit good water dispersion

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and significantly enhanced therapeutic efficacy of cisplatin when employed as nanocarriers.23

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However, the grafted polymers synthesized via surface initialized polymerisation (“grafting

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from”) are generally challenging to characterize.17 Analysis of the structures of polymers on the

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nanodiamonds’ surface is however critical, especially when drugs are covalently conjugated to

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the polymer chains and the number and structure of the conjugated drugs is essential to

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understand the biological activity. This particular shortcoming of the “grafting from”

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methodology can be addressed with the “grafting onto” method as pre-synthesized polymers,

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which can be comprehensively characterized prior grafting, are used for functionalization.

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However, high polymer grafting densities are usually difficult to reach using the “grafting onto”

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method, resulting in low colloidal stability and low drug loading.

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polymer/nanodiamond hybrids with high polymer grafting density and accessible polymer

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structural characterisation is of critical importance for biomedical applications.

Thus, the design of

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To date, various conjugation techniques including amidation reaction,22-25 esterification,26

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light-induced ligation17-19 as well as copper-catalyzed azide-alkyne cycloaddition (CuAAC),27

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have been employed to graft polymers onto the surface of nanodiamonds. Among these methods,


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azide-alkyne ligation exhibits high conjugation efficiency (0.54 mmol g-1),28 compared with the

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surface conjugation with oxyhexanol groups (0.13 mmol g-1)29 and in the range of surface

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loading (0.25-1.6 mmol g-1) reported for the conjugation of nanodiamonds with various

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molecules.30

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nanodiamond/polymer hybrids in biomedicine. Recently, some copper-free click reactions such

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as strain-promoted alkyne-azide cycloaddition (SPAAC) have been exploited to covalently

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immobilize copolymers containing prodrugs onto the surface of nanodiamonds.31 In addition,

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light-triggered strategies such as light-induced photo-enol chemistry,17, 19 have been reported as

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an elegant method for surface modification of nanodiamonds. Such strategies proceed under mild

10

reaction conditions, i.e., ambient temperature as well as catalyst-free conditions, and exhibit

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excellent potential in biological applications.

However,

the

use

of

copper

catalysis

limits

the

application

of

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The generation of imine, hydrazone and oxime bonds has attracted significant attention in

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biomedical applications due to their high reaction efficiency, bioorthogonality32 and dynamic

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nature of bonds.33 Imine bonds, which are stable under physiological conditions, yet experience

15

rapid hydrolysis under acidic conditions, have been extensively used for the design of pH-

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responsive drug delivery systems34-35 as well as functionalization of polymers.36 Recently, oxime

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bonds, which feature significantly higher hydrolytic stability than imines and hydrazones,37 are

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regarded as ideal candidates for various application such as hydrogel synthesis,38-40 surface

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patterning,41-42 polymer synthesis33 and post-functionalization43-45 as well as drug delivery46 as

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the ligation fast, occurs in aqueous solution, requires no catalysts and generates water as by-

21

product. Therefore, the oxime chemistry represents a versatile strategy for conjugation.

22

Herein, we report a glycopolymer/nanodiamond hybrid drug delivery system by grafting

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amonafide-conjugated glycopolymers onto the nanodiamonds via oxime ligation in order to


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achieve high polymer grafting density and accessible polymer structural characterisation. Our

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previous studies indicate that fructose-based nanoparticles have great potential as drug carriers in

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breast cancer therapy.24,

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2,3:4,5-di-O-isopropylidene-β-D-fructopyranose)-b-poly(3-vinylbenzaldehyde-co-methyl

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methacrylate) is prepared via RAFT polymerisation. Amonafide, which has demonstrated strong

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activity against many cancer cells,48 is conjugated to the polymer chains by formation of imine

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bonds between aldehyde and amine groups. The obtained amonafide-conjugated glycopolymers

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are further grafted onto the surface of aminooxy-functionalised nanodiamonds. Compared with

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“grafting from” strategies, oxime ligation enables the facile characterization of the grafted

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polymers. The molecular weight of Poly(1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-β-D-

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fructopyranose)-b-poly(3-vinylbenzaldehyde-co-methyl methacrylate) is characterised by Size

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Exclusion Chromatography (SEC). Successful drug conjugation is confirmed by nuclear

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magnetic resonance (NMR) spectrometry, while the polymer conjugation to nanodiamonds is

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evidenced by Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis

15

(TGA). In addition, a high polymer grafting density is obtained via the oxime ligation approach.

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The polymer grafting density of the hybrid is significantly higher than that obtained via

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amidation coupling reaction reported earlier.24 The therapeutic efficiency of the amonafide

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delivery system in both 2D and 3D breast cancer cell culture models indicated

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glycopolymer/nanodiamond hybrid materials can be promising platforms for breast cancer

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therapy. These results demonstrate that oxime ligation is an efficient strategy to synthesize

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glycopolymer/nanodiamonds hybrid materials with high polymer grafting density and accessible

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polymer structural characterisation.

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METHODS

47

Therefore, fructose-based glycopolymer Poly(1-O-methacryloyl-


5


1

Materials.

Nanodiamonds

(ND;

98%, Aldrich), trifluoroacetic acid (TFA; 99%, Aldrich), methyl

4

methacrylate (>99%, Aldrich), n-butylamine (>99%, Aldrich) and dichloromethane (DCM;

5

anhydrous, >99.8%, Aldrich) were used as received. 1,4-dioxane (99%, Ajax Finechem) was

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purified by reduced-pressure distillation. 3-Vinylbenzaldehyde (3-VBA; 97%, Aldrich) and

7

methyl methacrylate (>99%, Aldrich) were passed over basic aluminum oxide to remove

8

inhibitors. 2, 2’-Azobisisobutyronitrile (AIBN) was recrystallized twice from methanol before

9

use. Amine-functionalized nanodiamonds (ND-NH2) were prepared as reported previously.23 The

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RAFT agent 4-cyanopentanoic acid dithiobenzoate (CPADB) was synthesized according to a

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literature procedure.49 Amonafide was prepared by the procedure reported previously.50

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Synthesis of Poly(1-O-MAipFru)48. The synthesis of 1-O-methacryloyl-2,3:4,5-di-O-

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isopropylidene-β-D-fructopyranose followed a similar procedure as reported previously.47 The

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polymerization of the glycomonomer was carried out using the following procedure: in a

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Schlenk tube, 1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose (2 g, 6.1

16

mmol), AIBN (4 mg, 2.44 × 10-2 mmol) and CPADB (35 mg, 0.12 mmol) were dissolved in 1,4-

17

dioxane (4 mL). The tube was subsequently degassed by three freeze-pump-thaw cycles. The

18

polymerization was carried out at 70 ℃ and stopped after 8 h by cooling the solution in ice water

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(conversion: 97%). The polymer solution was poured into a large excess of diethyl ether for

20

precipitation. The viscous polymer was dried under vacuum for 24 h.

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Synthesis of Poly(1-O-MAipFru)48-b-Poly(3-VBA53-co-MMA43). The synthesis of the

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Poly(1-O-MAipFru)48-b-Poly(3-VBA53-co-MMA43) was conducted as follows: in a Schlenk

23

tube, Poly(1-O-MAipFru)48 (200 mg, 1.25 × 10-2 mmol), methyl methacrylate (250 mg, 2.5


6

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mmol), 3-vinylbenzaldehyde (330 mg, 2.5 mmol) and AIBN (0.41 mg, 2.50 × 10-3 mmol) were

2

dissolved in 1,4-dioxane (4 mL). The solution was then degassed by three freeze-pump-thaw

3

cycles. The polymerization was carried out at 70 ℃ and stopped after 24 h by cooling the

4

solution in ice water. The polymer solution was poured into a large excess of diethyl ether for

5

precipitation. The viscous polymer was dried under vacuum for 24 h. The conversion of MMA

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and 3-VBA are 22% and 27%, respectively.

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Removal of Thiocarbonylthio Group from Poly(1-O-MAipFru)48-b-Poly(3-VBA53-co-

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MMA43). The aminolysis of the Poly(1-O-MAipFru)48-b-Poly(3-VBA53-co-MMA43) was carried

9

out using the following procedure: in a 25 mL vial, Poly(1-O-MAipFru)48-b-Poly(3-VBA53-co-

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MMA43) (300 mg, 1.10 × 10-2 mmol) was dissolved in THF (6 mL), followed by adding methyl

11

acrylate (9.5 mg, 0.11 mmol) and n-butylamine (8 mg, 0.11 mmol). The solution was stirred at

12

ambient temperature for 48 h. After the reaction, the solution was air-dried in the fume hood and

13

poured into a large excess of hexane for precipitation. The viscous polymer was dried under

14

vacuum for 24 h.

15

Deprotection of Poly(1-O-MAipFru)48-b-Poly(3-VBA53-co-MMA43). The deprotection of

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the aminolysized Poly(1-O-MAipFru)48-b-Poly(3-VBA53-co-MMA43) was conducted as follows:

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in a 25 mL vial, Poly(1-O-MAipFru)48-b-Poly(3-VBA53-co-MMA43) (289 mg, 1.06 × 10-2

18

mmol) was dissolved in TFA/H2O (9:1, 3 mL) and stirred for 3 h. The solution was subsequently

19

dialyzed against with MQ water for purification. The deprotected polymer was then lyophilized.

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Conjugation of Amonafide (AMF) to Poly(1-O-MAFru)48-b-Poly(3-VBA53-co-MMA43).

21

Amonafide (43 mg, 0.15 mmol) and Poly(1-O-MAFru)48-b-Poly(3-VBA53-co-MMA43) (200 mg,

22

8.6 × 10-3 mmol) were dissolved in 2 mL anhydrous DMSO. The conjugation was carried out at


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110℃ for 36 h. After the reaction, the solution was concentrated and poured into a large excess

2

of diethyl ether for precipitation. After five precipitation cycles, the diethyl ether was colorless

3

and the precipitant was dried under vacuum for 24 h.

4

Grafting of (Boc-aminooxy)acetic Acid (Boc-AmoAcOH) to the Nanodiamond Surface.

5

ND-NH2 (80 mg) was added to dry dichloromethane (DCM, 2 mL) and sonicated for 1 h before

6

introducing (Boc-aminooxy)acetic acid (Boc-AmoAcOH) (160 mg, 0.84 mmol), N, N’-

7

dicyclohexylcarbodiimide (DCC, 50 mg, 0.24 mmol), and 4-dimethylaminopyridine (DMAP, 30

8

mg, 0.24 mmol). The solution was stirred for 72 hours followed by centrifugation to isolate the

9

nanodiamonds. The obtained nanodiamonds were subsequently washed by consecutive

10

washing/centrifugation cycles with DCM and then methanol for 5 times before drying under

11

vacuum. The removal of Boc groups was carried out under acidic conditions as follows. Boc-

12

AmoAcOH functionalized NDs (100 mg) were dispersed in 2 mL THF first, then 0.5 mL TFA

13

was added slowly under stirring. The deprotection was conducted at ambient temperature for 24

14

h. After reaction, TFA was removed by dialysis against milli-Q (MQ) water.

15

Oxime Ligation between Poly(1-O-MAFru)48-b-Poly(3-VBA53-co-MMA43)-AMF and ND-

16

NHAcomA. Attachment of Poly(1-O-MAFru)48-b-Poly(3-VBA53-co-MMA43)-AMF to the

17

surface of nanodiamonds (ND-NHAcomA) was conducted via an oxime ligation. 30 mg ND-

18

NHAcomA and 60 mg Poly(1-O-MAFru)48-b-Poly(3-VBA53-co-MMA43)-AMF were dissolved

19

in 2 mL anhydrous THF and stirred at room temperature for 48 h. The free polymers were

20

removed by centrifuged and washed with THF until the supernatant was colourless.

21

Size Exclusion Chromatography (SEC). The molecular weight and dispersity Ð of the

22

prepared polymers was analyzed via size exclusion chromatography (SEC). A Shimadzu


8

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Biomacromolecules

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modular system comprising a SIL-10AD auto-injector, DGU-12A degasser, LC-10AT pump,

2

CTO-10A column oven and a RID-10A refractive index detector was used. A Phenomenex 5.0-

3

µm bead-size guard column (50 × 7.5 mm2) followed by four Phenomenex PL (Styragel) linear

4

columns (500, 103, 104, and 105 Å pore size, 5 µm particle size) were employed for analysis. N,

5

N-dimethylacetamide [DMAc; HPLC grade, 0.05% w/v 2,6-di-butyl-4-methylphenol (BHT) and

6

0.03% w/v LiBr] with a flow rate of 1 mL/min at 50 ℃ was used as mobile phase. 50 µL of

7

polymer solution with a concentration of 2 mg/mL in DMAc was used for every injection. The

8

calibration was performed using commercially available narrow-disperse PMMA standards (0.5-

9

1000 kDa, Polymer Laboratories).

10

Nuclear Magnetic Resonance (NMR) Spectrometry. All NMR spectra were recorded using

11

a Bruker Avance Ш 300 spectrometer (300 MHz). All chemical shifts are recorded in ppm (δ)

12

relative to tetramethylsilane (δ=0 ppm), referenced to the chemical shifts of residual solvent

13

resonances (1H).

14 15

Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR).The infrared spectra of nanodiamonds were measured using ATR-IR (BRUKER, IFS 66/s).

16

Thermogravimetric Analysis (TGA). Thermogravimetric analysis was performed on a

17

Perkin-Elmer Thermogravimetric Analyzer (Pyris 1 TGA). Pre-dried samples were heated from

18

room temperature to 800 ℃ at a set temperature increase rate of 20 ℃/min using air as the

19

furnace gas.

20

Dynamic Light Scattering (DLS). Hydrodynamic diameters, Dh, were determined using a

21

Malvern Zetaplus particle size analyser (laser, angle = 173°) at a nanodiomand concentration of

22

100 µg mL-1. Samples were prepared in deionized water and sonicated for 30 min prior to the


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1

measurements. The ζ potential determinations were based on electrophoretic mobility of the

2

nanoparticles in the aqueous medium, which was performed using folded capillary cells in

3

automatic mode.

4

Transmission Electron Microscopy (TEM). TEM micrographs were obtained using a

5

JEOL1400 transmission electron microscope comprising of a dispersive X-ray analyzer and a

6

Gatan CCD facilitating the acquisition of digital images. The measurement was conducted at an

7

accelerating voltage of 80 kV. The samples were prepared by casting the solution (100 µg mL-1)

8

onto a copper grid. The grids were dried by air before measurement. No staining was used due to

9

the high contrast of nanodiamonds.

10

SRB Assay for Cytotoxicity. Human breast cancer MCF-7 and MDA-MB-231 cells were

11

seeded in 96-well plates (4000 cells per well for MCF-7 Cells and 8000 cells per well for MDA-

12

MB-231 cells) with 200 µL of culture medium Dulbecco’s modified Eagle’s medium (DMEM)

13

supplemented with 2.2 g L-1 NaHCO3, 10% (v/v) foetal bovine serum (FBS), 100 U/mL

14

penicillin and 100 µg mL-1 streptomycin in the incubator (5% CO2/95% air atmosphere at 37 °C)

15

for 24 h. The sample solution to be tested was sterilized via UV irradiation (20 min) before the

16

solution was serially halved via dilution in sterile Milli-Q water. The nanodiamond solutions

17

were then loaded into the plate at 100 µL per well. After incubation for 24 h, cells were treated

18

with trichloroacetic acid 10% w/v (TCA) and incubated at 4 ℃ for 40 min, and then washed five

19

times with Milli-Q water to get rid of the TCA solution. TCA- fixed cells were stained for 30

20

min with 0.4% (w/v) sulforhodamine B (SRB) dissolved in 1% acetic acid. SRB outside the cells

21

was removed by washing the plates with 1% acetic acid. The plates were left to air-dry overnight

22

followed by the addition of 100 µL 10 Mm Tris buffer per well to dissolve the dye in the cells.

23

The absorbance at 490 nm of each well was measured using a microtiter plate reader scanning


10

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Biomacromolecules

1

spectrophotometer to calculate cell viability [cell viability (%) = (test - blank) / (control - blank)

2

× 100].

3

3D Multicellular Tumor Spheroids (MCTS) Formation. The MCTs were prepared in a

4

procedure reported by us before.51 In brief, MCF-7 cells were suspended in DMEM media at a

5

density of 1.0 × 104 cells/mL. 200 µL of the cell suspension was seeded into each well of

6

ultralow attachment 96-well plate (Corning) and incubated for 3 days.

7

Acid phosphatase (APH) assay. The MCTs were incubated with free amonafide solution

8

(53.2 µg/mL) and amonafide conjugarted nanodiamonds (1 mg/mL) in 96 well suspension

9

culture plates. After 1，4 and 8 days, MCTs and entire supernatant were transferred into U shape

10

96-well microplates with a pipettor and then centrifuge for 5 min at RT at 400×g to spin down

11

spheroids, clusters and single cells. The spheroid pellet was washed by carefully replacing 150

12

µL of the supernatant with PBS. A final volume of 100 µL was obtained by repeating

13

centrifugation and discard supernatant. Then 100 µL of APH assay buffer was added to each well

14

and incubated for 90 min at 37 ℃. After incubation, supplement each well with 10 µL of 1 N

15

NaOH and transfer the supernatant to standard flat-bottomed 96-well microplates. The

16

absorption at 405 nm within 10 min was measured by a microplate reader.

17

RESULTS AND DISCUSSION

18

Synthesis and Coating of Amonafide-conjugated Glycopolymer on nanodiamonds

19

The synthesis of Poly(1-O-MAipFru)48 was carried out via RAFT polymerization (Scheme

20

1).52 Isopropylidene-protected glycomonomer was polymerized at 70 ℃ in the presence of 4-

21

cyanopentanic acid dithiobenzoate (CPADB) as a RAFT agent. The obtained glycopolymer was

22

used as Macro-CTA for chain extension with 3-Vinylbenzaldehyde (3-VBA). Hydrophobic


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Page 12 of 27

1

methyl methacrylate (MMA) monomer was added as comonomer during the chain extension in

2

order to decrease the steric hindrance during the reaction of 3-Vinylbenzaldehyde with the bulky

3

drug amonafide. Since the conversion of MMA and 3-VBA after 24 hours are close to 22% and

4

27%, the repeating unit of MMA and 3-VBA are around 43 and 53, respectively. After removal

5

of the thiocarbonylthio endgroup and the isopropylidene protecting groups, Poly(1-O-MAFru)48-

6

b-Poly(3-VBA53-co-MMA43) was conjugated with amonafide (AMF) via Schiff base reaction. (1)

N C

S [M]:[CPADB]:[AIBN]= 50 :1: 0.2 O

O

m O O O

1,4-Dioxane, 70 oC, 8 h

O

[MMA]:[3-VBA]:[CTA]:[AIBN] = 200: 200: 1: 0.2

OH S

O

O

O

1,4-Dioxane, 70 oC, 24 h

O O

O O

O

*

Poly(1-O-MAipFru)48 O

1-O-MAipFru

z

y-z

NH 2

O

* y

x

N

TFA/H2O (9:1) O

OO

N

OH OH

OH OH O

N

O

OH OH

DMSO, 110 oC, 36 h

O

H

O OH

N

OH

N

A Poly(1-O-MAFru)48-b-Poly(3-VBA 53-co-MMA 43)

O

Poly(1-O-MAFru)48-b-Poly(3-VBA53-co-MMA43)-AMF

H N

O

(2)

O

O

O

O

OO H

H

O

m*

m*

x

OH O

O

O

O H2 O Si C

O

O Si CH2 NH 2 3 m O DCM, DCC, DMAP, RT, 72 h

H N

H N

3

O

O

n

O O

O

ND-NH 2 THF, TFA, RT, 24 h

O H2 O Si C O

3

H N O

NH 2

B n

ND-NHAcomA

O

* y-z

(3) A +B

O THF, RT, 48 h

H2 O Si C O

3

H N

z

x

O

OO H

N

m* O OH OH

O O

H

O

N

n

OH OH O N N O

7 8

ND-Polymer-AMF

Scheme 1. Synthesis and Coating of Amonafide-conjugated Glycopolymer on Nanodiamonds.


12

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Biomacromolecules

1

The SEC traces of Poly(1-O-MAipFru)48, Poly(1-O-MAFru)48-b-Poly(3-VBA53-co-MMA43)

2

and Poly(1-O-MAFru)48-b-Poly(3-VBA53-co-MMA43)-AMF are shown in Figure 1. The

3

molecular weight distribution of Poly(1-O-MAipFru)48, and Poly(1-O-MAFru)48-b-Poly(3-

4

VBA53-co-MMA43) are narrow. The low dispersity indexes (Ð < 1.2) indicate that the

5

polymerization of glycomonomers and the co-polymerization of MMA and 3-VBA proceeded in

6

a controlled manner. After conjugation with amonafide (AMF), the molecular weight of Poly(1-

7

O-MAFru)48-b-Poly(3-VBA53-co-MMA43)-AMF increased and the SEC curve shifted to higher

8

molecular weight, which suggests the successful conjugation. The appearance of a small

9

shoulder in the SEC curve of Poly(1-O-MAFru)48-b-Poly(3-VBA53-co-MMA43)-AMF might be

10

due to the oxidization of thiol groups and formation of disulphide linkages between polymer

11

chains. The acid (TFA) used for fructose deprotection can cleave RAFT agents and convert

12

dithioesters into thiol groups at the end of polymer chains.

13 14

Figure 1. Normalized SEC curve of Poly(1-O-MAipFru)48, Poly(1-O-MAFru)48-b-Poly(VBA53-

15

co-MMA43) and Poly(1-O-MAFru)48-b-Poly(VBA53-co-MMA43)-AMF.


13


1

Page 14 of 27

The successful conjugation of AMF to the block copolymers was further confirmed using the

2

1

3

b-Poly(3-VBA53-co-MMA43)-AMF. As shown in Figure 2, the chemical shift of the aromatic

4

protons corresponding to the benzaldehyde can be found at 7.0 – 8.0 ppm, while that of CHO

5

group is at 10.0 ppm. After reaction, the characteristic signals of formed imine bonds at 8.5 ~ 9.0

6

ppm appeared and the chemical shifts of the conjugated AMF at 8.0 ppm can be clearly

7

identified, indicating the successful incorporation of AMF into the polymer. The ratio of

8

unreacted benzaldehyde groups and conjugated AMF moieties was calculated based on the

9

integral of those functional groups in the following equation:

10 11

H-NMR spectra of Poly(1-O-MAFru)48-b-Poly(3-VBA53-co-MMA43) and Poly(1-O-MAFru)48-

[(y-z) × Integral(m’+ n’+ o’+ p’) + z × Integral(m”+ n”+ o”+ p” + k” + a’ + b’ + c’ + d’ + e’)] : (y-z) × Integral(k’) = 8.52

12

Thus, the ratio of unreacted benzaldehyde groups and reacted benzaldehyde groups is 2.2 [(y-

13

z)/z = 2.2]. Since the total repeating unit of 3-VBA in the unconjugated polymer chains is 53 (y

14

= 53), that of benzaldehyde groups conjugated to amonafide is 17 (z = 17). Therefore, the mass

15

percentage of amonafide in the polymer chains is around 17% (Supporting Information E-2). The

16

presence of unreacted aldehyde functionalities is crucial as they will form the reactive anchor for

17

the reaction with the surface of nanodiamonds.


14

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Biomacromolecules

1 2

Figure 2. 1H-NMR Spectra of Amonafide (AMF), Poly(1-O-MAFru)48-b-Poly(VBA53-co-

3

MMA43) and Poly(1-O-MAFru)48-b-Poly(VBA53-co-MMA43)-AMF (300 MHz, d6-DMSO as

4

solvents).

5

Since unreacted benzaldehyde groups of Poly(1-O-MAFru)48-b-Poly(3-VBA53-co-MMA43)-

6

AMF (A) can react with aminooxyl groups, aminooxy-functionalized nanodiamonds (ND-

7

NHAcomA) (B) were further prepared as described in Scheme 1. Specifically, amine-

8

functionalized nanodiamonds (ND-NH2), prepared as reported before,23 were conjugated with

9

(Boc-aminooxy)acetic acid (Boc-AmoAcOH) via an amide coupling reaction. The solution was

10

stirred for 72 h followed by centrifugation to isolate the nanodiamonds. The obtained

11

nanodiamonds were subsequently washed by consecutive washing/centrifugation cycles with

12

DCM and then methanol for 5 times before drying under vacuum. After removal of the Boc

13

groups under acidic conditions (TFA), Poly(1-O-MAFru)48-b-Poly(3-VBA53-co-MMA43)-AMF


15


Page 16 of 27

1

were attached to the surface of nanodiamonds (ND-NHAcomA) via oxime ligation. The reaction

2

was carried at ambient temperature for 48 h. The free polymers were removed by centrifugation

3

and washing with THF until the supernatant was colourless. Though imine/oxime exchange

4

usually happens in the presence of oxyamine,53 this side reaction is suppressed in large parts

5

since the amount of benzaldehyde groups is significantly higher than those of amonafinde and

6

oxyamine in this system.

7 8

Figure 3. FTIR spectra of a) ND-NHAcomA-Boc, b) ND-NH2, c) Boc-AmoAcOH, d) ND-

9

Polymer-AMF, e) Poly(1-O-MAFru)48-b-Poly(VBA53-co-MMA43)-AMF, f) ND-NHAcomA.

10

The surface grafting reactions were monitored by FTIR. As show in Figure 3, the amino

11

groups of ND-NH2 (b) were observed at a wavenumber of 3500-3000 cm-1 (N-H stretch) and

12

1654 cm-1 (N-H bend), which are in agreement with values in literature.54 After conjugation with


16

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Biomacromolecules

1

(Boc-aminooxy) acetic acid (Boc-AmoAcOH) (c), two absorption peaks at 1617 cm-1 and 1563

2

cm-1 appeared (a), which are assigned to the formed amide groups. The removal of Boc groups

3

(f) is confirmed by the disappearance of the absorption peak at 1716 cm-1. The oxime ligation

4

between Poly(1-O-MAFru)48-b-Poly(3-VBA53-co-MMA43)-AMF (e) and ND-NHAcomA (f) is

5

confirmed by the appearance of –HC=N-O- groups at 1612 cm-1 and decreased absorption of –

6

CHO groups at 1693 cm-1.

7 8

Figure 4. TGA analysis of ND-NH2, ND-NHAcomA, and ND-Polymer-AMF.

9

The quantification of polymers conjugated to the surface of nanodiamonds was calculated via

10

thermogravimetric analysis (TGA). As depicted in Figure 4, the weight loss of ND-NH2 started

11

around 400℃. However, the thermo-stability of NDs is significantly enhanced by surface

12

decoration with aminooxy groups or glycopolymers, which is consistent with our former

13

reports.24 The thermo-degradation of polymer-coated NDs occurred rapidly once the temperature

14

reached 475℃.The weight loss correlating to the surface-grafted Poly(1-O-MAFru)48-b-


17


Page 18 of 27

1

Poly(VBA53-co-MMA43)-AMF is calculated by the weight difference (31%) between ND-

2

Polymer-AMF and ND-NHAcomA at 470 ℃. Further calculation indicate that the density of

3

polymer chains on the surface of ND-Polymer-AMF is close to 0.24 polymer chains / nm2.

4

Therefore, the mass percentage of conjugated amonafide in the overall drug carrier (ND-

5

Polymer-AMF) is about 5.27%. (Supporting Information E-8) The high density of polymer

6

chains on the surface of ND may be due to the high percentage of unreacted benzaldehyde

7

groups in the structure of Poly(1-O-MAFru)48-b-Poly(VBA53-co-MMA43)-AMF. As calculated

8

from the NMR spectra, the repeating unit of free benzaldehyde groups after drug conjugation is

9

36, which enabled the efficient reaction between benzaldehyde groups and aminooxyl groups.

10 11

Figure 5. Stability and size of nanodiamonds in aqueous solutions. (A) Storage of nanodiamonds

12

in MQ water, room temperature for 24 h, concentration: 200 µg mL-1; (B) TEM image of ND-

13

Polymer-AMF; (C) TEM image of ND-NH2; (D) Size and Zeta potential values of the

14

nanodiamonds measured by DLS. (1) ND-Polymer-AMF, (2) ND-NH2.

15

The size and zeta potential values of ND-Polymer-AMF in aqueous solution were measured by

16

TEM and DLS. As shown in Figure 5(D), ND-Polymer-AMF has an average hydrodynamic size

17

of 383 nm after dispersing in MQ water, which was further confirmed by TEM analysis [Figure

18

5(B)]. The larger size of ND-Polymer-AMF (383 nm) compared to that of unmodified ND-NH2

19

(186 nm) may result from the fact that polymers were conjugated to the surface of nanodiamond


18

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Biomacromolecules

1

clusters rather than single nanodiamonds during the oxime ligation in aqueous solution.

2

Moreover, the lower PDI of ND-Polymer-AMF (0.3) than that of ND-NH2 (0.8) indicates the

3

enhanced dispersibility in solution after polymer conjugation, due to the hydrophilic

4

glycopolymer coating on the surface of nanodiamonds. The negative charges of ND-Polymer-

5

AMF may originate from the carboxylic acid groups from RAFT agent CPADB. In addition,

6

consistent with what we reported before,24 the hydrophilic glycopolymer blocks increase the

7

suspension stability of nanodiamonds greatly. As shown in Figure 5(A), significantly less

8

precipitation of ND-Polymer-AMF was observed compared to unmodified ND-NH2 in aqueous

9

solution after storage for 24 h.

10

Cytotoxicity of ND-Polymer-AMF in 2D and 3D breast cancer cell culture models

11

The cytotoxicity of free amonafide, and ND-Polymer-AMF on breast cancer cell lines (MCF-7

12

and MDA-MB-231 cells) are shown in Figure 6. Fructose-coated nanodiamonds were found to

13

be non-toxic in the previously reported paper.24 The mass percentage of conjugated amonafide in

14

the overall drug carrier (ND-Polymer-AMF) is calculated by E-8 (Supporting Information). After

15

incubation for 24 h, ND-Polymer-AMF delivery systems resulted in higher cytotoxicity than free

16

amonafide drugs in both MCF-7 and MDA-MB-231 cell lines. In 2D MCF-7 cell culture models,

17

the IC50 value of ND-Polymer-AMF delivery system (7.19 µM) is smaller than that of free

18

amonafide (11.23 µM), suggesting an enhanced delivery efficiency of amonafide into cells by the

19

ND-Polymer-AMF delivery system. More significantly, promotion of cytotoxicity was observed

20

in the treatment of MDA-MB-231 cells. As shown in Figure 6(B), an approximately two-fold

21

increase in toxicity was achieved after conjugating amonafide to the surface of nanodiamonds.

22

Compared with the hydrophobic free amonafide, the ND-Polymer-AMF can improve the

23

dispersibility of drugs in the aqueous solution. Moreover, the fructose moieties on the outer


19


Page 20 of 27

1

surface of ND-Polymer-AMF have specific binding to GLUT5, which is an overexpressed sugar

2

transporter on the plasma membranes of MCF-7 and MDA-MB-231 cells, and enhance their

3

uptake by breast cancer cells. 24, 47, 51, 55

4

To further investigate the drug delivery efficiency of ND-Polymer-AMF, MCF-7 spheroids

5

cell culture models were used for studying the penetration of drug delivery systems. The cell

6

viability of MCF-7 cells was evaluated on day 1, day 4 and day 8, respectively, to track the cell

7

growth inhibition. Compared with the control, both free amonafide and ND-Polymer-AMF start

8

inhibiting the growth of MCF-7 cells after one day incubation. Significant growth inhibition

9

effect of free amonafide and ND-Polymer-AMF was observed on day 4 and day 8. However, the

10

ND-Polymer-AMF did not exhibit the same enhanced cytotoxicity in MCF-7 spheroid models

11

compared to free amonafide as it was observed in 2D cell culture models. The different results

12

obtained from 2D and 3D cell culture models are common as 3D models do not only measure the

13

cellular uptake, but also the diffusion of the nanoparticles either via the paracellular or

14

trancellular route.24, 55 Parameters affecting the cellular uptake such as size will also be favorable

15

for the penetration. However, penetration via the transcellular route requires a series of

16

endocytosis and exocytosis steps. Fast drug release during the penetration into the peripheral

17

cells will kill the outer layer and pause sustainable delivery of drugs to the inner part of

18

spheroids. Compared with 2D cell culture models, 3D multicellular spheroids models have in

19

vivo-mimic extracellular matrix and provide a model to bridge the gap between 2D cell assays

20

and in vivo studies. As shown in Figure 6, ND-Polymer-AMF can greatly inhibit the viability of

21

cancer cells and the growth of spheroids after 4 and 8 day incubations is negligible. These results

22

indicate that oxime ligation is a promising strategy for fabrication of polymer-diamonds hybrid

23

materials for breast cancer therapy.


20

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Biomacromolecules

1

2 3

Figure 6. Cytotoxicity of free amonafide, ND-Amonafide in 2D and 3D cell culture models. (A)

4

IC50 curves of free amonafide, ND-Amonafide in 2D MCF-7 cell culture models after 24h

5

incubation; (B) IC50 curves of free amonafide, ND-Amonafide in 2D MDA-MB-232 cell culture

6

models after 24h incubation; (C) Cell viability after 1 day, 4 days and 8 days treatment of free

7

amonafide, ND-Amonafide in 3D MCF-7 spheroid models. Data represent means ± SD, n = 10.

8

**, significant difference, P < 0.01 ***, significant difference, P < 0.001; ****, significant

9

difference, P < 0.0001.

10


21


1

Page 22 of 27

CONCLUSIONS

2

In the current study, poly(1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose)-

3

b-poly(3-vinylbenzaldehyde-co-methyl methacrylate), featuring aldehyde moieties along the

4

polymer chain, was prepared via RAFT polymerization. The molecular weight of the conjugated

5

polymers is characterised by Size Exclusion Chromatography (SEC). The anticancer drug

6

amonafide was conjugated to the polymer chains by the reaction between aldehyde and amine

7

groups. Successful conjugation was confirmed by Nuclear Magnetic Resonance (NMR) and 17%

8

mass percentage of amonafide in the polymer chains was calculated from the 1H-NMR spectra.

9

The grafting of polymers to the surface of nanodiamonds was characterized by Fourier transform

10

infrared spectroscopy (FTIR) and Thermogravimetric Aanalysis (TGA). The surface density of

11

polymer chains was quantitively determined by NMR and TGA assessments. The prepared drug

12

delivery system has good water dispersity and can effectively deliver amonafide into breast

13

cancer cell and significantly inhibit the cancer cell viability. The results suggest that oxime

14

ligation, which can modify the surface of nanodiamonds with high polymer grafting density and

15

accessible polymer structural characterisation, is a promising strategy for fabrication of polymer-

16

diamonds hybrid materials for breast cancer therapy.

17

ASSOCIATED CONTENT

18

The detailed calculations of the mass percentage of amonafide in the polymer chain of Poly(1-

19

O-MAFru)48-b-Poly(3-VBA53-co-MMA43)-AMF, the density of polymer chains on the surface of

20

ND-Polymer-AMF and the mass percentage of amonafide on the surface of ND-Polymer-AMF

21

can be found in Supporting Information.

22

Corresponding Authors

23

*E-mail:

[email protected]; [email protected]


22

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1 2 3

Biomacromolecules

Notes The authors declare no competing financial interest. ACKNOWLDGEMENTS:

4

J. Z. would like to acknowledge the China Scholarship Council (CSC) for scholarship support.

5

P. X. acknowledges funding from the Australian Research Council’s Discovery Early Career

6

Researcher Award (DE140100318). C.B.-K. acknowledges the Australian Research Council

7

(ARC) for funding in the context of a Laureate Fellowship. C.B.-K. and M.H.S. acknowledge the

8

German Research Council for support.

9

REFERENCES

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

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Stealth Polyglycerol, Targeting Peptide and Acid-Responsive Antitumor Drug. Adv. Funct. Mater. 2014, 24 (34), 5348-5357. 11. Liang, Y.; Ozawa, M.; Krueger, A., A General Procedure to Functionalize Agglomerating Nanoparticles Demonstrated on Nanodiamond. ACS Nano 2009, 3 (8), 2288-2296. 12. Zhang, F.; Lees, E.; Amin, F.; Rivera Gil, P.; Yang, F.; Mulvaney, P.; Parak, W. J., Polymer-coated nanoparticles: a universal tool for biolabelling experiments. Small 2011, 7 (22), 3113-3127. 13. Cha, I.; Hashimoto, K.; Fujiki, K.; Yamauchi, T.; Tsubokawa, N., Modification of dispersibility of nanodiamond by grafting of polyoxyethylene and by the introduction of ionic groups onto the surface via radical trapping. Mater. Chem. Phys. 2014, 143 (3), 1131-1138. 14. Lee, J. W.; Lee, S.; Jang, S.; Han, K. Y.; Kim, Y.; Hyun, J.; Kim, S. K.; Lee, Y., Preparation of non-aggregated fluorescent nanodiamonds (FNDs) by non-covalent coating with a block copolymer and proteins for enhancement of intracellular uptake. Mol. Biosyst. 2013, 9 (5), 1004-1011. 15. Zhang, X. Q.; Chen, M.; Lam, R.; Xu, X.; Osawa, E.; Ho, D., Polymer-Functionalized Nanodiamond Platforms as Vehicles for Gene Delivery. ACS Nano 2009, 3 (9), 2609-2616. 16. Sun, Y.; Olsén, P.; Waag, T.; Krueger, A.; Steinmüller-Nethl, D.; Albertsson, A.-C.; Finne-Wistrand, A., Disaggregation and Anionic Activation of Nanodiamonds Mediated by Sodium Hydride—A New Route to Functional Aliphatic Polyester-Based Nanodiamond Materials. Part. Part. Syst. Charact. 2015, 32 (1), 35-42. 17. Wuest, K. N. R.; Trouillet, V.; Goldmann, A. S.; Stenzel, M. H.; Barner-Kowollik, C., Polymer Functional Nanodiamonds by Light-Induced Ligation. Macromolecules 2016, 49 (5), 1712-1721. 18. Wuest, K. N. R.; Lu, H.; Thomas, D. S.; Goldmann, A. S.; Stenzel, M. H.; BarnerKowollik, C., Fluorescent Glyco Single-Chain Nanoparticle-Decorated Nanodiamonds. ACS Macro Lett. 2017, 6 (10), 1168-1174. 19. Wuest, K. N. R.; Trouillet, V.; Koppe, R.; Roesky, P. W.; Goldmann, A. S.; Stenzel, M. H.; Barner-Kowollik, C., Direct light-induced (co-)grafting of photoactive polymers to graphitic nanodiamonds. Polym. Chem. 2017, 8 (5), 838-842. 20. Stenzel, M. H., Hairy Core-Shell Nanoparticles via RAFT: Where are the Opportunities and Where are the Problems and Challenges? Macromol. Rapid Commun. 2009, 30 (19), 16031624. 21. Lai, H.; Chen, F.; Lu, M.; Stenzel, M. H.; Xiao, P., Polypeptide-Grafted Nanodiamonds for Controlled Release of Melittin to Treat Breast Cancer. ACS Macro Lett. 2017, 6 (8), 796-801. 22. Lu, M.; Wang, Y.-K.; Zhao, J.; Lu, H.; Stenzel, M. H.; Xiao, P., PEG GraftedNanodiamonds for the Delivery of Gemcitabine. Macromol. Rapid Commun. 2016, 37 (24), 2023-2029. 23. Huynh, V. T.; Pearson, S.; Noy, J.-M.; Abboud, A.; Utama, R. H.; Lu, H.; Stenzel, M. H., Nanodiamonds with Surface Grafted Polymer Chains as Vehicles for Cell Imaging and Cisplatin Delivery: Enhancement of Cell Toxicity by POEGMEMA Coating. ACS Macro Lett. 2013, 2 (3), 246-250. 24. Zhao, J.; Lai, H.; Lu, H.; Barner-Kowollik, C.; Stenzel, M. H.; Xiao, P., Fructose-Coated Nanodiamonds: Promising Platforms for Treatment of Human Breast Cancer. Biomacromolecules 2016, 17 (9), 2946-2955.


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Biomacromolecules

1

Delivery of Amonafide from Fructose-coated

2

Nanodiamonds by Oxime Ligation for Treatment of

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Human Breast Cancer

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Jiacheng Zhao,a Mingxia Lu,a Haiwang Lai,a Hongxu Lu,a Jacques Lalevée,b Christopher

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Barner-Kowollik,c,d Martina H. Stenzel,*,a and Pu Xiao*,a, b

6

a

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South Wales, Sydney, Australia; bInstitut de Science des Matériaux de Mulhouse IS2M, UMR

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CNRS 7361, ENSCMu-UHA, 15, rue Jean Starcky, 68057 Mulhouse Cedex, France; cSchool of

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Chemistry, Physics and Mechanical Engineering, Queensland University of Technology (QUT),

Centre for Advanced Macromolecular Design, School of Chemistry, The University of New

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2 George Street, Brisbane, QLD 4000, Australia; dMacromolecular Architectures, Institut für

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Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstr.

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18, 76128 Karlsruhe, Germany.

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TOC

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Biomacromolecules - ACS Publications - American Chemical Society

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