Amphiphilic Polysaccharide Block Copolymers for pH-Responsive

Subscriber access provided by UNIV OF NEWCASTLE

Article

Amphiphilic polysaccharide block copolymers for pH-responsive micellar nanoparticles Benjamin B. Breitenbach, Ira Schmid, and Peter R. Wich Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00771 • Publication Date (Web): 26 Jul 2017 Downloaded from http://pubs.acs.org on July 27, 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.

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.

Page 1 of 41

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

Biomacromolecules

Amphiphilic polysaccharide block copolymers for pH-responsive micellar nanoparticles Benjamin B. Breitenbach, Ira Schmid, Peter R. Wich Institut für Pharmazie und Biochemie, Johannes Gutenberg-Universität Mainz Staudingerweg 5, 55128 Mainz, Germany

KEYWORDS: polysaccharide, block copolymer, acetalated dextran, pH-sensitive, nanoparticles, microwave

ABSTRACT: A full polysaccharide amphiphilic block copolymer was prepared from end groupfunctionalized dextrans using copper mediated azide-alkyne click chemistry. Sufficient modification of the reducing end in both blocks was achieved by microwave-enhanced reductive amination in a borate-buffer/methanol solvent system. The combination of a hydrophilic dextran block with a hydrophobic acetalated dextran block results in an amphiphilic structure that turns

ACS Paragon Plus Environment

1

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

Page 2 of 41

water-soluble upon acid treatment. The material has a low CMC and self-assembles in water to spherical micellar nanoparticles. The formed nanoparticles have a narrow size distribution below 70 nm in diameter and disassemble in slightly acidic conditions. The amphiphilic polysaccharide system shows low toxicity and can stabilize the hydrophobic model drug curcumin in aqueous solutions over extended time periods.

INTRODUCTION Linear AB type block copolymers represent a well-studied compound class in polymer chemistry.1 Increasing interest in nanotechnology2 and associated applications in drug delivery boosted the use of block copolymers as suitable components for smart nanomaterials. The conjugation of blocks with hydrophilic and hydrophobic characteristics has become a popular method to create self-assembling structures. Their amphiphilic nature and capacity to selforganize into nanostructures enables manifold applications such as nano-sized catalytic environments3, 4, nanoparticles for drug delivery5-8 or solution stabilizing surfactants.9-11 Especially for drug delivery, smart polymers with stimuli-responsive release characteristics are of great interest. They allow rapid cargo release under specific conditions, e.g. in slightly acidic environment (pH 5-6), as it is found in tumor epithelial tissue or sites of inflammation.12 So far, a variety of artificial pH-responsive block copolymers have been designed, with blocks containing poly(β-amino esters)13, poly(L-histidines)14, 15 or modified polysaccharides16-18. Block copolymers containing polysaccharide moieties present a sustainable and promising alternative to synthetic polymer materials. They feature beneficial properties like degradability, biocompatibility and good solubility in aqueous solvents.5,

19, 20

Depending on the type of

polysaccharide they carry different functional groups in their backbone19, predominantly hydroxyl groups21 but also amine-22, carboxyl-23 or sulfate groups20, which allow a


2

Page 3 of 41

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

Biomacromolecules

functionalization with a variety of bioorthogonal conjugation methods. However, in order to link linear polysaccharides the limited availability of the reducing chain-end aldehyde19 and its slow reaction kinetics24, 25 resulted in a small amount of studies using polysaccharides as copolymer blocks so far. Also, quantification and structural analysis of the modified polysaccharide chainend by 1H-NMR is difficult because of the wide distribution and high intensity of all sugar proton signals, often overlaying the introduced linker signal. To date, most publications in this field focus on an amination reaction for the chain-end modification. However, most groups analyze this reaction only qualitatively by observing the disappearance of the reducing end group anomeric protons at δ = 6.7 ppm (α anomer) and δ = 6.3 ppm (β anomer).16, 26, 27 Only a limited amount in recent literature report a thorough and quantitative analysis of their polysaccharide building blocks and their degree of modification.20,

21

Recently, p-substituted anilines were

reported as valuable tool for organo-catalyzed derivatization of oligosaccharides.25 The aniline derivative has an ideal pKa for reductive amination and also allows identification and quantification by

1

H-NMR spectroscopy. End-group modification of polysaccharides by

reductive amination can exceed several days to complete and typically needs high molar amounts of small molecule linker.26, 27 Gu et al. were overcoming the issue of slow reaction kinetics by applying microwave irradiation, which reduces the reaction times and the necessary equivalents of linker molecules significantly.24 So far only non-stimuli-responsive block copolymers built from short oligosaccharide blocks have been reported.28, 29 For this project, we decided to focus solely on the natural polysaccharide dextran as biopolymer material for both blocks. It is a linear macromolecule with a low percentage of (1,3)branching and a molecular weight of 5 kDa. It is composed of 30 individual 1,6-linked glucose units and is produced by lactic bacteria leuconostoc and streptococcus species.

30, 31

Dextran is


3

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

Page 4 of 41

FDA approved in selected medicinal uses and has wide applications in bionanotechnology.17, 32, 33

Its low molecular weight fractions are known to be non-toxic, biodegradable and show low

immunogenicity.5, 34, 35 The good biocompatibility and available alcohol groups in the backbone for functionalization, make dextran a valuable, water-soluble building block for all kinds of functional nanomaterials. With the introduction of acid-labile acetal groups, Fréchet and coworkers extended the application of dextran from a hydrophilic to a hydrophobic polysaccharide building block and incorporated a pH-responsive solubility switch.33,

36

Acetalated dextran (AcDex) has been studied extensively as a biopolymer material with pHsensitivity37 for the delivery of hydrophobic38 or hydrophilic39,

40

payloads in emulsion-based

nanoparticle formulations. However, despite its utility, the use of AcDex as stimulus-responsive hydrophobic building block for alternative polymer architectures has been reported only a few times so far.32, 41 Reported examples using dextran as material for block copolymers applied the polysaccharide either for the hydrophilic42-46 or the hydrophobic16-18 block, but so far not for both blocks at the same time. Here, we present the synthesis of an acid-responsive full polysaccharide block copolymer. We use for both blocks modified dextrans that when linked together, can self-assemble into micellar structures in aqueous solutions. Combining microwave-enhanced reductive amination24 for the introduction of p-substituted anilines21, 25 at the reducing chain end and acetalation of backbone hydroxyl groups, we establish a quick and efficient synthesis of hydrophobic and hydrophilic polysaccharide building blocks. Copper-mediated click chemistry connects the two blocks and forms a pH-responsive polysaccharide amphiphile. To the best of our knowledge, this is the first report of an acid-degradable amphiphilic block copolymer composed only of polysaccharides. EXPERIMENTAL SECTION


4

Page 5 of 41

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

Biomacromolecules

Materials. Dextran T5 (‫ܯ‬௡ 5000 g·mol-1) was purchased from Pharmacosmos (Denmark). Sodium

cyanoborohydride,

N,N,N’,N’’,N’’-pentamethyldiethylenetriamine

(PMDTA),

4-

azidoaniline hydrochloride, 4-ethynylaniline, pyridinium p-toluensulfonate (PPTS), dry dimethylsulfoxide (DMSO), glycine and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma Aldrich (Germany). Copper(I)bromide was purchased from Alfa Aesar (Germany). Methanol and triethylamine (Et3N) were purchased from Carl Roth (Germany). Methanol was distilled and used without further purification. Aqueous buffers for DLS measurements were filtered through a Filtropur syringe filter (0.45 µm, Sarstedt, Germany). Due to the light sensitivity of 4-azidoaniline, all reactions involving this compound were kept under light exclusion.47 Dialysis membranes were purchased from Spectrum Labs (Spectra-Por® 6, MWCO 1000) and Carl Roth (T2: MWCO 6000-8000). Dulbecco’s modified eagle medium (DMEM), fetal calf serum (FCS), glutamine, phosphate buffered saline (PBS, for cell culture), pyruvate and penicillin/streptomycin were purchased from Invitrogen (Germany). Double distilled (dd) water was used if not mentioned otherwise.

Instrumentation. 1H-NMR and

13

C-NMR spectra were recorded using 300 MHz Bruker

Topspin fourier spectrometer. Infrared (IR) spectra were recorded using a Nicolet Avatar 330-IR (ATR-unit) spectrometer. Size-exclusion chromatography was performed at 25 °C using an Agilent 1260 Infinity system (1260 IsoPump with 1260 LAS injector), including a PSS Suprema Linear M column, equipped with a UV/VIS Dual 2487 detector (Waters, Germany) and a RI-101 detector (ERC). Calibration (using dextran standards provided by PSS) and measurements were carried out in water containing 0.1 M NaNO3 at a flow rate of 1 mL·min-1. Microwave reactions were carried out using a Discover Benchmate microwave synthesis system (CEM, Canada).


5

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

Page 6 of 41

Particle characterization. The critical micelle concentration (CMC) for Dex-b-AcDex block copolymer was determined using a Dataphysics DCAT 11 EC ring tensiometer equipped with a TV 70 temperature control unit, a LDU 1/1 liquid dosing and refill unit, as well as a RG 11 Du Noüy ring. Surface tension data was processed with SCAT v3.3.2.93 software. The CMC presented is a mean value of three experiments. All solutions for surface tension measurements were stirred for 300 s at a stir rate of 50%. After a relaxation period of 120 s, three surface tension values were measured. The mean values of the three measurements were plotted against the concentration. The slopes of the traces at high concentrations as well as in the low concentration range were determined by linear regression. The concentration at the intersection of the fits determines the CMC (Figure S-4). The Du Noüy ring was rinsed thoroughly with water and annealed in a butane flame. Mean diameter and the scattering intensity of the nanoparticle suspension were determined by dynamic light scattering. The measurements were conducted on a Malvern Zetasizer Nano ZS at 20 °C and a scattering angle of 90° at 20 °C. The nanoparticle samples were measured in disposable PS cuvettes from Carl Roth. Data was analyzed using the Zetasizer Software v.7.11 and processed with Microsoft Excel 2016. For TEM measurements, the nanoparticle solution was drop-casted on a 300-mesh copper carbon grid from Plano GmbH. The image acquisition was carried out with a transmission electron microscope Tecnai 12 (FEI, acceleration voltage: 120 kV, electron source: LaB6 BIO-TWIN cathode) equipped with a 4K CCD camera (Tietz).

Preparation of azide end-functionalized dextran (1). Dextran (200 mg, 0.04 mmol, 1 eq) was dissolved in 1.8 mL B(OH)3-buffer (0.1 M, pH 8.3) in a sealed microwave vial. The 4-


6

Page 7 of 41

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

Biomacromolecules

azidoaniline hydrochloride (68 mg, 0.4 mmol, 10 eq) was dissolved in 0.46 mL MeOH with 61 µL NEt3 (0.44 mmol, 11 eq) and added slowly while stirring vigorously at room temperature. NaCNBH3 (30.2 mg, 0.48 mmol, 12 eq) was added to the suspension in one portion and the vial was placed in the microwave. The reductive amination was performed for 4 h at 30 °C with maximum microwave power set to 100 W. Temperature was maintained by air cooling throughout the reaction using pressurized air (2 bar). The colorless product was precipitated in MeOH from a clear slightly yellow reaction solution, centrifuged down (15000xg, 20 min, 20 °C) and washed 3 times with MeOH. The resulting off-white pellet was dissolved in 5 mL H2O-d and dialyzed against H2O-d (MWCO 1 kDa) overnight to remove residual buffer salts. After freeze-drying, the purified product was obtained as off-white powder. Yield: 156 mg (78%), 92% modified chains (by 1H-NMR). FTIR (cm-1): 3365 (O-H), 2919 (C-H), 2113 (N3), 1639 (C=C, aromatic), 1515 (C=C, aromatic), 1411, 1338, 1265 (C-H, bending), 993(C-O). 1H-NMR (300 MHz, D2O) δ (ppm): 6.98 and 6.95 (d, Harom), 6.86 and 6.83 (d, Harom), 4.93 (s, Hanom), 3.963.86 (m, H-dex), 3.73-3.65 (m, H-dex), 3.56-3.45 (m, H-dex).

Preparation of alkyne end-functionalized dextran (2). Compound 2 was synthesized similar to compound 1 with slightly changed conditions. Dextran (200 mg, 0.04 mmol, 1 eq) was dissolved in 1.2 mL B(OH)3-buffer (0.1 M, pH 8.3) in a sealed microwave vial. 4-ethynylaniline (46 mg, 0.4 mmol, 10 eq) was dissolved in 0.79 mL MeOH and slowly added under vigorous stirring to the aqueous solution. The NaCNBH3 (30.2 mg, 0.48 mmol, 12 eq) was added and the vial placed in the microwave. The reaction was carried out with air-cooling at 50 °C for 4 h. The maximum power was set to 100 W. Work-up was equal to the purification of (1). Yield: 158 mg (79%), 73% modified chains (by 1H-NMR). FTIR (cm-1): 3363 (O-H), 2920 and 2985 (C-H),


7

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

Page 8 of 41

2104 (C≡C, weak), 1639 and 1608 (C=C, aromatic), 1416, 1344, 1271 (C-H, bending), 1014 (CO). 1H-NMR (300 MHz, D2O) δ (ppm): 7.48 and 7.45 (d, Harom), 6.85 and 6.82 (d, Harom), 5.03 (s, Hanom), 4.06-3.96 (m, H-dex), 3.83-3.75 (m, H-dex), 3.65-3.49 (m, H-dex).

Synthesis of acetalated Dex-alkyne (3). The acetalization was carried out according to a procedure previously described.33, 40 Compound 2 (158 mg, 0.031 mmol, 1 eq) was dissolved in 1.5 mL anhydrous DMSO in a dried flask under argon atmosphere. PPTS (5 mg, 0.026 mmol, 0.64 eq) was added and the yellow clear solution stirred for 5 minutes. Then 1.14 mL 2methoxypropene (12 mmol, 380 eq) was added dropwise and the solution stirred for 10 minutes. The reaction was quenched by adding 0.32 mL NEt3 and the product precipitated in H2O-d (pH 9, adjusted with TEA). The pellet was isolated by centrifugation (12000xg, 20 min, 20 °C) after 3 washing steps including redissolving in MeOH and subsequent precipitation in H2O-d (pH 9). The slightly yellow pellet was lyophilized and isolated as fluffy off-white solid. Yield: 166 mg, 66% acetals (by 1H-NMR), theor. mol. weight: 6069.39 g/mol (eq. S-1). FTIR (cm-1): 3427 (OH, weak), 2987 (C-H), 2937 (C-H), 2833 (C-H), 2104 (C≡C, weak), 1659 and 1608 (C=C, aromatic), 1466 (C-H, bending), 1373, 1381 (C-H, bending), 1057 (C-O). 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 7.27-7.16 (m, Harom), 7.66-7.59 (m, Harom), 5.48-4.90 (m, OH), 4.72 (s, Hanom), 3.79-3.411 (m, H-dex), 3.21-3.15 (m, H-dex), 1.31-1.29 (m, acetals).

Synthesis of Dex-b-AcDex amphiphile (4). Compound 1 (30 mg, 6 µmol, 1 eq), compound 3 (69 mg, 11.4 µmol, 1.5 eq) and PMDTA (3.7 µL, 18 µmol, 2 eq) were dissolved in 1 mL dry DMSO and the solvent was degassed by three freeze-thaw cycles. Cu(I)Br (1.7 mg, 12 µmol, 2 eq) was added under Ar atmosphere to the degassed solution and the reaction mixture stirred in


8

Page 9 of 41

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

Biomacromolecules

dark for 3 d at 50 °C. The dark green solution was added to a 10-time excess of H2O-dd (pH 9, adjusted by TEA), the unmodified AcDex residue was removed by centrifugation (12000xg, 25 min, 20 °C) and the supernatant was excessively dialyzed (MWCO 6-8 kDa) for 72 h against H2O-dd (pH 9) to completely remove residual copper and non-modified dextran material. The product was obtained as off white solid. Yield: 60 mg (90%). FTIR (cm-1): 3369 (O-H, weak), 2979 (C-H), 2923 (C-H), 2844 (C-H), 1652 (C=C, aromatic), 1454 (C-H, bending), 1205, 1153 (C-H, bending), 1016 (C-O). 1H-NMR (300 MHz, D2O) δ (ppm): 8.55 (s, triazole), 7.75 and 7.73 (m, Harom), 7.65 and 7.62 (m, Harom), 7.00-6.94 (m, Harom), 4.98 (s, Hanom), 4.19-3.91 (m, H-dex), 3.83-3.70 (m, H-dex), 3.60-3.32 (m, H-dex), 1.50 (s, acetals).

Scheme 1. Synthetic route to the amphiphilic Dex-b-AcDex block copolymer (4) by Cu(I)mediated click reaction of a hydrophilic azide-functionalized dextran block (1) with a hydrophobic alkyne-functionalized acetalated dextran block (3). Preparation of Dex-b-AcDex micellar nanoparticles (empty and drug-loaded). Selfassembly of block copolymer 4 was achieved by using a solvent replacement method (DMSOH2O system).16, 17 Dex-b-AcDex (5 mg) was dissolved in 200 µL DMSO (or 5 mM curcumin in


9

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

Page 10 of 41

DMSO), incubated to swell overnight and then slowly dropped into 1.8 mL of H2O-dd at pH 8 (adjusted with TEA). The solution was vortexed, sonicated (3 s) and then centrifuged (12000xg, 5 min, 20 °C) to remove any non-dissolved curcumin. The supernatant was dialyzed for 48 h (MWCO 6-8 kDa) with steady exchange of the water (every 2 h) to fully remove DMSO. Samples were analyzed by DLS and TEM. The final concentration of particle solution was determined to be 1.28 mg·mL-1 (empty) and 1.42 mg·mL-1 (curcumin-loaded) by lyophilization of 1 mL of micelle solution and weighing of the residual solid.

Degradation of particles under acidic conditions. Samples of micellar nanoparticles in H2Odd (130 µl) were diluted with NaAc-buffer (130 µl, pH 5.5) or H2O-dd (130 µl, pH 8 with TEA) and placed in a micro cuvette or HPLC vials. The degradation was monitored by DLS and by taking pictures of the nanoparticle solution after 0 h, 0.5 h, 1 h, 2 h, 4 h and 24 h.

Cell culture. HeLa cells were grown in Dulbecco’s Modified Eagle Medium (DMEM GlutaMAX™) supplemented with 10% (V/V) fetal calf serum (FCS), 1% pyruvate, and 1% penicillin-streptomycin. Cell incubations were performed in a humidified incubator at 37 °C with 5% CO2 atmosphere. All used buffers were either autoclaved, sterile filtered or already sterile when supplied and were preheated to 37 °C before usage. Cells were grown in 25 cm2 or 75 cm2 standard cell culture flasks.

In vitro cell viability (MTT assay). The cytotoxic effects of empty and curcumin-loaded micellar nanoparticles were evaluated by MTT assay48 using human HeLa cells. Cells were precultured in DMEM containing 10% FCS and 1% P/S and then seeded in sterile clear, flat


10

Page 11 of 41

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

Biomacromolecules

bottom 96-well cell culture microplates at a concentration of 15,000 cells per well and a volume of 100 µL. Cells were allowed to attach and grow overnight. Empty micellar nanoparticles and curcumin-loaded samples were prepared by mixing the micellar solutions (H2O-dd, pH 7.4) with DMEM in a concentration range from 10 to 500 µg·mL-1. As blank, H2O-dd was also diluted with DMEM in the same manner as the micellar solutions. The next day, DMEM was removed from HeLa cells and replaced by 100 µL of the sample solutions as well as the blank solutions. All measurements were carried out in triplets. The cells were incubated for 48 hours at 37 °C in 5% CO2. After 48 hours, 40 µL MTT solution (3 mg·mL-1 in DMEM) was added to each well and incubated at 37 °C for 30 minutes. After total removal of the medium, a mixture of 200 µL DMSO and 25 µL glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5) was added to each well and softly shaken for 15 minutes to dissolve the purple formazan salt. 50 µL of this concentrated, purple DMSO solution was added to a second clear, flat bottom 96-well microplate containing a mixture of 17 µL glycine buffer and 133 µL DMSO per well. Finally, the absorbance of the formazan was read using an Infinite 200 PRO (Tecan) plate reader at 595 nm. Furthermore, the background was measured at 670 nm and subtracted from the data obtained from the first read out. Cell viability was calculated with Microsoft Excel. Cell viability was normalized to the absorbance of the blank samples.

RESULTS AND DISCUSSION The synthesis of our amphiphilic block copolymer was achieved by end-on ligation of two linear dextran blocks with different solubility properties. Efficient site-selective activation of each


11

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

Page 12 of 41

dextran block was possible by addressing the free aldehyde at the reducing end of the last glucose unit with reductive amination using a microwave-assisted approach. Dextran modification. The main challenge for chain end modification of polysaccharides is the low concentration of free aldehyde in the open chain form (< 0.1 %)49 which requires very long reaction times or large excess of linker molecules. There are several elegant ways in literature to overcome this limitation and achieve a high endgroup-functionalization. Oxime ligation offers fast and chemoselective modification of the reducing end on dextran.50,

51

However, for this it is necessary to perform multi-step preparations of aminooxy-containing linker molecules. Zhang et al. reported the oxidation of terminal aldehydes to lactones that can be further exploited for ring-opening ligation reactions.52 Finally, the incorporation of aminebearing molecules by reductive amination offers an easy and inexpensive alternative, but requires high equivalents of linker molecules and long reaction times up to several days.26, 53 To overcome these limitations, dextrans 1 and 2 (Scheme 1) were chain end-modified combining a microwave-assisted approach by Gu and coworkers24 together with aniline-mediated reductive amination as previously described by Halila et al.25 This allowed a sufficient modification within 4 h using only ten equivalents of commercially available aniline derivatives. Furthermore, the introduction of an aromatic residue allows a direct quantification of the modification by 1HNMR (%mc - modified chains). Integration of the two aromatic signals at 6.98 ppm and 6.86 ppm for the aryl azide (Figure 1; H-a, a’ b, b’), or at 7.48 and 6.82 ppm for the alkyne derivative, relative to the anomeric proton of each glucose repeating unit at 6.98 ppm (Figure 1, H-c) reveals a degree of modification of 89-92%mc for Dex-N3 (1) and 76%mc for Dex-alkyne (2). It is noteworthy to say, that the achieved modification rates, while high but not being 100%, proved to be sufficiently high enough for the next reaction steps. After the click reaction to


12

Page 13 of 41

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

Biomacromolecules

combine both blocks, single unmodified dextran chains can be removed by precipitation and dialysis.

Figure 1. Comparing the 1H-NMR spectra of Dex (a), Dex-N3 1 (b) and Dex-alkyne 2 (c) (300 MHz, D2O) allows the quantification of end group modification by integrating the ratio of introduced aromatic protons H-a,a'/H-b,b' and the anomeric H-c of the dextran. Molecular weight of hydrophobic block AcDex-alkyne 3 (d) can be calculated from the 1H-NMR signal relation of generated acetone and methanol, resulting from cleaved off acetals after acidic treatment (300 MHz, D2O, DCl). Since arylazides, e.g. p-azidoaniline, are known to undergo photolysis with elimination of nitrogen forming azaepine side-products,47 FTIR was used as reaction control if the azide group


13

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

Page 14 of 41

was still present in the isolated product. The appearance of a signal at 2112 cm-1 indicates qualitatively the presence of an azide group that can be used for further click chemistry (Figure 3).

Acetalation of alkyne dextran (3). The introduction of acid-degradable acetals (Figure S-2) to cover the alcohol groups of the Dex-alkyne block turns the polysaccharide hydrophobic. By controlling the percentage of cyclic (slow degrading) or acyclic (fast degrading) acetals it is possible to install a solubility switch with a tunable time-dependent degradation rate.54 We adapted this concept for one block of our amphiphilic dextran copolymer. It was the goal to form self-assembled micellar nanoparticles that degrade and disassemble under acidic treatment. Therefore, it would be possible to revert the particle material to the original, fully water-soluble polysaccharides and release any encapsulated payload. Acetalation was carried out using a procedure established by the Fréchet group.33 The reaction of the alkyne-modified dextran with 2-methoxypropene produces a hydrophobic material that is only soluble in organic solvents, e.g. dichloromethane. The FTIR spectrum shows a significant decrease of the O-H vibration at 3583-3086 cm-1 after acetalation, confirming the consumption of free hydroxyl groups during the synthesis of compound 3 (Figure 3b). The proton NMR in DMSO-d6 also shows a new signal for the introduced acetals (Figure S-2, H-c, H-c’, 1.31-1.29 ppm). Cleaving off the acetals in D2O and DCl allows the calculation of the theoretical molecular weight (eq. S-1) and the amount and type of introduced acetals (cyclic or acyclic, Table S-1), based on the proton signals of the generated acetone (H-c, 2.13 ppm) and MeOH (H-d, 3.26 ppm) molecules (Figure 1). Coupling of hydrophilic with hydrophobic polysaccharide block: Dex-b-AcDex (4). We decided to use the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction for the synthesis of our block copolymer because of its high chemoselectivity and compatibility with


14

Page 15 of 41

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

Biomacromolecules

other functional groups, such as the hydroxyl groups in the polysaccharide backbone.16, 18, 20 Due to the different nature of both blocks, the reaction was carried out in DMSO, mediated with Cu(I)Br and PMDTA at 50 °C over 3 days. At higher temperatures, the triazole proton signal at 8.55 ppm became weaker and less product was formed, indicating the formation of side products. To ensure a full conversion of the higher modified azide-dextran block, we used 1.5 eq of the AcDex-alkyne block. Excess of unreacted AcDex was easily removed by precipitation in H2O-dd (pH 8 with TEA). Excess copper and unreacted dextran were removed with extensive dialysis over 3 days. The successful reaction and isolation of block copolymer 4 was confirmed with 1HNMR, FTIR and SEC. The coupled product carries the acetals in the hydrophobic block, as seen in the 1H-NMR spectra of Dex-b-AcDex in D2O (Figure 2a at 1.5 ppm and 3.2 ppm, H-g, h) in comparison to the spectra when treated with D2O/DCl (Figure 2b). Also, the aromatic protons of both aniline derivatives are visible at 7.00 ppm, 7.65 ppm and 7.75 ppm (Figure 2, H-a, a', b, b’, d, d’, e, e’). The successful click reaction was further confirmed by the formation of a triazole ring, resulting in the appearance of a singulett signal at 8.55 ppm (Figure 2, H-c). The disappearance of the azide peak at 2112 cm-1 in the FTIR spectrum (Figure 3) indicates a complete reaction of all azide-modified dextran. The lower retention time of copolymer 4 compared to the Dex-azide block 1 using aqueous size exclusion chromatography (Figure 4) supports the fact that a product with higher molecular weight was obtained. The hydrophobic block doesn’t dissolve in aqueous solution and can’t be compared under the same SEC conditions. The block copolymer did not elute at the same volume as a 10 kDa dextran standard. However, Zhang and Marchant pointed out that interactions between the hydrophobic segment of a block copolymer and the column matrix could lead to retardation in retention times not consistent with the molecular weight.52


15

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

Page 16 of 41

Figure 2. 1H-NMR spectra of Dex-b-AcDex block copolymer 4 in D2O (a) and D2O+DCl (b) (300 MHz). Spectrum a shows the characteristic signal of the acetal protons (H-g, H-h) as well as signals of both aromatic linker H-a-e' and the formed triazole proton H-c. Adding a few drops of DCl cleaves the acetals and forms methanol and acetone (H-i and H-j in spectrum b).


16

Page 17 of 41

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

Biomacromolecules

Figure 3. FTIR spectra of hydrophilic block Dex-N3 1 (a), hydrophobic block AcDex-alkyne 2 (b) and copolymer Dex-b-AcDex 4 (c). The azide group of Dex-N3 is visible at 2112 cm-1. The missing absorption from 3583 to 3086 cm-1 in the spectrum of the hydrophobic block AcDexalkyne 2 shows the consumption of hydroxyl groups after acetalation. The product Dex-b-AcDex 4 shows a mixture of the aromatic CH-vibration absorption band of Dex-N3 and AcDex-alkyne from 2982 to 2928 cm-1 and is missing the azide peak.

Figure 4. SEC elugrams of block copolymer Dex-b-AcDex (4) and the hydrophilic block DexN3 (1) in H2O with 0.1 M NaNO3. The block copolymer shows a smaller elution volume (8.67


17

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

Page 18 of 41

mL) than the single block Dex-N3 (8.87 mL) indicating an increase in molecular weight. Measurement of AcDex-alkyne (3) was not possible due to its insolubility in the elution solvent. The amphiphilic character of copolymer 4 was also supported by the fact, that already during the aqueous work-up after the click reaction the self-assembly of the material into micellar systems was observed using DLS and TEM (Table S-2, Figure S-4). The applied CuAAC click reaction can be a very powerful tool for an efficient bioorthogonal linkage of both polymer blocks. However one has to consider that residual metal catalyst can be toxic in micromolar concentrations to cells and organisms.55 Therefore, we carefully removed copper salts by extensive dialysis and verified the non-toxicity of compound 4 with a MTT assay (Figure 8). However, long-term diversification of our synthetic strategy will most likely include methods of copper-free click chemistries.51, 56

Self-assembly of Dex-b-AcDex micellar nanoparticles. Particles were prepared from lyophilized material using a standard solvent exchange method. By slowly removing the DMSO and increasing the polarity of the solvent, spherical nanoparticles formed in order to minimize the contact area of the hydrophobic region with the surrounding aqueous media. The obtained nanoparticles showed a narrow size distribution, an average hydrodynamic diameter (DH) of 69 nm for the largest particle population (by number) with a PdI of 0.107 and a neutral zeta potential (Table 1, Figure 5). The particle formation was investigated using dynamic light scattering (DLS). Considering an average chain length of about 27 nm for a fully stretched 10 kDa dextran57 our DLS results suggest the assembly of micellar nanoparticles, instead of ideal micelles. We postulate that the amphiphiles assemble into knot-like structures where the hydrophobic blocks entangle with several amphiphilic polymers to form a tightly packed core.


18

Page 19 of 41

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

Biomacromolecules

Electron microscopy (TEM) confirms the ability of the Dex-b-AcDex amphiphile to form micellar nanoparticles and shows filled spherical assemblies in the sub 100 nm range (Figure 5).

Table 1. Particle characterization of self-assembled Dex-b-AcDex particles (4) without (empty) and with cargo (cur-loaded). Z-Average (Z-Ave) describes the mean size of all particles in the sample; intensity describes the particle size distribution within the sample, depending on their scattering intensities; number represents the size of the particles forming the largest population in the sample.

particles

PdI

empty cur-loaded

0.107 0.098

Z-Ave (d.nm) 103.3 98.9

intensity (d.nm) 112.3 ± 39.38 109.8 ± 36.72

number (d.nm) 68.79 ± 21.31 68.71 ± 19.58

ζ-potential (mV) 2.83 ± 5.22 –6.11 ± 6.31


19

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

Page 20 of 41

Figure 5. DLS analysis and TEM images for empty micellar nanoparticles (A, top) and curcumin-loaded micellar nanoparticles (B, bottom). The self-assembled particles show an average size distribution (DH) of around 100 nm in diameter (69 d.nm for largest particle population by number). Curcumin-loaded nanoparticles show no significant change in size compared to empty particles. (Light gray artifacts in the background represent disassembled or unformed particle material, as a possible result of the TEM preparation) Self-assembly of micellar nanostructures is a dynamic process and micelle stability is strongly dependent on the amount of hydrophobic content within the amphiphile.58 The critical micelle concentration (CMC) is a good indicator for the particle stability. In general, lower CMC values suggest a higher thermodynamic stability of the particle system. To be useful for drug delivery application, micelles have to withstand high dilution in the blood stream after administration and therefore should have favorably a low CMC. The CMC of Dex-b-AcDex was determined to be


20

Page 21 of 41

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

Biomacromolecules

relatively low with a concentration of 12 mg·L-1 (Figure S-3). In comparison, Zhang et al. reported CMC values for AcDex-b-PEG around 82 mg·L-1 and 7.2 mg·L-1.16 Dex-PCL conjugates were reported with CMCs ranging from 6.8 to 50.4 mg·L-1 depending on the length of the hydrophobic block.44,

59

Modolon et al. reported a CMC value of 100 mg·l-1 for their full

oligosaccharide based amphiphile.28 This result shows, that our micellar nanoparticles show high stability compared to other reported saccharide-containing block copolymers.

Particle degradation under acidic conditions. A site-specific payload release is favorable for drug delivery applications. In particular, small pH changes can be utilized for a triggered drug release. Environments of slightly lower pH values (5-6) can be found for example in endosomal/lysosomal compartments within cells, but also in tumor tissue or sites of inflammation.12 To test the acid-degradability of our micellar system, we incubated our Dex-b-AcDex particles in sodium acetate buffer at pH 5.5 (Figure 6, vials B). Immediately measured (0 h) they show a maximum particle population at 70-80 d.nm with a narrow size distribution. After 2 h the micellar nanoparticles begin to lose their organized structure due to the cleavage of acetals and the associated loss of amphiphilic nature of the material. This can be visualized by the weakened intensity of back-scattered green laser light from the particle solution in vial B (Figure 6) and a slightly wider size distribution in DLS. In addition, the maximum particle diameter is shifting towards 50-60 nm. After 4 h, the size of the particles further decreases to 30-40 nm. At this point, the green laser beam is almost not visible anymore in vial B, due to the only limited amount of remaining particles that can scatter the laser light. After 24 h, only random aggregates smaller than 5 nm are visible in the DLS. This confirms that the acid-labile nanoparticle material


21

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

Page 22 of 41

turns water-soluble over time under slightly acidic conditions, causing a disassembly of the micellar structure. In comparison, particles incubated at pH 7 (samples A) are still stable after this time.

Figure 6. Degradation of Dex-b-AcDex micelles incubated in NaAc-buffer at pH 5.5 over 24 h, observed using DLS (top) and laser beam scattering (bottom). After 2 h, the particles in acidic conditions start to disassemble (vials B) resulting in smaller populations and show limited backscatter compared to particles incubated in neutral pH conditions (vials A). After 24 h full particle degradation can be observed in acidic conditions. Curcumin encapsulation in particles. To show that we can encapsulate and stabilize drugs in aqueous solution, we loaded the hydrophobic model compound curcumin into the core area of the micellar structures. Curcumin is a bright yellow diarylheptanoid compound and the main constituent of the turmeric plant (Curcuma longa, part of the ginger family). It is heavily discussed as possible natural product drug for various medical applications, including cancer


22

Page 23 of 41

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

Biomacromolecules

treatment or chronical diseases like cystic fibrosis.60,

61

Due to its poor water solubility,

encapsulation in nanoparticle carriers is necessary to achieve stabilization in aqueous environment and thus resulting in a higher bioavailability.62,

63

In our case, curcumin was

dissolved together with block copolymer 4 in DMSO and stirred overnight. After dialysis against H2O-dd, the formation of stable micelles was observed and the concentration of the curcuminloaded micelle solution determined to be 1.42 mg·mL-1. Particles with encapsulated drug were characterized with TEM and DLS (Figure 5, Table 1) but showed no significant increase in size compared to empty particles. The hydrophobic drug was encapsulated with a resulting loading ratio of 0.93 mol per mol carrier material (Figure S-5) (corresponding to an encapsulation efficiency of 23 % and a loading content of 3 %; see eq. S-2 and S-3). We compared, using the same dialysis technique, solutions of curcumin stabilized by the amphiphile (Figure 7, vial A) with attempts to dissolve pure curcumin in water (vial B). The aqueous suspension of pure curcumin shows no solubility of the hydrophobic drug and contains large visible aggregates at 0 h. After 24 h, vial B became clear with all curcumin precipitated on the bottom of the vial. In comparison, the polymer-stabilized solution (vial A) stays yellow and shows no precipitation. The long-term stability of the nano-formulation was confirmed (up to 4 months when storing in the fridge) (Figure 7).


23

Biomacromolecules

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

Page 24 of 41

Figure 7. Aqueous solution of curcumin stabilized by Dex-b-AcDex (A) and without copolymer addition (B). The micellar solutions (A) are stable for prolonged times (up to 4 months) compared to pure curcumin in water (B) that shows large aggregates and precipitates. To monitor the release of the hydrophobic model compound curcumin, we performed a dialysis experiment. A solution of freshly prepared curcumin-loaded nanoparticles was dialyzed against sodium acetate buffer at pH 5.5. The release of curcumin was very fast initially and leveled off over time (Figure S-6). After 24 h, approx. 95 % of the hydrophobic compound was released from the particles.

Toxicity of empty and drug loaded particles. The in vitro toxicity of empty and curcuminloaded micellar nanoparticles was evaluated by MTT assay (Figure 8). After 48 h, empty particles show no toxicity on HeLa cells up to high concentrations of 500 µg·mL-1. In comparison, curcumin-loaded particles show a concentration-dependent inhibition of cell growth starting at particle concentrations of around 250 µg·mL-1 (corresponding to a curcumin concentration of 20.41 µmol·L-1). These chemotherapeutic effects are comparable to literature known results using nanoparticles with curcumin concentrations of 10-30 µmol·L-1.64-66 Our in vitro experiments confirm the biocompatibility of the particle material and exemplify its potential as drug carrier with the successful stabilization and delivery of curcumin.


24

Page 25 of 41

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

Biomacromolecules

Figure 8. In vitro studies show no toxicity for empty Dex-b-AcDex micellar nanoparticles when incubated with HeLa cells for 48 h, whereas curcumin-loaded particles inhibit the growth of cancer cells.

CONCLUSION In this work, an amphiphilic, pH-responsive block copolymer composed only with polysaccharide blocks was synthesized using copper-mediated click chemistry. We optimized the conditions for a high end-group modification of both linear dextran blocks, combining microwave irradiation and aniline-mediated reductive amination methods. The resulting block copolymer has a low CMC and can self-assemble into micellar nanoparticles with a diameter of around 70 nm. The micelles are stable at neutral pH, but degrade under slightly acidic conditions. In vitro experiments confirm the biocompatibility of the fully polysaccharide particles. As a proof of concept for possible future therapeutic applications we showed, that the new biomaterial can stabilize the hydrophobic model drug curcumin in aqueous solutions. We think that fully polysaccharide-based block copolymers represent particular interesting systems, due to their high biocompatibility, degradability and low toxicity. In addition, different to many synthetic block copolymers, these natural biopolymers also offer remaining functional groups in the backbone of both blocks. This allows for a variety of modifications for future smart biopolymer materials


25

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

Page 26 of 41

with applications in drug delivery or surfactant chemistry. Ongoing studies focus on metal free ligation strategies, as well as on surface and core modifications of the polysaccharide micelles.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: FTIR of Dex-alkyne (2), 1H-NMR and acetal-content characterization of AcDex-alkyne (3), CMC plot, characterization and DLS plot of self-assembled particles during work up, data plot for curcumin loading efficiency.

AUTHOR INFORMATION Corresponding Author *Email: [email protected], Web: www.wichlab.com, Phone: +49 6131 39-25727 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (DFG) as part of the collaborative research center SFB 1066.


26

Page 27 of 41

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

Biomacromolecules

REFERENCES 1. Darling, S. B. Directing the self-assembly of block copolymers. Prog. Polym. Sci. 2007, 32 (10), 1152-1204. 2. Bockstaller, M. R.; Mickiewicz, R. A.; Thomas, E. L. Block Copolymer Nanocomposites: Perspectives for Tailored Functional Materials. Adv. Mater. 2005, 17 (11), 1331-1349. 3. Wang, Y.; Xu, H.; Ma, N.; Wang, Z.; Zhang, X.; Liu, J.; Shen, J. Block copolymer micelles as matrixes for incorporating diselenide compounds: a model system for a water-soluble glutathione peroxidase mimic fine-tuned by ionic strength. Langmuir 2006, 22 (13), 5552-5555. 4. Qu, R.; Shen, L.; Chai, Z.; Jing, C.; Zhang, Y.; An, Y.; Shi, L. Hemin-block copolymer micelle as an artificial peroxidase and its applications in chromogenic detection and biocatalysis. ACS Appl. Mater. Interfaces 2014, 6 (21), 19207-19216. 5. Zhang, N.; Wardwell, P. R.; Bader, R. A. Polysaccharide-based micelles for drug delivery. Pharmaceutics 2013, 5 (2), 329-352. 6. Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Biodegradable block copolymers as injectable drug-delivery systems. Nature 1997, 388 (6645), 860-862. 7. Petrelli, A.; Borsali, R.; Fort, S.; Halila, S. Redox tunable delivery systems: sweet block copolymer micelles via thiol-(bromo)maleimide conjugation. Chem. Commun. 2016, 52 (82), 12202-12205. 8. Cohen, J. A.; Beaudette, T. T.; Cohen, J. L.; Broaders, K. E.; Bachelder, E. M.; Frechet, J. M. Acetal-modified dextran microparticles with controlled degradation kinetics and surface functionality for gene delivery in phagocytic and non-phagocytic cells. Adv. Mater. 2010, 22 (32), 3593-3597. 9. Gauche, C.; Soldi, V.; Fort, S.; Borsali, R.; Halila, S. Xyloglucan-based diblock co-oligomer: synthesis, self-assembly and steric stabilization of proteins. Carbohydr. Polym. 2013, 98 (2), 1272-1280. 10. Kampmann, A.-L.; Luksin, M.; Pretzer, I.; Weberskirch, R. Formation of Well-Defined Polymer Particles in the Sub-100 nm Size Range by Using Amphiphilic Block Copolymer Surfactants and a Microemulsion Approach. Macromol. Chem. Phys. 2016, 217 (15), 1704-1711. 11. Goodwin, A. P.; Tabakman, S. M.; Welsher, K.; Sherlock, S. P.; Prencipe, G.; Dai, H. Phospholipid-dextran with a single coupling point: a useful amphiphile for functionalization of nanomaterials. J. Am. Chem. Soc. 2009, 131 (1), 289-296. 12. Kato, Y.; Ozawa, S.; Miyamoto, C.; Maehata, Y.; Suzuki, A.; Maeda, T.; Baba, Y. Acidic extracellular microenvironment and cancer. Cancer Cell Int. 2013, 13 (1), 89. 13. Tang, S.; Yin, Q.; Zhang, Z.; Gu, W.; Chen, L.; Yu, H.; Huang, Y.; Chen, X.; Xu, M.; Li, Y. Co-delivery of doxorubicin and RNA using pH-sensitive poly (beta-amino ester) nanoparticles for reversal of multidrug resistance of breast cancer. Biomaterials 2014, 35 (23), 6047-6059. 14. Lee, E. S.; Oh, K. T.; Kim, D.; Youn, Y. S.; Bae, Y. H. Tumor pH-responsive flower-like micelles of poly(L-lactic acid)-b-poly(ethylene glycol)-b-poly(L-histidine). J. Controlled Release 2007, 123 (1), 19-26. 15. Yin, H.; Lee, E. S.; Kim, D.; Lee, K. H.; Oh, K. T.; Bae, Y. H. Physicochemical characteristics of pH-sensitive poly(L-histidine)-b-poly(ethylene glycol)/poly(L-lactide)-bpoly(ethylene glycol) mixed micelles. J. Controlled Release 2008, 126 (2), 130-138. 16. Zhang, Z.; Chen, X.; Chen, L.; Yu, S.; Cao, Y.; He, C.; Chen, X. Intracellular pH-sensitive PEG-block-acetalated-dextrans as efficient drug delivery platforms. ACS Appl. Mater. Interfaces 2013, 5 (21), 10760-10766.


27

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

Page 28 of 41

17. Kuang, H.; Wu, Y.; Zhang, Z.; Li, J.; Chen, X.; Xie, Z.; Jing, X.; Huang, Y. Double pHresponsive supramolecular copolymer micelles based on the complementary multiple hydrogen bonds of nucleobases and acetalated dextran for drug delivery. Polym. Chem. 2015, 6 (19), 36253633. 18. Li, Q.; Liu, W.; Dai, J.; Zhang, C. Synthesis of polysaccharide-block-polypeptide copolymer for potential co-delivery of drug and plasmid DNA. Macromol. Biosci. 2015, 15 (6), 756-764. 19. Schatz, C.; Lecommandoux, S. Polysaccharide-containing block copolymers: synthesis, properties and applications of an emerging family of glycoconjugates. Macromol. Rapid Commun. 2010, 31 (19), 1664-1684. 20. Fajardo, A. R.; Guerry, A.; Britta, E. A.; Nakamura, C. V.; Muniz, E. C.; Borsali, R.; Halila, S. Sulfated glycosaminoglycan-based block copolymer: preparation of biocompatible chondroitin sulfate-b-poly(lactic acid) micelles. Biomacromolecules 2014, 15 (7), 2691-2700. 21. Ryno, L. M.; Reese, C.; Tolan, M.; O'Brien, J.; Short, G.; Sorriano, G.; Nettleton, J.; Fulton, K.; Iovine, P. M. Amphiphilic graft copolymers from end-functionalized starches: synthesis, characterization, thin film preparation, and small molecule loading. Biomacromolecules 2014, 15 (8), 2944-2951. 22. Ganji, F.; Abdekhodaie, M. J. Synthesis and characterization of a new thermosensitive chitosan-PEG diblock copolymer. Carbohydr. Polym. 2008, 74 (3), 435-441. 23. Upadhyay, K. K.; Le Meins, J. F.; Misra, A.; Voisin, P.; Bouchaud, V.; Ibarboure, E.; Schatz, C.; Lecommandoux, S. Biomimetic doxorubicin loaded polymersomes from hyaluronanblock-poly(gamma-benzyl glutamate) copolymers. Biomacromolecules 2009, 10 (10), 28022808. 24. Verma, M. S.; Gu, F. X. Microwave-enhanced reductive amination via Schiff's base formation for block copolymer synthesis. Carbohydr. Polym. 2012, 87 (4), 2740-2744. 25. Guerry, A.; Bernard, J.; Samain, E.; Fleury, E.; Cottaz, S.; Halila, S. Aniline-catalyzed reductive amination as a powerful method for the preparation of reducing end-"clickable" chitooligosaccharides. Bioconjugate Chem. 2013, 24 (4), 544-549. 26. Schatz, C.; Louguet, S.; Le Meins, J. F.; Lecommandoux, S. Polysaccharide-blockpolypeptide copolymer vesicles: towards synthetic viral capsids. Angew. Chem. Int. Ed. Engl. 2009, 48 (14), 2572-2575. 27. Zhang, A.; Zhang, Z.; Shi, F.; Xiao, C.; Ding, J.; Zhuang, X.; He, C.; Chen, L.; Chen, X. Redox-sensitive shell-crosslinked polypeptide-block-polysaccharide micelles for efficient intracellular anticancer drug delivery. Macromol. Biosci. 2013, 13 (9), 1249-1258. 28. de Medeiros Modolon, S.; Otsuka, I.; Fort, S.; Minatti, E.; Borsali, R.; Halila, S. Sweet block copolymer nanoparticles: preparation and self-assembly of fully oligosaccharide-based amphiphile. Biomacromolecules 2012, 13 (4), 1129-1135. 29. Kamitakahara, H.; Nakatsubo, F.; Klemm, D. New class of carbohydrate-based nonionic surfactants: diblock co-oligomers of tri-O-methylated and unmodified cello-oligosaccharides. Cellulose 2007, 14 (5), 513-528. 30. Ioan, C. E.; Aberle, T.; Burchard, W. Structure properties of dextran. 2. Dilute solution. Macromolecules 2000, 33 (15), 5730-5739. 31. Yi, L.; Ouyang, Y.; Sun, X.; Xu, N.; Linhardt, R. J.; Zhang, Z. Qualitative and quantitative analysis of branches in dextran using high-performance anion exchange chromatography coupled to quadrupole time-of-flight mass spectrometry. J. Chromatogr. A 2015, 1423, 79-85.


28

Page 29 of 41

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

Biomacromolecules

32. Lin, Z.; Li, J. Z.; He, H. Y.; Kuang, H. H.; Chen, X. S.; Xie, Z. G.; Jing, X. B.; Huang, Y. B. Acetalated-dextran as valves of mesoporous silica particles for pH responsive intracellular drug delivery. RSC Adv. 2015, 5 (13), 9546-9555. 33. Bachelder, E. M.; Beaudette, T. T.; Broaders, K. E.; Dashe, J.; Frechet, J. M. Acetalderivatized dextran: an acid-responsive biodegradable material for therapeutic applications. J. Am. Chem. Soc. 2008, 130 (32), 10494-10495. 34. Mehvar, R. Dextrans for targeted and sustained delivery of therapeutic and imaging agents. J. Controlled Release 2000, 69 (1), 1-25. 35. Cees J De Groota, M. J. A. V. L., Welmoed N.E Van Dijk-Wolthuisc, Jenny A Cadéec, Josée A Plantingab, Willem Den Ottera, Wim E Henninkc. In vitro biocompatibility of biodegradable dextran-based hydrogels tested with human fibroblasts. Biomaterials 2001, 22 (11), 1197-1203. 36. Cui, L.; Cohen, J. L.; Chu, C. K.; Wich, P. R.; Kierstead, P. H.; Frechet, J. M. Conjugation chemistry through acetals toward a dextran-based delivery system for controlled release of siRNA. J. Am. Chem. Soc. 2012, 134 (38), 15840-15848. 37. Bachelder, E. M.; Pino, E. N.; Ainslie, K. M. Acetalated Dextran: A Tunable and AcidLabile Biopolymer with Facile Synthesis and a Range of Applications. Chem. Rev. 2017, 117 (3), 1915-1926. 38. Ornelas-Megiatto, C.; Shah, P. N.; Wich, P. R.; Cohen, J. L.; Tagaev, J. A.; Smolen, J. A.; Wright, B. D.; Panzner, M. J.; Youngs, W. J.; Frechet, J. M.; Cannon, C. L. Aerosolized antimicrobial agents based on degradable dextran nanoparticles loaded with silver carbene complexes. Mol. Pharmaceutics 2012, 9 (11), 3012-3022. 39. Cohen, J. L.; Shen, Y.; Aouadi, M.; Vangala, P.; Tencerova, M.; Amano, S. U.; Nicoloro, S. M.; Yawe, J. C.; Czech, M. P. Peptide- and Amine-Modified Glucan Particles for the Delivery of Therapeutic siRNA. Mol. Pharmaceutics 2016, 13 (3), 964-978. 40. Cohen, J. L.; Schubert, S.; Wich, P. R.; Cui, L.; Cohen, J. A.; Mynar, J. L.; Frechet, J. M. Acid-degradable cationic dextran particles for the delivery of siRNA therapeutics. Bioconjugate Chem. 2011, 22 (6), 1056-1065. 41. Duong, H. T.; Hughes, F.; Sagnella, S.; Kavallaris, M.; Macmillan, A.; Whan, R.; Hook, J.; Davis, T. P.; Boyer, C. Functionalizing biodegradable dextran scaffolds using living radical polymerization: new versatile nanoparticles for the delivery of therapeutic molecules. Mol. Pharmaceutics 2012, 9 (11), 3046-3061. 42. Stanciu, M. C.; Nichifor, M. New biocompatible amphiphilic diblock copolymer based on dextran. Eur. Polym. J. 2015, 71, 352-363. 43. Mocanu, G.; Nichifor, M.; Stanciu, M. C. New shell crosslinked micelles from dextran with hydrophobic end groups and their interaction with bioactive molecules. Carbohydr. Polym. 2015, 119, 228-235. 44. Lee, R. S.; Li, Y. C.; Wang, S. W. Synthesis and characterization of amphiphilic photocleavable polymers based on dextran and substituted-varepsilon-caprolactone. Carbohydr. Polym. 2015, 117, 201-210. 45. Nichifor, M.; Mocanu, G.; Stanciu, M. C. Micelle-like association of polysaccharides with hydrophobic end groups. Carbohydr. Polym. 2014, 110, 209-218. 46. Chen, S.; Alves, M.-H.; Save, M.; Billon, L. Synthesis of amphiphilic diblock copolymers derived from renewable dextran by nitroxide mediated polymerization: towards hierarchically structured honeycomb porous films. Polym. Chem. 2014, 5 (18), 5310-5319.


29

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

Page 30 of 41

47. Sundberg, R. J.; Suter, S. R.; Brenner, M. Photolysis of 0-substituted aryl azides in diethylamine. Formation and autoxidation of 2-diethylamino-1H-azepine intermediates. J. Am. Chem. Soc. 1972, 94 (2), 513-520. 48. Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays Journal of lmmunological Methods 1983, 65, 55-63. 49. Zhang, Y. L.; Dou, X. W.; Jin, T. Synthesis and self-assembly behavior of amphiphilic diblock copolymer dextran-block-poly(ε-caprolactone) (DEX-b-PCL) in aqueous media. eXPRESS Polym. Lett. 2010, 4 (10), 599-610. 50. Bondalapati, S.; Ruvinov, E.; Kryukov, O.; Cohen, S.; Brik, A. Rapid End-Group Modification of Polysaccharides for Biomaterial Applications in Regenerative Medicine. Macromol. Rapid Commun. 2014, 35, 1754-1762. 51. Novoa-Carballal, R.; Muller, A. H. Synthesis of polysaccharide-b-PEG block copolymers by oxime click. Chem. Commun. 2012, 48 (31), 3781-3783. 52. Zhang, T.; Marchant, R. E. Novel Polysaccharide Surfactants: Synthesis of Model Compounds and Dextran-Based Surfactants. Macromolecules 1994, 27 (25), 7302-7308. 53. Yalpani, M.; Brooks, D. E. Selective chemical modifications of dextran. Journal of Polymer Science, Polymer Chemistry Edition 1985, 23 (5), 1395-1405. 54. Broaders, K. E.; Cohen, J. A.; Beaudette, T. T.; Bachelder, E. M.; Frechet, J. M. Acetalated dextran is a chemically and biologically tunable material for particulate immunotherapy. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (14), 5497-5502. 55. Egorova, K. S.; Ananikov, V. P. Which Metals are Green for Catalysis? Comparison of the Toxicities of Ni, Cu, Fe, Pd, Pt, Rh, and Au Salts. Angew. Chem. Int. Ed. Engl. 2016, 55 (40), 12150-12162. 56. Becer, C. R.; Hoogenboom, R.; Schubert, U. S. Click chemistry beyond metal-catalyzed cycloaddition. Angew. Chem. Int. Ed. Engl. 2009, 48 (27), 4900-4908. 57. Matthias Rief, F. O., Berthold Heymann and Hermann E. Gaub. Single Molecule Force Spectroscopy on Polysaccharides by Atomic Force Microscopy. Science 1997, 275, 1295-1297. 58. Zhang, T. H.; Marchant, R. E. Novel polysaccharide surfactants: The effect of hydrophobic and hydrophilic chain length on surface active properties. J. Colloid Interface Sci. 1996, 177 (2), 419-426. 59. Sun, H.; Guo, B.; Li, X.; Cheng, R.; Meng, F.; Liu, H.; Zhong, Z. Shell-sheddable micelles based on dextran-SS-poly(epsilon-caprolactone) diblock copolymer for efficient intracellular release of doxorubicin. Biomacromolecules 2010, 11 (4), 848-854. 60. Maheshwari, R. K.; Singh, A. K.; Gaddipati, J.; Srimal, R. C. Multiple biological activities of curcumin: a short review. Life Sci. 2006, 78 (18), 2081-2087. 61. Egan, M. E.; Pearson, M.; Weiner, S. A.; Rajendran, V.; Rubin, D.; Glockner-Pagel, J.; Canny, S.; Du, K.; Lukacs, G. L.; Caplan, M. J. Curcumin, a major constituent of turmeric, corrects cystic fibrosis defects. Science 2004, 304 (5670), 600-602. 62. Jiang, Y.; Liang, M.; Svejkar, D.; Hart-Smith, G.; Lu, H.; Scarano, W.; Stenzel, M. H. Albumin-micelles via a one-pot technology platform for the delivery of drugs. Chem. Commun. 2014, 50 (48), 6394-6397. 63. Ma, Z.; Haddadi, A.; Molavi, O.; Lavasanifar, A.; Lai, R.; Samuel, J. Micelles of poly(ethylene oxide)-b-poly(epsilon-caprolactone) as vehicles for the solubilization, stabilization, and controlled delivery of curcumin. J. Biomed. Mater. Res., Part A 2008, 86 (2), 300-310.


30

Page 31 of 41

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

Biomacromolecules

64. Sahu, A.; Bora, U.; Kasoju, N.; Goswami, P. Synthesis of novel biodegradable and selfassembling methoxy poly(ethylene glycol)-palmitate nanocarrier for curcumin delivery to cancer cells. Acta Biomater. 2008, 4 (6), 1752-1761. 65. Mohanty, C.; Acharya, S.; Mohanty, A. K.; Dilnawaz, F.; Sahoo, S. K. Curcuminencapsulated MePEG/PCL diblock copolymeric micelles: a novel controlled delivery vehicle for cancer therapy. Nanomedicine (Lond) 2010, 5 (3), 433-449. 66. Sun, J.; Bi, C.; Chan, H. M.; Sun, S.; Zhang, Q.; Zheng, Y. Curcumin-loaded solid lipid nanoparticles have prolonged in vitro antitumour activity, cellular uptake and improved in vivo bioavailability. Colloids Surf., B 2013, 111, 367-375.


31

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

Synthetic route to the amphiphilic Dex-b-AcDex block copolymer (4) by Cu(I)-mediated click reaction of a hydrophilic azide-functionalized dextran block (1) with a hydrophobic alkyne-functionalized acetalated dextran block (3). 92x37mm (300 x 300 DPI)


Page 32 of 41

Page 33 of 41

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

Biomacromolecules

Comparing the 1H-NMR spectra of Dex (a), Dex-N3 1 (b) and Dex-alkyne 2 (c) (300 MHz, D2O) allows the quantification of end group modification by integrating the ratio of introduced aromatic protons H-a,a'/H-b,b' and the anomeric H-c of the dextran. Molecular weight of hydrophobic block AcDex-alkyne 3 (d) can be calculated from the 1H-NMR signal relation of generated acetone and methanol, resulting from cleaved off acetals after acidic treatment (300 MHz, D2O, DCl). 127x90mm (300 x 300 DPI)


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

1

H-NMR spectra of Dex-b-AcDex block copolymer 4 in D2O (a) and D2O+DCl (b) (300 MHz). Spectrum a shows the characteristic signal of the acetal protons (H-g, H-h) as well as signals of both aromatic linker Ha-e' and the formed triazole proton H-c. Adding a few drops of DCl cleaves the acetals and forms methanol and acetone (H-i and H-j in spectrum b). 209x176mm (300 x 300 DPI)


Page 34 of 41

Page 35 of 41

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

Biomacromolecules

FTIR spectra of hydrophilic block Dex-N3 1 (a), hydrophobic block AcDex-alkyne 2 (b) and copolymer Dexb-AcDex 4 (c). The azide group of Dex-N3 is visible at 2112 cm-1. The missing absorption from 3583 to 3086 cm-1 in the spectrum of the hydrophobic block AcDex-alkyne 2 shows the consumption of hydroxyl groups after acetalation. The product Dex-b-AcDex 4 shows a mixture of the aromatic CH-vibration absorption band of Dex-N3 and AcDex-alkyne from 2982 to 2928 cm-1 and is missing the azide peak. 190x165mm (300 x 300 DPI)


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

SEC elugrams of block copolymer Dex-b-AcDex (4) and the hydrophilic block Dex-N3 (1) in H2O with 0.1 M NaNO3. The block copolymer shows a smaller elution volume (8.67 mL) than the single block Dex-N3 (8.87 mL) indicating an increase in molecular weight. Measurement of AcDex-alkyne (3) was not possible due to its insolubility in the elution solvent. 949x752mm (72 x 72 DPI)


Page 36 of 41

Page 37 of 41

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

Biomacromolecules

DLS analysis and TEM images for empty micellar nanoparticles (A, top) and curcumin-loaded micellar nanoparticles (B, bottom). The self-assembled particles show an average size distribution (DH) of around 100 nm in diameter (69 d.nm for largest particle population by number). Curcumin-loaded nanoparticles show no significant change in size compared to empty particles. (Light gray artifacts in the background represent disassembled or unformed particle material, as a possible result of the TEM preparation) 106x137mm (600 x 600 DPI)


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

Degradation of Dex-b-AcDex micelles incubated in NaAc-buffer at pH 5.5 over 24 h, observed using DLS (top) and laser beam scattering (bottom). After 2 h, the particles in acidic conditions start to disassemble (vials B) resulting in smaller populations and show limited backscatter compared to particles incubated in neutral pH conditions (vials A). After 24 h full particle degradation can be observed in acidic conditions. 1011x782mm (72 x 72 DPI)


Page 38 of 41

Page 39 of 41

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

Biomacromolecules

Aqueous solution of curcumin stabilized by Dex-b-AcDex (A) and without copolymer addition (B). The micellar solutions (A) are stable for prolonged times (up to 4 months) compared to pure curcumin in water (B) that shows large aggregates and precipitates. 1028x328mm (72 x 72 DPI)


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

In vitro studies show no toxicity for empty Dex-b-AcDex micellar nanoparticles when incubated with HeLa cells for 48 h, whereas curcumin-loaded particles inhibit the growth of cancer cells. 967x662mm (72 x 72 DPI)


Page 40 of 41

Page 41 of 41

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

Biomacromolecules

1057x427mm (72 x 72 DPI)


Amphiphilic Polysaccharide Block Copolymers for pH-Responsive

Recommend Documents