Squalenoylation of Chitosan: A Platform for Drug Delivery

Page 1 of 32

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

Squalenoylation of Chitosan: a Platform for DrugDelivery? Elise Lepeltier1,*, Brigitta Loretz1, Didier Desmaële2, Josef Zapp3, Jennifer Herrmann4, Patrick Couvreur2, Claus-Michael Lehr1,5 1

Drug Delivery (DDEL), Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),

Helmholtz Centre for Infection Research (HZI), Saarland University, 66123, Germany 2

Faculté de Pharmacie, Institut Galien Paris Sud, Université Paris Sud, UMR CNRS 8612,

92296, France 3

Institut für Pharmazeutische Biologie, Saarland University, 66123, Germany

4

Microbial Natural Products, Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),

Helmholtz Centre for Infection Research (HZI), Saarland University, 66123, Germany 5

Department of Pharmacy, Saarland University, Saarbrücken, Germany

KEYWORDS: squalenoylation, chitosan derivatives, nanoparticles, self-assembly The present study describes the synthesis of chitosan-squalene (chitosan-SQ), a unique amphiphilic chitosan derivative, enabling the efficient formation of nanoparticles in acetate buffer by self-assembly. The influence of different parameters on the nanoparticle size such as percentage of substitution, pH of the acetate buffer, concentration in chitosan-SQ and time of stirring was studied. It could be demonstrated that this new polymer was non-toxic to cells,

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 32

biodegradable and preserved the anti-infective properties of the initial chitosan.1 Moreover, chitosan-SQ showed good carrier properties allowing the encapsulation of both hydrophilic and hydrophobic model drug compounds.

1. Introduction Nanoparticles emerged over the last decade as a major research initiative for drug delivery2-4: dimension comparable to microorganisms (between bacteria and virus), protection of the drug from degradation, higher bioavailability by facilitating transport across epithelial barriers and modifying pharmacokinetics, biodistribution profile, increase of cellular internalization and finally even the possibility of active targeting by surface functionalization of such carriers. To summarize, nanoparticles are able to increase the efficacy of the drug and to reduce its toxicity. However, despite the impressive blooming of interest in the research field of nanomedicine over the last decades, only few are on the market.5, 6 One serious limitation is the lack of adequate materials to make such nanocarriers, which must be biocompatible, biodegradable and easily manufactured in high purity and large scale. The core idea of this work was to explore nanoparticulate drug carrier based on a derivative of chitosan. This natural, biodegradable and strongly positively charged polysaccharide has been reported to interact with the bacterial cell wall as well as with biofilms,1, 7 thus adding antiinfective properties to its interesting spectrum of multifunctional features, including bioadhesion and penetration enhancement. Because of its cationic charges and its bioadhesivity, administration to mucosal epithelia in particular of the respiratory tract (e.g. nose, lung) is probably favored.8-10


2

Page 3 of 32

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

Ionic gelation with sodium tripolyphosphate (TPP) or complex coacervation are frequently reported in the literature for the preparation of drug-loaded chitosan nanoparticles.11-16 However, the main problems of these techniques are the colloidal stability of such suspensions (addition of stabilizers may be necessary) and the need of using very dilute solutions. Chitosan can be also chemically modified in order to become an amphiphilic compound with the aim to obtain nanoparticles by simple self-assembly: linoleic acid-modified chitosan,17, 18 glycol chitosan bearing 5-cholanic acid19, 20 or oleoyl-chitosan21 have been already reported. In these cases, the modified chitosan nanoparticles were prepared by O/W emulsion method17, 18, 21 or by suspending the amphiphilic polymer in a phosphate-buffered saline solution under gentle shaking for 6h, followed by sonication and filtration.19, 20 In line with the “squalenoylation” concept discovered by Couvreur´s group22, we chose to covalently bind squalene (SQ) to chitosan. Squalene is a biomolecule, precursor of cholesterol and it has been previously shown to render self-assembling properties, enabling the formation of nanoparticles, when covalently bound to some hydrophilic drug molecules (siRNA, nucleosides, penicillin…).22 Squalenoyl chitosan was synthesized in a single-step reaction, with a well-defined degree of substitution. Interestingly enough, we observed that the thus obtained polymer spontaneously formed particles, by just adding the chitosan-SQ in an acetate buffer under stirring during 1h. In such way, stable particles were obtained in one step and without using any organic solvent. The self-assembly was observed even with only ~1% of squalenoylation. Several model drugs were also encapsulated in this new polymer and quantified: hydrophilic compounds (caffeine, fluorescein), hydrophobic compound (Nile red) and a macromolecule (pDNA). The new


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 32

chitosan-SQ showed the same favorable toxicity as plain chitosan and therefore appeared to hold interesting potential for the design of nanomedicines. 2. Experimental Section Material. Ultrapure chitosan UPCL 114 (purchased from Novamatrix) with a mass average molar mass MW = 90 kDa, ĐM = 2.8 and a deacetylation percentage > 90% was used in all the experiments. This material was analysed by GPC ( HLC-8320GPC, Tosoh, Japan), equipped with an online viscometer (ETA-2010, PSS, Germany), on SUPREMA 1000 and 30 columns (PSS) at a flow rate of 1 mL/min at 35 °C in 1 M sodium nitrate and Pullulan standards were used for the universal calibration. Purified water is produced by Milli-Q water purification system (Merk Millipore, Billerica, MA). Acetic acid, ethanol, sodium cyanoborohydride, sodium hydroxide, acetic acid-d4 and sulfuric acid-d2 solution (96-98 wt. % in D2O), caffeine, fluorescein sodium salt and Nile red were purchased from Sigma-Aldrich. pUC 18 plasmid DNA was obtained by transfection of E. coli XL-1 cells with pUC 18 for propagation and isolated using the QIAGEN Plasmid Mini Kit, Qiagen Inc., CA, USA. The quantity and quality of purified pDNA were evaluated by spectrophotometric analysis at 260 and 280 nm. The molecular weights markers (1kb DNA ladder) were purchased from PEQLAB Biotechnologie GmbH. A549 cells No. ACC 107 were purchased from DSMZ GmbH (Braunschweig, Germany), L929 cells No ATCC-CCL-1 were purchased from ATCC. Cell culture medium (RPMI 1640) was purchased from PAA Laboratories GmbH (Pasching, Austria), and fetal bovine serum (FBS) was purchased from Lonza (Basel, Switzerland).

Synthesis of the 1,1’,2-trisnorsqualenaldehyd. A solution of 2,3-oxidosqualene23 (3.73 g, 8.7 mmol) in diethyl ether (20 mL) was added dropwise to a suspension of periodic acid hydrate


4

Page 5 of 32

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

(2.37g ,10.4 mmol) in 150 mL of diethyl ether. The reaction mixture was stirred for 1 h at room temperature. The mixture was filtered and the solid was thoroughly washed with diethyl ether. The filtrate was treated with brine (20 mL) and the phases were separated. The aqueous phase was extracted with diethyl ether (450 mL). The combined organic layers were dried over MgSO4 filtered and concentrated under reduced pressure. The crude aldehyde was purified by chromatography over silica gel eluting cyclohexane/ethyl acetate 4:1 to give trisnorsqualenaldehyde (2.77 g, 83 % yield) as a colorless oil. IR (neat)  : 2924-2853, 2750-2700, 1728 cm-1; 1H NMR (CDCl3, 300 MHz), δ 9.74 (1H, t, J = 1,8 Hz, CHO), 5.16-5.07 (5H, m, =CH), 2.50 (2H, td, J = 7.3 Hz, J = 1.8 Hz, OHCCH2), 2.31 (2H, t, J = 7.3 Hz, OHCCH2CH2), 2.08-1.98 (16H, m, =CHCH2CH2C(CH3)), 1.67 (3H, s, CH3), 1.61 (3H, s, CH3), 1.60 (12H, s, CH3) ppm; 13C NMR (CDCl3, 75 MHz); δ 202.6 (CH, CHO), 135.1 (1C, =C(CH3)), 134.8 (1C, =C(CH3)), 134.7 (1C, =C(CH3)), 132.8 (1C, =C(CH3)), 131.1 (1C, =C(CH3)), 125.4 (1CH, HC=C(CH3)), 124.5 (1CH, HC=C(CH3)),124.4 (1CH, HC=C(CH3)), 124.2 (2CH, HC=C(CH3)), 42.1 (1CH2), 39.7 (2CH2), 39.5 (1CH2), 31.9 (1CH2), 28.3 (2CH2), 26.,7 (1CH2), 26.6 (1CH2), 26.5 (1CH2), 25.6 (1CH3, HC=C(CH3)2), 17.6 (1CH3, HC=C(CH3)CH2), 16.1 (1CH3, HC=C(CH3)CH2), 16.0 (1CH3, HC=C(CH3)CH2), 15.9 (2CH3, HC=C(CH3)CH2) ppm.

Synthesis of the chitosan-SQ. The chitosan derivatives were obtained by reductive amination adapting the procedure described by Desbrières.24 Fifty milligrams of chitosan were dissolved in 3 mL of 0.2 M acetic acid (AcOH). After complete dissolution, 2 mL of ethanol (EtOH) were slowly added. The pH was then adjusted to 5 (with NaOH 1 M solution) to avoid the precipitation of the macromolecule, the optimal reaction pH range being between 4 and 6. Four different starting molar ratio of aldehyde-SQ were used: 2:1, 9:1, 42:1 and 109:1. The


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 32

corresponding amount of aldehyde was dissolved in 0.5 mL of EtOH and added dropwise to the chitosan solution. An excess of sodium cyanoborohydride NaCNBH3 (1.5 moles per glucosamine unit) was then added to reduce the intermediate imine. The mixture was stirred for 24 h at room temperature and the alkylated chitosan was precipitated with EtOH (30 mL). The gel-like precipitate was washed with EtOH/water mixtures with increasing EtOH content from 70% v/v to 100% and recovered by centrifugation at 10000 rpm for 15 min (at RT). The compound was finally dried overnight in a vacuum oven at 25 °C and then dissolved in a large amount of water with some acetic acid (to remove the EtOH) and freeze-dried during 2 days (Alpha 2-4, Martin Christ GmbH, Osterode, Germany) to give the squalenyl chitosan derivatives (45 ± 3 mg, 81 ± 5% yield).

Chemical characterization. Infra-red spectra were recorded in solid state with a Spectrum 400 FT-IR/FT-NIR spectrometer (PerkinElmer). NMR measurements were performed on a Bruker Fourier 300 (trisnorsqualenaldehyde) or a Bruker Avance III 500 equipped with a TCI cryo probe (polymers). Solvents were CDCl3 (trisnorsqualenaldehyde) or D2O with 5% (v/v) acetic acid-d4 and sulfuric acid-d2 (polymers). Measurements in aqueous solution run with a presat water suppression sequence. Elemental analysis was determined using a Leco CHN-900 analyzer. The final molar ratios (Glucosamine:SQ) have been calculated from the degree of substitution %DS, according to this formula: %𝐷𝑆 =

𝑛𝐶 ⁄𝑛𝑁 − 6 × 100 33 − 6

nC/nN = 6 if there is no SQ coupled to the glucosamine unit. nC/nN = 33 if there is one SQ coupled to each glucosamine unit.


6

Page 7 of 32

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

Nanoparticles preparation. The chitosan-SQ compounds with different final molar ratio 3:1, 13:1, 46:1 and 52:1 were added under stirring (400 rpm) in 2 mL of acetate buffer at pH = 4, 5.3, 6.5 or in water. The concentration was of 1 mg/mL most of cases, 5 mg/mL and 10 mg/mL were used to study the influence of the concentration on the size. The time of stirring was 1 hour, excepted when it was pointed out.

Dynamic Light Scattering (DLS) and Zeta Potential Measurements. The characterization (size, Polydispersity Index (PDI) for standard deviation of size and zeta potential) of particles was studied by Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, U.K.) equipped with a 4 mW He−Ne laser employing a wavelength of 633 nm and a backscattering angle of 173° at 25 °C. The reported size is the z-average diameter (intensity based) of 3 measurements.

SEM. 5 L of each chitosan-SQ suspension (1 mg/mL) were deposited on carbon discs (12 mm) mounted on pin stubs (12 mm) and dried overnight. As a reference, chitosan solution (1 mg/mL) was also observed. Images were obtained on a Zeiss Evo HD 15 scanning electron microscope (5 kV, Secondary Electrons detector, Göttingen, Germany) and were representative of the sample visualized.

MTT and LDH assays. LDH and MTT assay were performed to study the in vitro cytotoxicity of the chitosan-SQ 52:1 and 46:1 nanoparticles on two model cell lines. The A549 and L929 cells were seeded in a 96-wells plate at a density of 10 000 cells per well in 200 μL of cell culture medium: liquid cultured condition (LCC). Once confluent, nanoparticles in acetate buffer


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 32

pH = 5.3 were applied to the cells and incubated for 4 h at 37 °C with gently shaking. As reference, HBSS in cell medium (negative control, 0% of cytotoxicity), pH = 5.3 acetate buffer with cell medium as “vehicle control” (1/3 of acetate buffer, 2/3 of cell medium) and 0.1% (w/w) Triton X-100 solution (ICN, Eschwege, Germany, positive control, 100% of cytotoxicity) in HBSS buffer were applied. To ensure the no interference of nanoparticles to the test system, nanoparticles without any cells were also tested. All the experiments were conducted in duplicate, with n = 6 per sample. LDH Assay. After 4 h incubation of the above-mentioned 96-well plates, 100 μL of the supernatant was transferred to another 96-well plate. The 100 μL of LDH test solution (Roche Cytotoxicity LDH kit) was added to each well, and the plate was incubated in dark for 5 min at RT. The absorbance at 492 nm was read by a Tecan microplate reader. MTT Assay. After the 4 h incubation of the above-mentioned 96-well plates, the test solution was removed, and cells were washed once with HBSS buffer. The fresh HBSS buffer with 10% (vol) of MTT reagent (5 mg/mL) was added and incubated further for 4 h at 37 °C with gently shaking. The solution was removed and cells were dissolved in 100 L of DMSO and incubated 10 min at 37 °C with gentle shaking and protected from light. The absorbance at 550 nm was read by a Tecan microplate reader.

Antimicrobial assays. Chitosan-SQ 52:1 and 46:1 nanoparticles (1 mg/mL) were tested on Staphylococcus aureus subsp. aureus str. Newman and Escherichia coli (Tolc-deficient) in standard microdilution assays. Cell suspensions containing ca. 5x106 cfu/mL was prepared from mid-log cultures in Mueller-Hinton broth (pH = 7.3 at 25 °C) and samples were tested in serial dilution in a range from 0.06 to 128 g/mL. Bacteria were grown for 16 h at 37 °C and MIC


8

Page 9 of 32

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

(minimum inhibitory concentration) values were determined by visual inspection of the plates. As controls, pH = 5.3 acetate buffer and chitosan in solution in the same buffer were tested. The experiments were conducted in duplicate. Loading of model drugs. Three model compounds available in the laboratory were used to test the loading properties of the new amphiphilic chitosan nanoparticles: the caffeine, the fluorescein (hydrophilic) and the Nile red (lipophilic). First of all, two ways of loading were tested with the caffeine, during particles generation and post-formation: (i) 4.4 mg of caffeine is dissolved first in 2 mL of pH = 5.3 acetate buffer and then 4 mg of chitosan-SQ 52:1 is added (1:1 molar ratio of glucosamine and caffeine molecules) under stirring during 1h; (ii) 4 mg of chitosan-SQ 52:1 is first added to 1 mL of acetate buffer under stirring during 1h and then 1 mL of a caffeine solution in acetate buffer is added and mixed for 30 min. The fluorescein and the Nile red were loaded during the formation of particles. 0.01 mg/mL, 0.02 mg/mL and 0.05 mg/mL solutions of fluorescein (in pH = 5.3 acetate buffer) and of Nile red (in EtOH) were added to a 2 mg/mL chitosan-SQ 52:1 solution in acetate buffer (2 mL) during 1h at 400 rpm (0.0025% w/w, 0.005 %w/w and 0.025 %w/w dye/glucosamine). The suspensions were washed at least twice by centrifugation at 11000 rpm, at 4°C and during 15 min.

Loading quantification of model drugs. The caffeine was quantified by HPLC25 whereas with the fluorescein and the Nile red, the intensity of fluorescence was read by a Tecan microplate reader. HPLC. Pump: Dionex P580 pump; Autosampler: Dionex ASJ 100 automated sample injector; detector: UVD 170 S detector; column oven: Dionex STH 585 column oven; software: chromeleon 6.50 SP2 build 9.68. A 125×4 mm LiChrospher® 100/RP-18 column (Merck-


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 32

Hitachi, Darmstadt, Germany) with a 4×4-mmLiChrospher® 100/RP-18 guard column (5 μm) (Merck-Hitachi, Darmstadt, Germany) was used as stationary phase for all substances. The mobile phase was a pH = 2.6 buffer/acetonitrile; 90:10 (v/v) (pH = 2.6 buffer :1 L contains 1.15 mL of phosphoric acid, 4.08 g KH2PO4 anhydrous and deionised water.). The retention time observed was 6.2 ± 0.2 min, the detection wavelength was 262 nm, the flow rate was 1.2 mL/min and the injection volume was 50 L. The calibration curve was done with 8 different concentrations of caffeine in water: from 0.2 mg/mL to 0.005 mg/mL (r2 = 0.9997). Before injection, the samples were filtered through a cellulose acetate 0.2 m membrane, and the supernatant was analyzed. The amount of caffeine inside the particles was determined indirectly (amount inside = total amount-amount in the supernatant). As a control, the solutions of caffeine in water with and without filtration were analyzed to exclude interaction with the cellulose membrane. Fluorescence. The loading of the fluorescein and of the Nile red was determined by measuring the intensity of the emitted fluorescence of the supernatants after 2 washing (fluorescein: excitation= 494 nm and emission = 521 nm; Nile red: excitation = 530 nm and emission = 570 nm). Calibration curves of fluorescein in acetate buffer and Nile red in ethanol were done with five different concentrations: from 0.01 mg/mL to 0.002 mg/mL (r2 = 0.9887). The drug loading rate (LR) and the encapsulation efficiency (EE) were calculated according to the following equations: 𝐿𝑅 = 𝐸𝐸 =

weight of model drug in nanoparticles × 100 weight of nanoparticles

weight of model drug in nanoparticles × 100 initial amount of model drug in the system

Each sample was assayed in triplicate and results are reported as the mean ± standard deviation.


10

Page 11 of 32

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

Confocal Laser Scanning Microscopy (CLSM). One drop of the fluorescent suspensions was deposited on a slide and investigated by CLSM (LSM 710, Carl Zeiss, Oberkochen, Germany), equipped with an argon/neon laser and a 63× water immersion objective. Images were captured using FITC channel (excitation, 488 nm; band-pass filter, 500−530 nm) and rhodamine channel (excitation, 561 nm; band-pass filter, 574−638 nm). The measurements and image analysis were performed using Zeiss Zen Black software.

Polyplexes formation. An appropriate amount of pUC18 (N/P = 2, 5, 10, 20: ratio between nitrogen from polymer and phosphate from pDNA) was mixed with chitosan-SQ 52:1 (1 mg/mL) in pH = 5.3 acetate buffer for 10 minutes and incubated for 30 min at RT. As a control, polyplexes formed from chitosan and pDNA with an N/P ratio of 10 was used.

Agarose gel electrophoresis. To study the DNA binding ability, agarose gel electrophoresis was used. The polyplexes with appropriate N/P ratio (2, 5, 10 and 20) were prepared and four different categories of samples were done: (i)10 µL of polyplex suspension (containing 1 µg of pDNA) were mixed with 2 µL of loading dye, and loaded into a 0.7 wt% agarose gel containing 0.5 µg/mL ethidium bromide; (ii) 10 µL of polyplex suspension were mixed with 2 L of DNAseI during 1h at 37°C, then 1.4 L of EDTA (20 mM, pH = 8) is added to stop the digestion and samples were mixed with 2 µL of loading dye and loaded into the agarose gel; (iii) 10 µL of polyplex suspension were mixed with 2 L of DNAseI during 1h at 37°C, then 1.4 L of EDTA (20 mM, pH = 8) is added to stop the digestion and 3 L of heparin is incubated for 30 min at RT to study a possible release of the pDNA from the polyplexes. Finally samples were mixed


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 32

with 2 µL of loading dye and loaded into the gel; (iv) 10 µL of polyplex suspension were incubated with 0.5 mL of chitosanase (from Streptomyces purchased from Sigma-Aldrich, 135.2 U/mL) during 3h at 50°C and then samples were mixed with 2 µL of loading dye and loaded into the gel. Chitosanase catalyzes the endohydrolysis of β-1,4-linkages between D-glucosamine (GlcN-GlcN) residues in chitosan. The enzyme from Streptomyces has been reported to also hydrolyze the GlcNAc-GlcN linkage in partially acetylated chitosan. As a control, 1 g of pDNA was incubated with 2L of DNAseI (10000 U/mL) during 1h at 37°C, then 1.4 L of EDTA (20 mM, pH = 8) is added to stop the digestion. Electrophoresis was run in 0.5 % Tris/Borate/EDTA buffer at 50 V for 75 min. pDNA retardation was analyzed using UV illuminator, Fusion FX7 imaging system (Peqlab, Erlangen, Germany), to show the bands of the pDNA.

3. Results and Discussion 3.1. Synthesis of the squalenoylated chitosan

1,1’,2-Trisnor squalenaldehyde (SQCHO) was obtained from squalene according to the method of Van Tamelen.23,

26

Squalenoylated chitosan was prepared by reductive amination using

cyanoborohydride (Scheme 1) as described by Desbriere24 with four different initial molar ratios between the glucosamine units from the chitosan (Mw = 90 kDa and deacetylation percentage > 90 %) and SQCHO: 2:1, 9:1, 42:1 and 109:1. The mean yield for the four different syntheses was of 71 % ± 15%.


12

Page 13 of 32

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

HO

HO O

HO HO

NH2

O O HO

O HO NH2 n

OH

HO HO AcO

H

HO O

O HO

O O

NH3 AcO

O

OH

HO

N

NH3

n

AcO

SQ

AcO

OH

HO

NH2 AcO

HO O

O HO

O

O HO AcO

NH3

O HO

HO O

NH3

HO O

AcO

EtOH, AcOH

HO

NH3

O

HO HO

NH2

O

HO O

O

HO

NaCNBH3

HO

HO

NH3

n

SQ

SQ =

AcO

Scheme 1. Covalent coupling of the SQCHO with chitosan by reductive amination.

Fourier Transform Infrared (FTIR) analysis was carried out to confirm conjugation with squalene (Figure 1). The various synthesized samples were studied, in comparison with SQ and chitosan. The FTIR spectra highlighted the presence of the characteristic peaks at 2800-3000 cm-1 arising from the C-H stretching of the SQ27 and the peaks at 1000-1100 cm-1 from the C-O-C vibration of the sugar.28 The absence of carbonyl band at 1720 cm-1 established that the different final samples didn’t contain free SQCHO. The intensity of the peak at 2800-3000 cm-1 showed qualitatively the amount of SQ: the peaks are more intense with the chitosan-SQ 2:1.


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 32

Figure 1. IR spectra of the different chitosan-SQ derivatives. The both black squares show the typical peaks from the C-H stretching in the molecules of SQ and the C-O-C vibration bonds of chitosan respectively.

The 1H NMR is the method of choice to quantify the final ratio between the SQ and the glucosamine units. Unfortunately, the SQ-conjugated chitosan was completely insoluble in most NMR solvents including CDCl3, DMSO-d6, MeOD or D2O, D2O-DCl, D2O-acetic acid-d4, leading either to a precipitate or to a unsuitable opalescence colloidal suspension (see Figure S1). In the event, the only solvent allowing the complete dissolution of the chitosan-SQ was found to be sulfuric acid-d2. Protons from the SQ and the chitosan could be observed in the same H1 NMR spectra (see Figure S2). However, it was clear that extensive degradation occurred in this solvent, making impossible to attribute the different peaks observed and to quantify the amount of SQ. Therefore, elemental analyses were finally performed to estimate the percentage of substitution of the chitosan derivatives by comparing the C and N molar ratio (Table 1). The results showed that the coupling reaction was successful and the final molar ratio between glucosamine units and SQ could be determined (see details in Table 1). To simplify the understanding in the rest of the manuscript, the different compounds will be called according to the final glucosamine:SQ molar ratios: 3:1, 13:1, 46:1 and 52:1.

Table 1. Elemental analysis (%) of chitosan derivatives and the corresponding molar ratio between carbons and nitrogens (nC/nN).


14

Page 15 of 32

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

Starting molar ratio

C

H

N

nC/nN

(glucosamine:SQ)

Final molar ratio

Yield of

(glucosamine:SQ)

substitution

Chitosan

39.68

7.59

7.85

6.1

-

-

Chitosan-SQ 109:1

37.60

6.89

6.73

6.52

52:1

>100%

Chitosan-SQ 42:1

38.51

6.92

6.83

6.6

46:1

92%

Chitosan-SQ 9:1

38.21

7.54

5.56

8.1

13:1

72%

Chitosan-SQ 2:1

55.97

8.62

4.3

15.2

3:1

64%

3.2. Nanoparticles preparation and characterization

The ability of the different amphiphilic chitosan-SQ conjugates to elicit the formation of particles by self-assembly was investigated by directly stirring the material during several hours, in aqueous solution. Immediately an opalescence (Tyndall effect) could be observed, characteristic of the light diffusion through a suspension of nano-objects.

3.2.1 Nanoparticles prepared by self-assembly in acetate buffer: effect of the pH

The hydrophobic modified chitosan is an associating polyelectrolyte, and its aggregation behavior depends on two effects: the attraction of associating groups inducing aggregation and repulsion related to the presence of charged units from the amine of sugar (pKa of about 6.229) and counterions hampering aggregation. As a consequence, the pH determines the charging degree of the modified chitosan and should exert a strong effect on its self-assembly behavior.30 In water, large aggregates were observed because at neutral pH, a reduction in the charges on chains promoted aggregation. At low pH, the amines from the chitosan got protonated and


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 32

became positively charged.29 Therefore, sodium acetate buffers at different pH (6.5, 5.3 and 4.0) were used in order to have a buffered acidic medium. At pH = 6.5, the different submicrometer particles were polydisperse, and large aggregates were also observed. The size range and the polydispersity index (PDI) were considerably improved when the pH became more acidic (Figure S3). This can be explain by electrostatic repulsion from the positively charged glucosamines: for example, considering the Chitosan-SQ 13:1, at pH = 6.5, the size of the particles was of 3145 nm with a PDI = 0.8, at pH = 5.3, the size was of 2487 nm with a PDI = 0.48, and at pH = 4.0, the size was of 2817 nm with a PDI = 0.38. The smallest particles with the best PDI were observed at pH = 5.3 and 4: for example, with the chitosan-SQ 52:1, in the pH = 5.3 acetate buffer, particles with a diameter of 657 ± 11 nm and a PDI ≤ 0.2 were obtained. Thus, for physico-chemical reasons we chose acetate buffer at pH = 5.3 to perform the different experiments. The self-assembling properties of the squalene coupled to small polar heads have already been shown: notably with several nucleosides31, doxorubicine32, penicillin33 or fondaparinux34 but it is the first time that squalenoylation was performed on a large carbohydrate polymer. Apparently, coupling this polyisoprenyl tail to chitosan enables the self-assembly of this modified polymer in aqueous acetate buffer, leading to the spontaneous formation of particles. As we know from the squalenoylation of small molecules, this powerful self-assembling property is mainly due to the hydrophobic interactions between the lipid chains and the - stacking between the carboncarbon double bonds.35

3.2.2 Characterization of the particles


16

Page 17 of 32

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

After the complete dispersion of the squalenoylated chitosan in the acetate buffer at pH 5.3 (1 mg/mL), the different suspensions were characterized by dynamic light scattering (DLS). For the four different chitosan-SQ samples, the zeta potential of the particles was higher than +20 mV, due the protonated amino groups of the chitosan at the surface of the particles. Interestingly, a decrease in the amount of SQ per molecule of chitosan caused a decrease of the size and the PDI of particles (Table 2). So the smallest and most monodispersed particles were obtained with the chitosan-SQ 46:1 and 52:1 whose the particles had a diameter of 692 ± 13 nm and 657 ±11 nm respectively and a PDI of 0.2.

Table 2. Measurements of particle size, polydispersity index (PDI) and zeta potential according to the molar ratio of SQ in acetate buffer at pH = 5.3, immediately after preparation. Molar ratio

Size [nm]

PDI

(glucosamine:SQ)

Zeta Potential [mV]

3:1

8847 ± 1921

1

+22 ± 2

13:1

2487 ± 846

0.5

+30 ± 1

46:1

692 ± 13

0.2

+28 ± 1

52:1

657 ± 11

0.2

+29 ± 2

The effect of the hydrophobic modification of the chitosan on its self-aggregation has been already discussed by Philippova.30 Two mechanisms may occur in water: intra and intermolecular self-aggregation, and for both mechanisms in most cases an increase in the content of hydrophobic substituents will result in the compression and densification of aggregates due to the enhanced attraction between associating groups. However, Zhang36


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 32

reported the opposite with n-octyl-N-trimethylchitosan. It was indeed observed in this case that the diameter of intermolecular aggregates increased with an increase in the grafting density of hydrophobic appendages on the polymer. In our case, the results even showed that the diameter of the particles is proportional to the degree of substitution with an R2 value of 0.99 by linear regression analysis (Figure S4). A low degree of substitution led to a strong electrostatic repulsions of high charge density and disfavored intra-aggregation but if the degree of cationic charging decreased strongly (as it was the case with the Chitosan-SQ 3:1 and 13:1), this effect led to an enlargement of aggregates. According to these results, the chitosan-SQ 46:1 and 52:1 were chosen for the rest of the study. The morphology of the different particles was observed by Scattering Electron Microscopy (SEM) showing, as observed by DLS, large aggregates with the chitosan-SQ 3:1 but becoming less polydisperse and and more spherical in shape when the amount of SQ decreased (Figure 2 and S5). The sizes obtained by SEM were smaller than in suspension: for example with the chitosan-SQ 52:1 nanoparticles, the mean size was of 220 nm ± 45 nm. This result was expected because the particles were dried before observation. On the contrary, the DLS gives access to the hydrodynamic diameter of the particles and it is known that the chitosan particles strongly swell in aqueous solution.37


18

Page 19 of 32

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 2. Representative SEM pictures of chitosan-SQ 13:1 particles on the left and of chitosanSQ 52:1 particles on the right: a spherical shape was clearly observed.

The stability of the suspensions at 4 °C was followed by DLS (Figure S6). The suspensions were stable for > 20 days with even a small tendency for size and PDI of the particles to decrease after few days. The particles were formed by a static self-assembly mechanism in which the formation of the ordered structure required energy (stirring) but the particles remained stable once they are formed.38 The influence of the concentration on the particle size was studied with the chitosan-SQ 52:1 (Table 3): higher concentrations caused larger particles. Self-assembly is driven by the minimization of Gibbs free energy, and is governed by the process of nucleation and growth.39 In our case the results suggested that the particles size was only slightly affected by the chitosan-SQ concentration: an increase by a factor 10 of the concentration led only to an increase of a factor 1.3 of the particle size. So, if the concentration in chitosan-SQ became higher, an increase in particles number was probably preferred rather than an increase in particles size.

Table 3. Characterization of chitosan-SQ 52:1 particles according to the concentration in acetate buffer at pH = 5.3 (after three days stored at 4 °C).

Concentration

Size [nm]

PDI

1

566 ± 10

0.2

2

602 ± 11

0.3

[mg/mL]


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

5

624 ± 13

0.2

10

722 ± 6

0.3

Page 20 of 32

Finally, the influence of the stirring time, during the preparation of the chitosan-SQ 46:1 particles, on the particles size was followed (Figure S7): the longer the stirring time (1 h, 4 h or 6 h) the larger the particles. But after 2 days stored at 4 °C, whatever the stirring time, the same size was obtained: d = 699 nm ± 5 nm (PDI = 0.2). It means that aggregation was promoted when the stirring time increased, but after two days at 4 °C, the suspension reached its thermodynamic equilibrium again.

3.3. Evaluation of the chitosan-SQ as drug carrier 3.3.1 Biocompatibility

The cytotoxicity of the chitosan-SQ 46:1, chitosan-SQ 52:1 as well as plain chitosan were studied with lactate deshydrogenase

(LDH) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-

diphenyltetrazolium bromide) colorimetric assays on two different cell lines: A549, adenocarcinomic human alveolar basal epithelial cells and L929, mouse fibroblast cells. Both cell lines are good models to study the biocompatibility of new material, A549 cells being used in the context of a possible non-invasive lung administration of the particles via aerosolisation (will be studied in future studies). LDH is a soluble cytoplasmic enzyme that is present in almost all cells and is released into the extracellular space when the plasma membrane is damaged. According to this assay, for both cell lines, neither chitosan-SQ particles nor plain chitosan showed cytotoxicity up to 1 mg/mL (Figure S8).


20

Page 21 of 32

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

However, regarding the MTT assays, both cell lines were more sensitive than in the LDH assays. Indeed, at 1 mg/mL, highest concentration tested, the cell viability was around 70 % on L929 cell line and less that 20 % on A549 cell line for the plain chitosan and chitosan-SQ 46:1 and 52:1 particles. According to the MTT assays, Chitosan-SQ 46:1 and 52:1 appeared safe (cell viability > 80%) for a maximum concentration of ~0.75 mg/mL on L929 cells and for a maximum concentration of ~0.25 mg/mL on A549 cells. No difference of cell viability was however observed between chitosan-SQ particles and plain chitosan (Figure S8). This difference in results between MTT and LDH assays on A549 cells may be relied to the fact that the LDH activity assay is suitable for determining cytotoxicity by membrane damage, but is not adapted for determining the extent of increased and decreased cell numbers due, for example, to cell cycle alterations without membrane damage.40

3.3.2. Antimicrobial properties

The antimicrobial effect of chitosan is known and depends on various factors such as the molecular weight and the cationic charge density of the polymer (a higher positive charge density leads to strong electrostatic interaction) and can also differ between Gram-negative and Gram-positive bacteria due to the difference of their cell envelope composition.41 The stability of the chitosan-SQ 46:1 and 52:1 nanoparticles was first studied in the growth medium used for susceptibility studies (Mueller-Hinton broth, pH = 7.3 at 25 °C) and the same size range was observed (d = 520 ± 61 nm and d = 574 ± 8 nm respectively). The antimicrobial activity of nanoparticles was then evaluated on E. coli and S. aureus and the MIC values obtained were the same as for the chitosan solution (Table 4). We could have expected an


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 32

increase in the antimicrobial activity of the particles, due to hydrophobic interactions between the SQ and cell wall proteins, but with other chitosan lipo-derivatives such as(N-/2(3)-(dodec-2enyl)succinoyl) it has been shown that a substitution degree of at least 3 mol% was required to observe an improve activity.42 In our case, the degree of substitution (1.9 mol% and 2.2 mol%) was probably not sufficient to generate an increase of the antimicrobial activity.

Table 4. MIC values of chitosan and chitosan-SQ 46:1 and 52:1 particles on Gram-negative (E. coli) and Gram-positive bacteria (S. aureus). The acetate buffer at pH = 5.3 was used as a control, and no activity could be observed.

E. coli (TolC-

S. aureus str.

deficient)

Newman

no inhibition

no inhibition

Chitosan

>128 µg/mL

64 µg/mL

Chitosan-SQ 46:1

128 µg/mL

64 µg/mL

Chitosan-SQ 52:1

>128 µg/mL

64 µg/mL

Acetate buffer 6.4 % (v/v)

3.4. Encapsulation of small molecules 3.4.1. Caffeine loading

To characterize the loading capacity of this new particulate system, a first model drug was explored: caffeine is a hydrophilic compound and easily quantifiable by HPLC. The experiments


22

Page 23 of 32

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

were done with a 1:1 molar ratio between the caffeine and the chitosan-SQ 52:1, and two ways of loading were investigated: (i) the model drug and the amphiphilic chitosan were mixed together and added to the acetate buffer (the drug can be inside the particles or at the surface) and (ii) the particles were first prepared and then the model drug was added to the suspension (the drug would be mainly at the surface). After preparation, particle size measurements by DLS indicated 652 ± 9 nm and 696 ± 22 nm for (i) and (ii) respectively. Compared with the blank particles (657 ± 11 nm), no significant difference could be observed. When the caffeine and the polymer were mixed in the same time, the quantification showed an encapsulation efficacy (EE) of 11 % ± 3 % and a drug loading of 12 ± 3 %, whereas, when the caffeine was added after the preparation of the particles, an encapsulation efficacy of 10 ± 1% and a drug loading of 10 ± 1 % were measured. The finding that the respective amount of drug quantified showed no significant difference between these two ways of loading suggested that the drug was mainly at the surface of the particles in both cases. For the following section, the particles were formed in mixing in the same time the chitosan-SQ and the model drug.

3.4.2. Dye loading

Two other model drugs were tested to study the loading capability of chitosan-SQ nanoparticulate system: fluorescein, a hydrophilic green dye, and Nile red, a hydrophobic red dye. After preparation by premixing of the two components (0.02 mg/mL of dye with 2 mg/mL of chitosan-SQ and washing), the suspensions were imaging by fluorescence microscopy: Figure 3. In both cases, the particles were fluorescent, demonstrating that the dyes were efficiently encapsulated in the particles regardless of their hydrophobic/hydrophilic behavior. Moreover, the


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 32

Nile red dye was known to become fluorescent only in a lipid environment 43 indicating that this dye was supposed to be located in the squalene region.

Figure 3. Representative fluorescent images of fluorescein loaded nanoparticles on the left and Nile red loaded nanoparticles on the right.

The size of the different dye-loaded nanoparticles was also measured and larger particles could be observed with the fluorescein (d = 826 ± 24 nm) than with the Nile red loaded particles (d = 724 ± 25 nm). The encapsulation efficacy was then determined by measuring the intensity of the emitted fluorescence from the supernatants after two washings. Three different starting amounts of model drugs were tested: 0.02 mg/mL, 0.05 mg/mL and 0.2 mg/mL. The encapsulation efficiency (EE) and loading rate (LR) were depicted in Table 5.


24

Page 25 of 32

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

Table 5. Encapsulation efficiency (EE) and loading rate (LR) for the chitosan-SQ 52:1 particles according to the initial concentration of fluorescein and Nile red: 0.02 mg/mL, 0.05 mg/mL and 0.2 mg/mL. EE±SD

LR±SD

EE±SD

LR±SD

Fluorescein

Fluorescein

Nile red

Nile red

0.02 mg/mL

89 ± 0.4 %

1.8 ± 0.01 %

35 ± 1 %

0.7 ± 0.02 %

0.05 mg/mL

92 ± 0.2 %

4.6 ± 0.01 %

86 ± 3 %

4.3 ± 0.2 %

0.2 mg/mL

96 ± 0.1 %

19.1± 0.01

31 ± 5 %

6.2 ± 1.0 %

Concentration

The nanoparticles loaded with fluorescein showed a better EE and LC than the ones encapsulating Nile red dye. This result could tentatively correlate to the higher lipophilicity of the latter molecule that must concentrate in the SQ compartment whom substitution is limited to ~2 %, thus, only a small fraction of the Nile red could be loaded. Moreover, in the acetate buffer (pH = 5.3), chitosan is positively charged, and caffeine (pKa~14.0), fluorescein (pKa

Squalenoylation of Chitosan: A Platform for Drug Delivery

Recommend Documents