Accepted Manuscript Title: Solid lipid nanoparticles for oral drug delivery: Chitosan coating improves stability, controlled delivery, mucoadhesion and cellular uptake Author: Yangchao Luo Zi Teng Ying Li Qin Wang PII: DOI: Reference:

S0144-8617(15)00033-8 http://dx.doi.org/doi:10.1016/j.carbpol.2014.12.084 CARP 9604

To appear in: Received date: Revised date: Accepted date:

15-11-2014 24-12-2014 29-12-2014

Please cite this article as: Luo, Y., Teng, Z., Li, Y., and Wang, Q.,Solid lipid nanoparticles for oral drug delivery: chitosan coating improves stability, controlled delivery, mucoadhesion and cellular uptake, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2014.12.084 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights: SLN was prepared by a combined technique of solvent-diffusion and hot homogenization

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Chitosan coating stabilized SLN in simulated gastric condition

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Chitosan coating significantly improved mucoadhesive property of SLN

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Chitosan formed a thick layer around SLN in gastric condition as clearly observed by TEM

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Surfactants but not chitosan coating determined cell uptake of SLN

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Solid lipid nanoparticles for oral drug delivery: chitosan coating improves

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stability, controlled delivery, mucoadhesion and cellular uptake

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Yangchao Luoa, b, *, Zi Tengb, Ying Lib, Qin Wangb, *

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USA.

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b

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Park, MD 20742, USA

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Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269,

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Department of Nutrition and Food Science, University of Maryland, College

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*Corresponding authors.

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Current address:

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Dr. Yangchao Luo,

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Assistant Professor, Department of Nutritional Sciences, University of

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Connecticut, 3624 Horsebarn Road Extension, U-4017, Storrs, CT 06269-4017,

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USA. Phone: (860) 486-2180. Fax: (860) 486-3674. Email:

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[email protected]

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Dr. Qin Wang,

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Associate Professor, Department of Nutrition and Food Science, University of

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Maryland, 0112 Skinner Building, College Park, MD, 20742, USA. Phone : (301)

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405-8421. Fax : (301) 405-3313. Email : [email protected]

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Abstract The poor stability of solid lipid nanoparticles (SLN) under acidic condition

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resulted in large aggregation in gastric environment, limiting their application as

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oral delivery systems. In this study, a series of SLN was prepared to investigate

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the effects of surfactant/cosurfactant and chitosan coating on their

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physicochemical properties as well as cellular uptake. SLN was prepared from

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Compritol 888 ATO using a low-energy method combining the solvent-diffusion

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and hot homogenization technique. Poloxamer 188 and polyethylene glycol (PEG)

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were effective emulsifiers to produce SLN with better physicochemical properties

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than SLN control. Chitosan-coated SLN exhibited the best stability under acidic

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condition by forming a thick layer around the lipid core, as clearly observed by

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transmission electron microscope. The intermolecular interactions in different

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formulations were monitored by Fourier transform infrared spectroscopy. In vitro

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drug delivery assays, cytotoxicity, and cellular uptake of SLN were studied by

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incorporating coumarin 6 as a fluorescence probe. Overall, chitosan-coated SLN

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was superior to other formulations and held promising features for its application

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as a potential oral drug delivery system for hydrophobic drugs.

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Keywords: solid lipid nanoparticles; chitosan; controlled release; mucoadhesion;

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cellular uptake.

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1. Introduction In recent decades, various controlled delivery systems have been introduced and developed from natural biomaterials, including proteins (Elzoghby, Samy &

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Elgindy, 2012; Luo & Wang, 2014a), polysaccharides (Liu, Jiao, Wang, Zhou &

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Zhang, 2008; Luo & Wang, 2013; Luo & Wang, 2014c), and physiological lipids

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(Mehnert & Mader, 2001; Muller, Mader & Gohla, 2000), with aims to improve

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solubility, stability and bioavailability of poorly absorbed drugs and nutrients.

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Among these delivery systems, solid lipid nanoparticles (SLN) have received

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increasing interest in recent years, due to their superior characteristics and

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advantages over biopolymer-based colloidal nanoparticles (Harde, Das & Jain,

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2011). For example, SLN is able to effectively encapsulate hydrophobic

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molecules with higher drug payload than biopolymers-based nanoparticles.

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Avoidance of chemical crosslinking and toxic solvent during preparation opens a

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wide range of applications for SLN as drug/nutrient carriers with low toxicity or

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side effects. More importantly, the ease of large scale production and sterilization

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of SLN provides feasibility and suitability for industrialization with expanded

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applications (Shegokar, Singh & Muller, 2011).

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Generally, SLN can be fabricated by a number of different methods, including

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high energy (high pressure homogenization, ultrasound, etc.) and low energy

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methods (solvent diffusion, solvent emulsification, microemulsion, etc.) (Mehnert

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et al., 2001). Although high energy method is suitable for large scale production

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of SLN, it is reported, more often than not, that the quality of SLN produced by

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high energy method is usually compromised with microparticles due to lipid 4 Page 4 of 40

aggregation (Jenning, Lippacher & Gohla, 2002) and thermal degradation of

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bioactive compounds (Mehnert et al., 2001). In contrast, low energy method has

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attracted increasing attention nowadays. Solvent diffusion and solvent

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emulsification are the two most investigated low energy techniques to produce

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SLN, with the major difference being the solvent solubility. Water miscible

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solvents, such as ethanol and acetone, are used in solvent diffusion method to

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allow the fast diffusion of solvent into the aqueous phase, leading to the

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formation nano-scale lipid dispersion; while the water immiscible solvents, such

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as cyclohexane and dichloromethane, are required in the latter technique to

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induce the formation of oil/water emulsion, and lipid nanoparticles are obtained

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during the subsequent solvent evaporation process. In addition, the lipid

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composition and surfactant types are also vital parameters in determining the

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physicochemical properties of prepared SLN.

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In order to increase the absorption rate and improve the delivery efficacy of SLN, surface modification and biopolymer coating have been recently applied to

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develop SLN with enhanced mucoadhesive properties and sustained release

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profile. Polyethylene glycol (PEG) was recently conjugated onto stearic acid to

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fabricate SLN with better mucoadhesive property (Yuan, Chen, Chai, Du & Hu,

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2013). The PEGylated SLN exhibited higher permeability through in vitro mucus-

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secreting Caco-2/HT29 coculture monolayer and improved in vivo absorption of

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encapsulated drug with prolonged blood circulation time, showing 2-fold higher

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relative bioavailability than non-modified SLN. In addition to covalent conjugation,

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biopolymer coating is a promising alternative to improve the mucoadhesive

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property with avoidance of potential toxicity of chemical residues during

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conjugation. Biopolymers with well-known mucoadhesive properties, such as chitosan and alginate (George & Abraham, 2006), are the excellent candidates to

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coat SLN. Recently, Fangueiro and coworkers successfully prepared alginate-

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coated SLN via multiple emulsion process, using alginate solution as an external

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aqueous phase (Fangueiro, Andreani, Egea, Garcia, Souto & Souto, 2012).

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Chitosan-coated SLN has also been recently reported to effectively promote the

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in vivo absorption of encapsulated insulin by prolonging the retention time of

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insulin on the mucosa due to the enhanced mucoadhesive property (Fonte,

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Nogueira, Gehm, Ferreira & Sarmento, 2011).

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In this work, we developed a new and simple process to produce chitosan-

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coated SLN using combined solvent-diffusion and hot homogenization

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techniques. The first objective of present study was to investigate the suitability

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of this process in producing SLN and characterize the physicochemical

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properties as affected by different formulations and chitosan coating. Specifically,

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the particle size, zeta potential, morphology, and stability in gastric condition of

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as prepared SLN were studied. The mucoadhesive properties of SLN with

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different formulations were also investigated using quartz crystal microbalance

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(QCM), which has been proven as an effective tool to study the mucoadhesion of

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nanoparticles (Teng, Li, Luo, Zhang & Wang, 2013; Teng, Li, Niu, Xu, Yu &

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Wang, 2014). The second objective was to explore the drug delivery applications

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of as prepared SLN, using coumarin-6 as a hydrophobic model drug and

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fluorescent probe. The cytotoxicity and cellular uptake of SLN was also

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investigated using Caco-2 cell model.

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2. Materials & Methods 2.1. Materials

Compritol 888 ATO was generously provided as a free sample by Gattefosse

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Company. Chitosan with low molecular weight (Cat# 448869, M.W.

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approximately 50-190 kDa based on viscosity), poloxamer 188, and PEG

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(MW=300) were purchased from Sigma-Aldrich. Dulbecco’s modified Eagle

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medium (DMEM), fetal bovine serum (FBS), 100× nonessential amino acids,

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100× penicillin and streptomycin, 0.25% trypsin (w/v) with EDTA, Hanks’

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balanced salt solution (HBSS), phosphate buffer saline (PBS), and

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methylthiazolyldiphenyltetrazolium bromide (MTT) were all purchased from Life

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Technologies Corp. The 96 well cell culture plates were obtained from Corning

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Inc. All other reagent and chemicals were of analytical grade and purchased from

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VWR International. All chemicals were used without further purification.

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2.2. Preparation and characterization of SLN The SLN in the present study was prepared using a combined technique by

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adopting both solvent-diffusion method (Hu, Jiang, Du, Yuan, Ye & Zeng, 2005)

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and hot homogenization process (Sarmento, Mazzaglia, Bonferoni, Neto,

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Monteiro & Seabra, 2011). Briefly, 50 mg lipid (Compritol 888 ATO) was

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completely dissolved in 1 mL solvent containing equal amount of acetone and

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ethanol (1:1 v/v), which was then heated to 70 ºC in a water bath. The resultant

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clear organic phase was quickly mixed with 20 mL of aqueous phase containing

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emulsifiers preheated to the same temperature. The emulsifiers consisted of

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either Poloxamer 188 alone or the combination of Poloxamer 188 and PEG, at

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the total concentration of 2.5 mg/mL (weight ratio of emulsifier to lipid was 1:1,

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w/w). Subsequently, the mixture was emulsified at 24,500 rpm for 5 min at 70 ºC.

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Then, the SLN was obtained by cooling samples in an ice water bath to quickly

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crystallize the lipid. The resultant SLN samples were designated as SLN A, B,

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and C, as tabulated in Table 1.

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Chitosan coating was applied in SLN formulation D (Table 1) with aims to

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improve physicochemical properties. Briefly, chitosan (0.5 mg/mL) was dissolved

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at 0.1% acetic acid and filtered using a 0.45 μm Acrodisc membrane (Pall Co.,

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Newquay, UK) to remove any impurities. Chitosan solution preheated to 70 ºC

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was used as the aqueous phase, and the preparation procedures were the same

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as described above.

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2.3. Physicochemical characterizations of SLN

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2.3.1. Appearance

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The freshly prepared samples were subject to the appearance measurement.

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The absorbance of the samples at 500 nm was determined to compare their

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turbidity (Luo, Zhang, Pan, Critzer, Davidson & Zhong, 2014b). The digital photos

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were also presented to visually observe the difference between the samples.

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2.3.2. Particle size and zeta potential

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Particle size was expressed as the hydrodynamic diameter measured by a

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dynamic light scattering instrument (DLS, BI-200SM, Brookhaven Instruments

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Corp., Holtsville, NY, USA) equipped with a 35 mM HeNe laser beam at a

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wavelength of 637 nm. The zeta potential was calculated from electrophoretic

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mobility, determined by a laser Doppler velocimeter (Zetasizer Nano ZS90,

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Malvern, UK).

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2.3.3. Fourier transform infrared (FTIR) spectroscopy

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The freeze-dried SLN samples and native lipid were analyzed for their

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intermolecular interactions by infrared spectra, using a Jasco FT/IR 4100

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spectrometer (Jasco Inc., Easton, MD, USA). The spectra were acquired from

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wavelength 800 to 4000 cm-1 at a resolution of 4 cm-1. At least 125 scans were

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performed for each sample.

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2.3.4. Stability in simulated gastric condition

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The simulated gastric fluid was purchased from Ricca Chemical (Pocomoke city, MD), which contained 0.2% (w/v) sodium chloride in 0.7% (v/v) hydrochloric

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acid. The freshly prepared samples were diluted 10 times with simulated gastric

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fluid, and the mixture was incubated at 37 ºC for 1 h. Then, the particle size and

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zeta potential of incubated samples were determined following the same

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procedures as described in Section 2.3.2. The morphological observation of the

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SLN samples after incubation at gastric fluid were also performed using

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transmission electron microscope (TEM) as described in Section 2.3.5.

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2.3.5. Morphological observation

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The morphological observation of the samples were performed using a TEM (JEM 2100 LaB6, JEOL, Japan), operated at 120 kV. Briefly, SLN samples were

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first diluted 10 times with pure water. One drop of diluted sample was placed on

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a 200-mesh carbon-coated copper grid, stained with 2% phosphotungstic acid

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solution, and dried at room temperature. Representative images of each sample

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were reported.

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2.3.6. Mucoadhesive property

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Freshly prepared SLN samples were subject to mucoadhesive property evaluation by QCM using a Q-Sense E1 microbalance (Q-Sense Co., Linthicum,

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MD, USA), according to our previous work (Teng et al., 2013). The gold-coated

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AT-cut quartz crystals sitting in the sample chamber had a fundamental

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frequency of 4.96 MHz (QSX-301, Q-Sense Co., Linthicum, MD, USA). In QCM

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analysis, the mucin and SLN samples were sequentially injected into the sample

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chamber by a peristaltic pump (Ismatec Reglo, Glattbrugg, Switzerland) and

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formed two thin layers on the gold crystal. The frequency change of the crystal

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was recorded by Qtools 3 software (Q-Sense Co., Linthicum, MD, USA), and the

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third overtone was converted into deposited mass (ng/cm2 crystal surface), using

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Sauerbrey model. Specifically, mucin (400 µg/mL) was dissolved in 10 mM PBS

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(pH 7.0), followed by filtration through a 1.2 µm membrane. All SLN samples (A-

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D) were freshly prepared and also filtered prior to measurement. Pure PBS (10

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mM, pH 7.0) was firstly injected into the chamber until a stable frequency was

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obtained. Then, mucin was injected followed by a second rinse with PBS. After

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the achievement of another constant frequency, the SLN sample was injected

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until a third plateau of frequency was reached. A final rinse of PBS was applied

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to remove any loosely bound samples. The temperature of the chamber was kept

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at 37 °C during the measurement. Mucoadhesive property was calculated as the

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mass ratio between deposited SLN and mucin.

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2.4. Encapsulation and release applications

Coumarin-6 was used as a hydrophobic model drug to investigate the

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encapsulation and controlled release properties of as prepared SLN. Coumarin-6

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was firstly dissolved in 1 mL of ethanol/acetone mixture (0.1 mg/mL, equivalent

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to 0.2% loading of SLN) and the preparation of SLN was then carried out by

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following the same procedure as described in Section 2.2. Afterward, the SLN

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samples were centrifuged at 6,000 g for 30 min to precipitate free coumarin-6,

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which was collected and re-dissolved in ethanol for calculation of encapsulation

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efficiency (EE) using the equation below. The fluorescent intensity of coumarin 6

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was determined using a multilabel microplate reader (Victor X3, PerkinElmer

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2030), with excitation at 485 nm and emission at 535 nm. Courmarin-6 was

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quantified according to an appropriate standard curve (R2=0.9996).

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EE (%) 

Total courmarin  free coumarin 100% Total courmarin

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After removal of free coumarin 6, the release profile was determined

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according to a previous method using dialysis membrane (Venkateswarlu &

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Manjunath, 2004). Briefly, 3 mL of SLN sample was placed into a dialysis bag

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(molecular weight cutoff 10 kDa), which was then placed in a beaker containing

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200 mL of PBS (pH 7.0) and incubated at 37 °C under mild stirring. At designated 11 Page 11 of 40

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time intervals, 200 µL was withdrawn from the beaker and measured by

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fluorescent microplate reader as described above.

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2.5. Cytotoxicity and cellular uptake

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The freshly prepared SLN samples were subject to cytotoxicity and cellular

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uptake evaluations. Caco-2 cells were cultivated in DMEM supplemented with

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10% FBS and 1% penicillin–streptomycin at 37 °C in a humidified environment

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with 5% CO2. The medium was changed every other day, and the cells were

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subcultured after reaching 80–90% confluence. Caco-2 cells between passages

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6 and 10 were used in this study.

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For cytotoxicity test, different concentrations of SLN were obtained by diluting

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with DEME. Samples with dilution of 10, 50, 100 times were used. Caco-2 cells

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were seeded in 96-well microplate at a density of 3×104 cells/well. After seeding,

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cells were incubated for another 24 h to allow cell attachment. Then, cells were

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treated with DMEM containing different samples, with seven replicates for each

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treatment. After 24 h of incubation, MTT assay was conducted as previously

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described (Luo, Teng, Wang & Wang, 2013). The cytotoxicity was expressed as

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the normalized viability compared with untreated control cells. For cellular uptake

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experiment, the coumarin 6 encapsulated SLN samples were used, with the

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coumarin 6 being a fluorescent probe to express the cellular uptake of SLN. The

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SLN samples diluted with 50 times using HBSS were used due to their

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appropriate fluorescent intensity. Briefly, Caco-2 cells were seeded in 96-well

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microplate at a density of 3×104 cells/well and incubated for 3-4 days until a

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monolayer was formed. The SLN samples were then added into each well and

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incubated for another 4 h. The cells were washed by PBS for two times to

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remove any free nanoparticles and then treated with 100 μL of lysis buffer (0.5%

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Triton X-100 in 0.2 M NaOH solution) to allow permeabilization of cell membrane

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and expose the internalized nanoparticles. Cellular uptake of SLN was expressed

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as the percentage of fluorescent intensity in cells versus the original intensity

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present in feed medium.

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2.6 Statistical analysis

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The experiments were performed at least in triplicate. The results were expressed as mean ± standard deviation. The one-way analysis of variance

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(ANOVA) was conducted with Tukey’s multiple-comparison test to compare the

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significant difference among samples, using SPSS package (SPSS 13.0 for

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windows, SPSS Inc., Chicago, IL, USA). The significance level (P) was set as

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0.05.

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3. Results and Discussion

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3.1. Physicochemical characterizations of SLN

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To develop an ideal formulation of SLN for drug delivery applications,

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surfactants/cosurfactants and surface modification play essential roles in

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emulsifying lipid molecules into aqueous phase and obtaining stable products

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against aggregation. In the present study, different formulations (Table 1) with or

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without surfactant/cosurfactant and chitosan coating were developed and

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characterized, as shown in Table 2. The SLN control (sample A) prepared

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without any surfactant or cosurfactant exhibited the largest particle size of 427.7

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nm, being significantly larger than other formulations. With the aid of surfactant

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(poloxamer 188) and cosurfactant (PEG), the particle size showed gradual and

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decrease to 323.7 nm (SLN B) and 252.5 nm (SLN C), respectively. The zeta

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potential of SLN control was -31.8 mV, which increased to -26.3 mV after

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surfactant was added (SLN B), and it increased significantly further (-15.9 mV) in

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SLN C when PEG was added as cosurfactant during preparation. When melted

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lipid phase was homogenized in the chitosan-containing aqueous phase, the

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particle size of SLN D slightly increased (P>0.05) compared with SLN C and the

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zeta potential changed reversely from negative (-15.9 mV) to positive value (26.1

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mV), providing evidence of chitosan coating. The PDI values of all SLN samples

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were smaller than 0.3, which suggested the narrow distribution of particle size

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(Luo et al., 2014b).

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By taking the advantage of the combination of solvent-diffusion and hot

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homogenization, it was able to prepare SLN with a higher yield than solvent

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diffusion technique alone (Hu, Yuan, Zhang & Fang, 2002) and avoid high energy

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input method (high pressure) for hot homogenization technique alone (ALHaj,

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Abdullah, Ibrahim & Bustamam, 2008). Various solid lipids, including triglycerides,

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hard fat types, glyceryl-based lipids, pure saturated fatty acids, etc., have been

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studied for their capability to produce SLN (Mehnert et al., 2001). Among all lipids,

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ATO has been shown to be superior to others, in terms of its enhanced drug

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loading capacity, slower release rate, better in vivo absorption and higher

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bioavailability of encapsulated drug (Paliwal et al., 2009). In the present study,

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poloxamer 188 and PEG were selected as a surfactant and cosurfactant,

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respectively, to emulsify ATO in aqueous phase, and they were found effective in

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producing stable SLN by significantly reducing both particle size and PDI.

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Although a higher zeta potential is considered to provide stronger electrostatic

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repulsion and thus better stability of nanoparticles, the steric stabilization should

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also be considered when surfactant is present. The adsorption of surfactants

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onto the surface of SLN resulted in the shift in the shear plane of the particle and

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therefore caused a reduction in zeta potential. Similar observations were also

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previously reported (Chen et al., 2006; Muller et al., 2000; Yan et al., 2010).

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3.1.3. FT-IR spectra of SLN

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The inter-molecular interactions were monitored by FT-IR, as shown in Fig. 2.

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Chitosan showed a typical spectrum with three characteristic peaks at 3352,

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1649, 1591 cm-1, being O-H stretch, C=O stretch from amide I, N–H bending and

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C–N stretching from amide II, respectively (Luo, Zhang, Cheng & Wang, 2010;

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Luo, Zhang, Whent, Yu & Wang, 2011). The SLN control exhibited several

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characteristic peaks with strong intensity of C-H stretching at 2915 and 2849, and

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C=O stretching at 1739 cm-1 from carboxyl group, respectively, which are the

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typical bands of lipid mixture in the formulation of Compritol 888 ATO (Rahman,

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Zidan & Khan, 2010). Another characteristic peak at 1173 cm-1 in SLN sample,

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which was assigned to alkyl groups present in glyceryl behenate, the major

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constituent in ATO 888 formulation. The broad peak observed at 3409 cm-1 was

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due to the weak intensity of O-H stretch and revealed strong hydrophobic nature

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of lipid. After the SLN was emulsified with the presence of poloxamer, the

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absorption bands at 2915 and 2849 cm-1 did not shift, but the intensity of both

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peaks were greatly reduced and the absorption band at 1173 cm-1 disappeared.

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This may be explained by the increased hydrophilic nature and the shielding

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effect of the amphiphilic surfactant. The shift of O-H band from 3409 to 3478 cm-1

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suggested the interaction between poloxamer and lipid due to hydrogen bonding.

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Meanwhile, a new characteristic peak with strong intensity at 1106 cm-1 was

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observed as a result of strong stretching of C-O-C linkage from poloxamer. After

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the addition of PEG as a cosurfactant, the FT-IR spectra were not significantly

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changed, except that the peak of O-H stretch shifted to 3393-1 cm, explaining that

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the hydrogen bonds in the system were reinforced. After the SLN was coated by

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chitosan, two characteristic peaks of amide I and amide II bonds were shown at

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1636 and 1513 cm-1, being greatly shifted compared with original chitosan. This

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indicated the ionic interactions between the amine groups in chitosan and

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carboxylic group in lipid.

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3.1.4. Morphological observation

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The morphology of freshly prepared SLN samples was observed under TEM

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as shown in Fig. 3. The SLN control (A), prepared without any surfactants,

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exhibited spherical shape with rough surface and some aggregation from large

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particles (Fig. 3a). The SLN B and C, prepared with surfactants, showed particles

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with smooth surface but irregular shape (Fig. 3b and 3c). The contrast of dark

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and bright areas in a particle may be attributed to the heterogeneous distribution

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of lipids and surfactants throughout the particles that caused uneven staining.

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Interestingly, the particle size of SLN C was greater than SLN B in TEM images,

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which observation disagreed with the DLS data as shown in Table 2. Such

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difference may be, in part, due to the fact that SLN C contained PEG, a more

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hydrophilic molecule, as co-surfactant which was not tightly packed onto the

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surface of SLN by this low energy method and easily diffused out after drying

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during TEM sample preparation process. As water was gradually evaporated

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during drying process, the hydrophobic interactions among lipid molecules were

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further strengthened, which may weaken the interactions between co-surfactant

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and lipid molecules causing the diffusion of PEG and formation of irregular shape

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under TEM observation. In Fig. 3d, a thick and dark layer was clearly observed

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around the particles, evidencing the successful coating of chitosan on the surface

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of SLN. Meanwhile, the particles were more homogeneous and spherical than

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non-coated SLN samples.

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In the solvent diffusion method, the diffusion process of organic phase (water miscible) into aqueous phase is very rapid upon mixing two phases together.

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During this process, surfactants and cosurfactants are moved and adsorbed onto

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the surface of melted lipid droplets, resulting in spontaneous formation of

366

nanoemulsion at high temperature. As the system cools down, the lipid becomes

367

solid and forms SLN. However, this method is a low-energy method, which may

368

not be as effective as high-energy technique in producing well separated SLN,

369

unless the appropriate surfactants/emulsifiers are used. He et al. used solvent

370

diffusion to prepare SLN with polyvinyl alcohol as a surfactant and observed

371

similar morphologies, showing quasi-sphere or cylinder as well as irregular shape

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17 Page 17 of 40

with bimodal particle size distribution (Hu et al., 2002). Our study demonstrated

373

that chitosan coating prevented the particle aggregation and greatly improved the

374

surface morphology of SLN prepared by low-energy method.

375

377

3.2. Stability of SLN in simulated gastric condition.

To investigate the stability of SLN samples under gastric condition, the

cr

376

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372

particle size and zeta potential were determined after 1 h incubation in simulated

379

gastric fluid. The results were tabulated in Table 2. The SLN control formed large

380

aggregates and precipitated immediately after dispersing into gastric fluid, and

381

the zeta potential was no longer measureable due to severe precipitation. The

382

SLN B exhibited significant increase in its dimension to 1132 nm, which was

383

almost three times larger than its original size. Meanwhile, a dramatic reduction

384

of zeta potential was also observed for SLN B and C, with the absolute value

385

close to zero. The SLN C was able to maintain its particle size from being

386

aggregated, although its zeta potential also decreased close to zero. The SLN D

387

not only maintained its particle size, and only a slight reduction was found in zeta

388

potential, which may be attributed to the screening effects of salt content in SGF.

389

Furthermore, the morphology of SLN samples after gastric incubation was also

390

observed under TEM, which generally agreed with DLS measurement. The SLN

391

control was excluded for TEM observation because of the existence of large

392

precipitates. The SLN B (Fig. 4a) exhibited large aggregates with some small

393

particles scattered, while the SLN C (Fig. 4b) showed relatively smaller particles

394

with some being irregular in shape. Conversely, the SLN D (Fig. 4c) maintained

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18 Page 18 of 40

its spherical shape with homogeneous dimensions during the incubation in

396

simulated gastric condition, and the core-shell structures were clearly observed

397

for each nanoparticle. As shown in the magnified image of one particle (Fig. 4d),

398

the solid lipid core with crystalline structures was clearly seen and coated by a

399

thick layer on its surface.

The stability and integrity of an oral drug delivery system in gastric condition

cr

400

ip t

395

is of great significance to ensure its in vivo efficacy. Though SLN holds a

402

promising potential for oral drug delivery due to its resistance to digestive

403

enzymes, its poor stability in acidic conditions (such as stomach) limits practical

404

applications. Several studies reported that the SLN was easily centrifuged and

405

collected at acidic condition of pH 1.2 because of acid-induced aggregation (Hu

406

et al., 2005; Paliwal et al., 2009). This was because the carboxyl groups were

407

protonated and the lipid molecules were no longer charged at this condition,

408

explaining the severe aggregation due to attractive forces, such as hydrophobic

409

interactions and van der Waals force. However, such collected SLN was hard to

410

re-disperse into water due to strong severe aggregation unless probe-type

411

sonication or high concentration of surfactants was being used (Yuan, Miao, Du,

412

You, Hu & Zeng, 2008). In our study, chitosan coated SLN was able to maintain

413

the particle dimensions and charges at simulated gastric condition, suggesting its

414

potential applications as an oral drug delivery system.

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401

415 416

3.3. Mucoadhesive property

19 Page 19 of 40

The mucoadhesion was determined by QCM method, which is a well

418

standardized methodology and more accurate than turbidity analysis method

419

(Wiecinski, Metz, Mangham, Jacobson, Hamers & Pedersen, 2009). The

420

mucoadhesion was expressed as the mass ratio between deposited mass of

421

mucin and SLN. As shown in Fig. 5, the curve for SLN control exhibited a straight

422

line with unchanged mass during SLN deposition process, indicating that SLN

423

control had negligible muchadhesive property. This may be due to the strong

424

hydrophobicity and poor solubility in aqueous buffer, resulting in the weak

425

interactions with mucin under the studied condition. Nevertheless, all other SLN

426

formulations demonstrated varying mucoadhesive properties. The SLN B had a

427

very limited affinity to mucin (SLN B/mucin mass ratio 0.13), while the

428

mucoadhesion of SLN C and SLN D was significantly improved by 112%

429

(SLN/mucin mass ratio 0.27) and 482% (SLN/mucin mass ratio 0.74),

430

respectively.

cr

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Mucoadhesive property is an important factor for the development of oral drug

Ac ce p

431

ip t

417

432

delivery systems. It determines how the particles will be retained, as strong

433

mucoadhesion suggests intimate contact with absorption site, thus ensuring the

434

effective absorption. Several molecular interactions are involved in the

435

mucoadhesion, including ionic, hydrogen, Van der Waals, and hydrophobic

436

interactions (Smart, 2005). Since mucin is a highly glycosylated and negatively

437

charged protein, the positively charged molecules will have much stronger affinity

438

than negatively charged nanoparticles. The low mucoadhesion of SLN control

439

and strong affinity of chitosan-coated SLN suggested that the ionic interaction is

20 Page 20 of 40

the driving force between mucin and nanoparticles, compared to hydrophobic

441

interactions. This observation agreed with our previous studies (Teng et al., 2013;

442

Teng et al., 2014), showing that mucoadhesion of negatively charged protein

443

nanoparticles was significantly improved after protein molecules were surface

444

modified by cationic moieties. Furthermore, the hydrophilic nature of chitosan

445

coating would also greatly strengthen the hydrogen bonds between the

446

nanoparticles and mucin molecules and enhance the mucoadhesion. Fonte and

447

coworker reported that chitosan-coated SLN significantly enhanced intestinal

448

absorption of encapsulated insulin (Fonte et al., 2011), which may also be

449

attributed to the strong mucoadhesive property of chitosan.

450

3.4. Drug delivery applications

d

451

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440

To explore the drug delivery application of prepared SLN, coumarin 6 was

453

selected as a model hydrophobic drug for evaluation of EE and release profile of

454

different SLN formulations. As depicted in Fig. 6A, the SLN control had the

455

lowest EE of coumarin 6, which was significantly improved in all other

456

formulations. The chitosan-coated SLN D showed the highest EE of 94%, though

457

no significant difference was observed compared to SLN B and C. With the

458

addition of surfactant and/or cosurfactant into the system, the lipid and coumarin

459

6 were better dispersed in the aqueous phase, thus facilitating the hydrophobic

460

and hydrogen interactions between these two molecules and achieving higher

461

EE. Because the coumarin 6 was encapsulated into lipid core by hydrophobic

462

interaction, chitosan coating showed little improvement in the EE. From the

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21 Page 21 of 40

results, it was postulated that the dispersibility and stability of SLN, rather than

464

surface coating, played a key role in determining the EE of hydrophobic drug.

465

Our results agreed with previous studies showing that chitosan coating did affect

466

the EE of hydrophobic drugs in lipid-based nanocarriers (Garcia-Fuentes, Prego,

467

Torres & Alonso, 2005; Sandri et al., 2010). The controlled release profile was

468

shown in Fig. 6B. All the SLN formulations exhibited a bi-phase release profile,

469

with a burst release in first 3 h followed by a sustained release. The SLN control

470

was excluded for release study due to large precipitation when dispersed in the

471

PBS release medium. Among all formulations, chitosan-coating significantly

472

reduced the release rate, and the cumulative release at the end of 70 h was only

473

12%, compared to 17 and 22% of SLN B and C, respectively. SLN is known for

474

their exceptional capability for controlled release of encapsulated hydrophobic

475

bioactive compounds, owing to their solid status at room or body temperature

476

and strong hydrophobic interactions (Muller et al., 2000). Coincident with our

477

results, a previous study also pointed out that chitosan coating was beneficial to

478

further slower the release rate (Dharmala, Yoo & Lee, 2008). Together with the

479

excellent gastric stability, strong muoadhesive property, and slow release at

480

neutral pH (intestinal condition, the major absorption site), chitosan coated SLN

481

demonstrated promising potential to enhance absorption of hydrophobic drugs in

482

small intestine by oral administration.

Ac ce p

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463

483 484

3.5. Cytotoxicity and Cellular uptake

22 Page 22 of 40

The cytotoxicity and cellular uptake of SLN samples were investigated using

486

Caco-2 cells. All the SLN formulations showed no toxicity to the Caco-2 cells up

487

to 72 h (data not shown). Then, the cellular uptake was determined using

488

coumarin 6 encapsulated SLN samples, as shown in Fig. 7. After 4 h incubation,

489

the cellular uptake of SLN control was less than 10%, being the minimal uptake

490

among all SLN formulations. This may be explained by the poor stability of SLN

491

control in the cell uptake buffer medium. High concentration of various salts in

492

the buffer screened the negative charges on the surface of SLN control sample

493

and thus induced the aggregation and hindered the cell uptake of coumarin 6

494

from those bulky precipitates. The cell uptake of the other three formulations was

495

dramatically increased to 35, 42, 44% for SLN B, C, and D, respectively. As

496

previously presented, addition of surfactant and/or cosurfactant significantly

497

improved the stability of SLN samples, and therefore these surfactants-emulsified

498

SLN samples were able to disperse homogenously in the buffer medium.

499

Addition of cosurfactant PEG significantly improved the cell uptake compared to

500

SLN B, which was only emulsified by Poloxamer 188. In addition to the stability,

501

particle size and surface charge are also proven to affect the cell uptake of

502

nanoparticles (He, Hu, Yin, Tang & Yin, 2010). The least cell uptake of SLN A

503

could be also attributed to its largest particle size and highest negative surface

504

charge. The fact that SLN C and SLN D with similar particle size but opposite

505

surface charge exhibited similar cell uptake suggested that particle size and

506

stability, rather than surface charge, played a predominant role in determining the

507

cell uptake of SLN.

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23 Page 23 of 40

Although SLN has been widely studied as drug delivery systems for

509

hydrophobic and macromolecular drugs, their cellular uptake behavior and

510

underlying mechanisms are not fully understood. Several factors are known to

511

affect the cellular uptake of SLN, including types of solid lipids (Yuan et al., 2008),

512

cell lines tested (Miglietta, Cavalli, Bocca, Gabriel & Gasco, 2000), surfactants

513

(Schöler, Olbrich, Tabatt, Müller, Hahn & Liesenfeld, 2001), surface coating

514

(Sarmento et al., 2011), as well as particle size (Gaumet, Vargas, Gurny & Delie,

515

2008). Chitosan coating has been shown as a promising approach for

516

overcoming the phagocytosis of insulin loaded SLN by mononuclear phagocyte

517

system and thus prolonging insulin blood levels (Sarmento et al., 2011). Another

518

study also suggested that chitosan coating enhanced the intestinal absorption of

519

insulin-loaded SLN through increasing Caco-2 cell monolayer permeation rate

520

(Fonte et al., 2011). However, more research is needed to fully explore the

521

underlying mechanisms of cellule uptake of chitosan-coated SLN.

523 524

cr

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Ac ce p

522

ip t

508

4. Conclusion

In this study, a series of SLN were successfully prepared via a low-energy

525

method by combining the solvent-diffusion and hot homogenization techniques.

526

Surfactant and cosurfactant played important roles in determining the particle

527

size, stability, cellular uptake of SLN. The SLN control prepared without

528

surfactant or cosurfactant exhibited the largest particle size, the least stability,

529

and the fastest release as well as the minimal cellular uptake. Surfactants and

530

chitosan coating contributed to a better stability through mechanisms of steric

24 Page 24 of 40

stabilization and electrostatic repulsion, respectively. Chitosan coating was

532

shown to further improve the stability of SLN in simulated gastric condition by

533

forming a distinct and thick layer around the SLN against its aggregation, as

534

clearly observed under TEM. Chitosan-coated SLN was superior to other

535

formulations for the development of oral drug delivery system for hydrophobic

536

drugs and nutrients.

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25 Page 25 of 40

537 538

References

539

ALHaj, N. A., Abdullah, R., Ibrahim, S., & Bustamam, A. (2008). Tamoxifen drug loading solid lipid nanoparticles prepared by hot high pressure homogenization techniques. American Journal

541

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Chen, H., Chang, X., Du, D., Liu, W., Liu, J., Weng, T., Yang, Y., Xu, H., & Yang, X. (2006).

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Podophyllotoxin-loaded solid lipid nanoparticles for epidermal targeting. Journal of

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Controlled Release, 110(2), 296-306.

Dharmala, K., Yoo, J. W., & Lee, C. H. (2008). Development of Chitosan-SLN Microparticles for

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Elzoghby, A. O., Samy, W. M., & Elgindy, N. A. (2012). Protein-based nanocarriers as promising

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Experimental factorial design applied to mucoadhesive lipid nanoparticles via multiple

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Fonte, P., Nogueira, T., Gehm, C., Ferreira, D., & Sarmento, B. (2011). Chitosan-coated solid lipid

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nanoparticles enhance the oral absorption of insulin. Drug Delivery and Translational

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Garcia-Fuentes, M., Prego, C., Torres, D., & Alonso, M. J. (2005). A comparative study of the

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Gaumet, M., Vargas, A., Gurny, R., & Delie, F. (2008). Nanoparticles for drug delivery: the need

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drugs: Alginate and chitosan - a review. Journal of Controlled Release, 114(1), 1-14.

Harde, H., Das, M., & Jain, S. (2011). Solid lipid nanoparticles: an oral bioavailability enhancer vehicle. Expert Opinion on Drug Delivery, 8(11), 1407-1424.

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George, M., & Abraham, T. E. (2006). Polyionic hydrocolloids for the intestinal delivery of protein

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He, C., Hu, Y., Yin, L., Tang, C., & Yin, C. (2010). Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials, 31(13), 3657-

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Hu, F. Q., Jiang, S. P., Du, Y. Z., Yuan, H., Ye, Y. Q., & Zeng, S. (2005). Preparation and

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characterization of stearic acid nanostructured lipid carriers by solvent diffusion method in

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an aqueous system. Colloids and Surfaces B-Biointerfaces, 45(3-4), 167-173. Hu, F. Q., Yuan, H., Zhang, H. H., & Fang, M. (2002). Preparation of solid lipid nanoparticles with

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clobetasol propionate by a novel solvent diffusion method in aqueous system and

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physicochemical characterization. Int J Pharm, 239(1-2), 121-128.

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Jenning, V., Lippacher, A., & Gohla, S. H. (2002). Medium scale production of solid lipid

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nanoparticles (SLN) by high pressure homogenization. Journal of Microencapsulation, 19(1),

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1-10.

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Liu, Z. H., Jiao, Y. P., Wang, Y. F., Zhou, C. R., & Zhang, Z. Y. (2008). Polysaccharides-based nanoparticles as drug delivery systems. Advanced Drug Delivery Reviews, 60(15), 1650-1662.

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Luo, Y., & Wang, Q. (2013). Recent advances of chitosan and its derivatives for novel

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Luo, Y., & Wang, Q. (2014a). Zein-based micro- and nano-particles for drug and nutrient delivery: A review. Journal of Applied Polymer Science, 131(16), 40696. Luo, Y., Zhang, B., Cheng, W.-H., & Wang, Q. (2010). Preparation, characterization and evaluation of selenite-loaded chitosan/TPP nanoparticles with or without zein coating.

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Carbohydrate Polymers, 82(3), 942-951.

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Luo, Y., Zhang, B., Whent, M., Yu, L. L., & Wang, Q. (2011). Preparation and characterization of

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zein/chitosan complex for encapsulation of alpha-tocopherol, and its in vitro controlled

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Luo, Y., Zhang, Y., Pan, K., Critzer, F., Davidson, P. M., & Zhong, Q. (2014b). Self-Emulsification of

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Alkaline-Dissolved Clove Bud Oil by Whey Protein, Gum Arabic, Lecithin, and Their

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Combinations. Journal of Agricultural and Food Chemistry, 62(19), 4417-4424.

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Luo, Y. C., Teng, Z., Wang, T. T. Y., & Wang, Q. (2013). Cellular uptake and transport of zein

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nanoparticles: Effects of sodium caseinate. Journal of Agricultural and Food Chemistry,

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Luo, Y. C., & Wang, Q. (2014c). Recent development of chitosan-based polyelectrolyte

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Macromolecules, 64, 353-367.

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Mehnert, W., & Mader, K. (2001). Solid lipid nanoparticles: Production, characterization and applications. Advanced Drug Delivery Reviews, 47(2-3), 165-196. Miglietta, A., Cavalli, R., Bocca, C., Gabriel, L., & Gasco, M. R. (2000). Cellular uptake and

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cytotoxicity of solid lipid nanospheres (SLN) incorporating doxorubicin or paclitaxel. Int J

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Pharm, 210(1-2), 61-67.

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Muller, R. H., Mader, K., & Gohla, S. (2000). Solid lipid nanoparticles (SLN) for controlled drug

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delivery - a review of the state of the art. European Journal of Pharmaceutics and

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Biopharmaceutics, 50(1), 161-177. Paliwal, R., Rai, S., Vaidya, B., Khatri, K., Goyal, A. K., Mishra, N., Mehta, A., & Vyas, S. P. (2009).

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Effect of lipid core material on characteristics of solid lipid nanoparticles designed for oral

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lymphatic delivery. Nanomedicine, 5(2), 184-191.

Rahman, Z., Zidan, A. S., & Khan, M. A. (2010). Non-destructive methods of characterization of

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risperidone solid lipid nanoparticles. European Journal of Pharmaceutics and

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Biopharmaceutics, 76(1), 127-137.

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Sandri, G., Bonferoni, M. C., Gokce, E. H., Ferrari, F., Rossi, S., Patrini, M., & Caramella, C. (2010). Chitosan-associated SLN: in vitro and ex vivo characterization of cyclosporine A loaded

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ophthalmic systems. Journal of Microencapsulation, 27(8), 735-746. Sarmento, B., Mazzaglia, D., Bonferoni, M. C., Neto, A. P., Monteiro, M. D., & Seabra, V. (2011).

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615

Effect of chitosan coating in overcoming the phagocytosis of insulin loaded solid lipid

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nanoparticles by mononuclear phagocyte system. Carbohydrate Polymers, 84(3), 919-925.

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618

Schöler, N., Olbrich, C., Tabatt, K., Müller, R. H., Hahn, H., & Liesenfeld, O. (2001). Surfactant, but

621

not the size of solid lipid nanoparticles (SLN) influences viability and cytokine production of

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macrophages. International Journal of Pharmaceutics, 221(1–2), 57-67.

623

Shegokar, R., Singh, K. K., & Muller, R. H. (2011). Production & stability of stavudine solid lipid

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nanoparticles-From lab to industrial scale. International Journal of Pharmaceutics, 416(2),

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461-470.

626 627

Smart, J. D. (2005). The basics and underlying mechanisms of mucoadhesion. Adv Drug Deliv Rev, 57(11), 1556-1568.

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Teng, Z., Li, Y., Luo, Y. C., Zhang, B. C., & Wang, Q. (2013). Cationic beta-Lactoglobulin

629

Nanoparticles as a Bioavailability Enhancer: Protein Characterization and Particle Formation.

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Biomacromolecules, 14(8), 2848-2856. Teng, Z., Li, Y., Niu, Y. G., Xu, Y. H., Yu, L. L., & Wang, Q. (2014). Cationic beta-lactoglobulin

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631

nanoparticles as a bioavailability enhancer: Comparison between ethylenediamine and

633

polyethyleneimine as cationizers. Food Chemistry, 159, 333-342.

636

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635

Venkateswarlu, V., & Manjunath, K. (2004). Preparation, characterization and in vitro release kinetics of clozapine solid lipid nanoparticles. Journal of Controlled Release, 95(3), 627-638. Wiecinski, P. N., Metz, K. M., Mangham, A. N., Jacobson, K. H., Hamers, R. J., & Pedersen, J. A.

an

634

cr

632

(2009). Gastrointestinal biodurability of engineered nanoparticles: Development of an in

638

vitro assay. Nanotoxicology, 3(3), 202-202.

M

637

Yan, F., Zhang, C., Zheng, Y., Mei, L., Tang, L., Song, C., Sun, H., & Huang, L. (2010). The effect of

640

poloxamer 188 on nanoparticle morphology, size, cancer cell uptake, and cytotoxicity.

641

Nanomedicine, 6(1), 170-178.

te

Yuan, H., Chen, C.-Y., Chai, G.-h., Du, Y.-Z., & Hu, F.-Q. (2013). Improved Transport and

Ac ce p

642

d

639

643

Absorption through Gastrointestinal Tract by PEGylated Solid Lipid Nanoparticles. Molecular

644

Pharmaceutics, 10(5), 1865-1873.

645

Yuan, H., Miao, J., Du, Y. Z., You, J., Hu, F. Q., & Zeng, S. (2008). Cellular uptake of solid lipid

646

nanoparticles and cytotoxicity of encapsulated paclitaxel in A549 cancer cells. Int J Pharm,

647

348(1-2), 137-145.

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30 Page 30 of 40

B

C

D

cr

ip t

A

3

us

2.5

Turbidity

2

an

1.5 1

0 A

B

C

D

d

649

M

0.5

Fig. 1. Digital photos and turbidity of solid lipid nanoparticles with different formulations.

651

SLN A, prepared without any surfactants; SLN B, prepared with poloxamer 188; SLN C,

652

prepared with poloxamer 188 and PEG; SLN D, prepared with chitosan coating.

Ac ce p

653

te

650

31 Page 31 of 40

M

an

us

cr

ip t

653

654

657

d

te

656

Fig. 2. FT-IR of solid lipid nanoparticles with different formulations

Ac ce p

655

32 Page 32 of 40

an

us

cr

ip t

657

M

658

Fig. 3. Transmission electron microscopy photographs of freshly prepared solid lipid

660

nanoparticles. (a) SLN A, prepared without any surfactants; (b) SLN B, prepared with

661

poloxamer 188; (c) SLN C, prepared with poloxamer 188 and PEG; (d) SLN D, prepared

662

with chitosan coating. Scale bar in each image represent 500 nm.

te

Ac ce p

663

d

659

33 Page 33 of 40

an

us

cr

ip t

663

M

664

Fig. 4. Transmission electron microscopy photographs of solid lipid nanoparticles after

666

incubation under simulated gastric condition for 1 h. (a) SLN B, prepared with poloxamer

667

188; (b) SLN C, prepared with poloxamer 188 and PEG; (c) SLN D, prepared with

668

chitosan coating; (d) higher magnification of selected area of image (c), as indicated by

669

the black lines. Scale bars represent 500 nm in image (a), (b), (c), and 200 nm in image

670

(d).

Ac ce p

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d

665

34 Page 34 of 40

cr

Mass (ng/cm2)

ip t

671

SLN Control (A)

us

SLN (B) SLN (C)

PBS

an

SLN (D)

Mucin

PBS

SLN Sample

PBS

M

Time (min:sec)

672

Fig. 5: Mucoadhesive properties of solid lipid nanoparticles investigated by Quartz

674

crystal microbalance (QCM). SLN A, prepared without any surfactants; SLN B, prepared

675

with poloxamer 188; SLN C, prepared with poloxamer 188 and PEG; SLN D, prepared

676

with chitosan coating.

te

Ac ce p

677

d

673

35 Page 35 of 40

us

cr

ip t

677

679

Ac ce p

te

d

M

an

678

680

Fig. 6. Encapsulation and controlled release properties of prepared solid lipid

681

nanoparticles. SLN B, prepared with poloxamer 188; SLN C, prepared with poloxamer

682

188 and PEG; SLN D, prepared with chitosan coating. Data sharing different letters was

683

significant different.

684

36 Page 36 of 40

us

cr

ip t

684

an

685

Fig. 7. Cellular uptake of solid lipid nanoparticles by Caco-2 cells. SLN A, prepared

687

without any surfactants; SLN B, prepared with poloxamer 188; SLN C, prepared with

688

poloxamer 188 and PEG; SLN D, prepared with chitosan coating. Data sharing different

689

letters was significant different.

d

te Ac ce p

690

M

686

37 Page 37 of 40

690

Graphic Abstract

M

an

us

cr

ip t

691

695

te

694

Ac ce p

693

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692

38 Page 38 of 40

Table 1. Formulations of solid lipid nanoparticles. Lipid phase

Aqueous phase (20 mL water) Pluronic® F-127

Sample Lipid (mg)

Solvent (mL)

Chitosan PEG (µL)

(mg)

(mg)

50

1

0

0

B

50

1

50

0

C

50

1

50

D

50

1

50

0 0

20

10

us

20

an

696

0

cr

A

ip t

695

Ac ce p

te

d

M

697

39 Page 39 of 40

697

Table 2. Particle size, polydispersity index (PDI) and zeta potential of solid lipid

699

nanoparticles. Zeta Sample*

Particle size

PDI

ip t

698

potential 427.7 ± 52.3a

0.24 ± 0.02a

B

323.7 ± 61.5ab

0.17 ± 0.02bc

C

252.5 ± 24.8b

0.13 ± 0.03c

-15.9 ± 2.1b

D

269.0 ± 8.0b

0.21 ± 0.03ab

26.1 ± 0.5a

703

us

an

Large Aggregates

B

1132.8 ± 250.4a

0.14 ± 0.01b

-0.2 ± 0.0b

C

234.8 ± 32.8b

0.25 ± 0.04a

-0.3 ± 0.1b

D

277.9 ± 4.1b

0.27 ± 0.01a

13.2 ± 1.1a

d

M

A

* Results were expressed as mean ± standard deviation. Data sharing different superscript letters was significantly different within the same parameter among freshly prepared samples or samples incubated at SGF for 1 h.

Ac ce p

700 701 702

-26.3 ± 2.2c

te

After incubation at SGF for 1 h

-31.8 ± 3.0c

cr

A

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