Development and characterization of the voriconazole loaded lipid-based nanoparticles

Accepted Manuscript Title: Development and characterization of the voriconazole loaded lipid-based nanoparticles Author: Petra Furedi ¨ Zs´ofia Edit P´apay Krist´of Kov´acs Borb´ala Dalmadi Kiss Krisztina Lud´anyi Istv´an Antal Imre Klebovich PII: DOI: Reference:

S0731-7085(16)30761-0 http://dx.doi.org/doi:10.1016/j.jpba.2016.09.047 PBA 10884

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

28-8-2016 28-9-2016 30-9-2016

Please cite this article as: Petra Furedi, ¨ Zs´ofia Edit P´apay, Krist´of Kov´acs, Borb´ala Dalmadi Kiss, Krisztina Lud´anyi, Istv´an Antal, Imre Klebovich, Development and characterization of the voriconazole loaded lipid-based nanoparticles, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2016.09.047 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.

Development and characterization of the voriconazole loaded lipidbased nanoparticles

Petra Füredi, Zsófia Edit Pápay, Kristóf Kovács, Borbála Dalmadi Kiss, Krisztina Ludányi, István Antal, Imre Klebovich*

Semmelweis University, Department of Pharmaceutics, H-1092 Budapest Hőgyes Endre St 7. , Hungary *[email protected] phone and fax: +36 1 217 0914

Graphical abstract

Highlights  All of the novel VCZ-LNP formulations inhibit the reproduction of fungus.  FTIR revealed the chemical structure of VCZ was not affected by HPH.  The developed VCZ-LNP can serve as the base of ocular drug delivery system.

Abstract The number of topical fungal infections is growing,

mostly owing to

immunosuppressive therapy. Several topical fungal infections, such as eye mycoses, can be treated by local administration of antimycotic drugs. One major group of the antifungal agents is triazole, such as voriconazole (VCZ), which is used as the first line treatment of aspergillosis. A disadvantage of VCZ is its low water solubility making the drug difficult to administer in a liquid preparation. The lipid-based nanoparticles (LNP) have attracted increasing attention due to their advantageous properties. Contrarily to the conventional carrier systems, LNP can improve the poor solubility of topically used drugs, such as VCZ. Therefore, LNP represents promising alternatives to traditional carrier systems. The aim of the study was to formulate VCZ loaded lipid-based nanoparticles (VCZLNP) by high pressure homogenization (HPH). The developed LNPs were characterized by particle size analysis, IR spectroscopy, differential scanning calorimetry, dialysis test and antifungal efficacy studies. The particle size of the optimized nanoparticles from the selected lipid base, Witepsol® W35, was 182 ± 4.1 nm after five cycles of homogenization at 600 bar. The antifungal study confirmed that the optimized VCZ-LNP inhibited the fungus reproduction. Keywords: high pressure homogenization, particle size analysis, FTIR spectroscopy, differential

scanning

calorimetry

(DSC),

dialysis

test,

antifungal

study

1. Introduction The need to combat systemic and topical fungal infections is ever more important. This is underlined by the fact that the number of such infections has increased in the past years due to the prolonged use of immunosuppressants administered to treat transplant recipients or patients with HIV infection. To answer the necessity medicinal sciences related to their treatment has undergone significant changes, mostly owing to the new antifungal agents [1, 2]. One such example is the introduction of the antifungal agent voriconazole (VCZ), a second generation triazole derivative with potent broad-spectrum activity against various fungi. The action of mechanism of voriconazole involves its binding to fungal cytochrome P-450, leading to the inhibition of ergosterol synthesis, which is a vital step in cell membrane synthesis. VCZ is generally used as the first line treatment of invasive fluconazole-resistant aspergillosis, candidiasis and keratomycosis [3, 4]. In addition to its beneficial pharmacological activity, VCZ has a number of side effects, including vomiting, diarrhea, headache and unpredictable interactions with drugs and foods [5-7]. In clinical practice there is a general preference for local administration versus the systemic route because among others local application of the drug decreases undesirable effects. Therefore ophthalmic dosage forms are preferable to apply in fungal keratitis that follow after ocular trauma or contact lens wearing. As VCZ is not available as an eye-drop but is available in oral and parenteral dosage forms as brand name Vfend®. Dupuis et al. described that VCZ was suitable for treating the fungal infection of cornea. The authors used the parenteral solution of VCZ as an eye drop during their clinical research. Their observations revealed that ophthalmic administration is an adequate route to treat the fungal infection of the cornea [8, 9]. The study indirectly also points out the lack of a VCZ eye-drop on the market and thus the need for such a pharmaceutical preparation. A possible explanation of the lack of the dosage form might be the fact that VCZ exhibits low water solubility making the drug difficult to administer in liquid preparation. The marketed intravenous dosage form contains sulfobutylether-beta-cyclodextrin (SBECD) to complex VCZ in aqueous media [10]. Corneal epithelial cytotoxicity was observed above the concentration of 0.08 mg/ml after 1 day exposure to SBECD by the study of Sobolewska and coworkers [11]. Their results suggest that a new ocular drug delivery system for VCZ has great potential.

In the past decades several colloidal carrier systems for ophthalmic administration such as liposomes and different types of nanoparticles based on solid lipids have been evaluated. The mentioned lipid-based nanoparticles (LNP) can improve both the water solubility and the bioavailability of drugs. Out of the investigated systems solid lipid nanoparticles (SLN) offer unique properties, such as the few hundred nanometers particle size and high drug loading capacity because of their large surface area. Such SLN formulations have proved their viability in dosage forms intended for various routes of administration (oral, parenteral, dermal, ocular) and have led to the solution of various solubility-related problems of poorly soluble drugs (ofloxacin, tetrandrine, cyclosporine, gatifloxacin) in ophthalmic drug delivery systems [12-15]. Furthermore, SLN can improve the effectiveness of several drugs (ketoconazole, baicalin, tobramycin) because it can provide protection for the drug against the degradation and hence prolong exposure of the drug by controlled release [16-18]. Therefore, SLNs can be good candidates to encapsulate VCZ, potentially increase its water solubility and its therapeutic efficacy in the treatment of ophthalmic fungal infection. Various methods (microemulsion, ultrasonication, high pressure homogenization (HPH) exist for the preparation of LNP dispersion. Both of the techniques are suitable for producing lipid-based nanoparticles. The main advantages of the HPH method are that the scaling up can be achieved and generally the yield has very narrow particle size distribution (polydispersity index <0.2) [19]. The published VCZ loaded LNP were developed by microemulsion or ultrasonication technique [20-22]. Khare et al. used stearic acid as a lipid material to form ophthalmic gel with VCZ-SLN and compared the above mentioned techniques. Their results demonstrated that SLN produced by ultrasonication method is more suitable than microemulsion method because it was able to sustain the drug release for up to 12 hours. The optimized VCZ-SLN particle size was 274 ± 4.01 nm [22]. Andrade et al. prepared Compritol® and sorbitan trioleat based nanostructured carrier system (NLC), which is the second generation of LNP by microemulsion method. The particle size of the designed VCZ loaded NLC was almost the same value (281 ± 28.9 nm) as the results of Khare and coworkers [21]. Outcome of the studies suggests that the LNP particle size mostly depend on the applied technique and less on the selected lipids. Our research team previously described a novel nanoformulation of voriconazole. The aim of above mentioned investigation was to increase the water solubility of VCZ without using conventional surface active agents. We applied human serum albumin (HSA) during the drug formulation, because it is biocompatible and well tolerated by the human organism

without serious side-effects, such as toxicity or allergic reaction. The water solubility of the HAS bound VCZ was over two times greater, than the substance’s itself. The developed albumin nanoparticle can serve as the base of liquid parenteral dosage form containing voriconazole [23]. The objective of the study was to formulate and evaluate solid lipid nanoparticles of VCZ (VCZ-SLN) and to determine the critical parameters of preparation. In order to achieve these aims the effect of the applied solid lipid, the concentration of active pharmaceutical ingredient (API) and the concentration of lipid on the produced nanoparticles were investigated. Nevertheless, our ultimate aim was to develop a novel formulation of ophthalmic dosage form, which can be promising alternative treatment for topical fungal infections. Contrary to our anteriorly defined VCZ loaded albumin nanoparticles in case of VCZ-SLN conventional surface active agent such as polysorbate 80 was utilized. In order to the cornea can well tolerate the developed VCZ-SLN, the applied concentration of surface active agents was minimized [24, 25]. Furthermore, our aim was to optimize the high pressure homogenization method to prepare VCZ-SLN, to apply the optimized technology in the preparation of the VCZ-SLN and characterize the VCZ-SLN by particle size, particle size distribution, loading capacity and in vitro dissolution. 2. Materials and methods 2.1 Materials and reagents Voriconazole was purchased from BrightGene Bio-Medical Technology Co. Ltd., (China). Phosphate buffered saline (PBS), L-α-Phosphatidylcholine (PPC) and dialysis tubing cellulose membrane were purchased from Sigma-Aldrich (Hungary). Compritol® 888 ATO (glyceryl behante) was a kind gift from Gattefossè (France). Ethanol 96%, Witepsol® W35, polysorbate 80 and stearic acid were procured from Molar Chemical Ltd., (Hungary). All other reagents used in this study were of analytical grade. Amicon® Ultarcentrifugal filter devices with regenerated cellulose membrane (molecular weight cut off 30 kDa) were procured from Millipore Ireland Ltd. (Ireland). 2.2 Preparation of VCZ-SLN VCZ-SLN were prepared by high pressure homogenization. Firstly, PPC (0.03 g) was dissolved in 0.5 g of ethanol and 0.0075 g of polysorbate 80 solution. In the preparation methods utilized Witepsol® W35, stearic acid and Compritol® 888 ATO were chosen as lipids. The weight of the applied lipid (0.25, 0.50, 0.75 g) was molten at 10 °C above their melting

points and voriconazole was dissolved therein to obtain a clear lipid phase. Later surface active agents were added to the molten lipid and to this mixture 10 ml of hot MilliQ water (70 °C) was added under constant stirring (magnetic stirrer). The mixture was prehomogenized for 3 minutes by Ultra-Turrax homogenizer (IKA® Works Inc., Germany) at 20,000 rpm. The prehomogenized mixture was then further homogenized to form VCZ-SLN using an Avestin Emulsiflex B15 instrument (Avestin Europe GmbH, Germany) utilizing 600 bar (26,106 psi) inlet pressure for 3, 4 or 5 cycles. In the present study, the threshold parameters for the preparation of the VCZ-SLN dispersion were selected by studying the effect of the number of homogenization cycles, fraction and type of lipids, particle size, polydispersity index (PDI) and entrapment efficiency (EE). Efforts were directed toward minimizing the particle size and PDI and maximizing the entrapment efficiency. 2.3 Characterization of VCZ-SLN 2.3.1 Particle size measurement The mean particle size of the undiluted SLN dispersions and the PDI were obtained by photocorrelation spectroscopy using Malvern Zetasizer Nano Zs (Malvern Istruments Ltd, UK). Temperature and equilibration time were set to 25 °C and 120 s respectively, and cell type was PCS115 glass cuvette during the particle size measurement. 2.3.2 Differential scanning calorimetry (DSC) Accurately weighted samples (2 mg) were placed in non-hermetically sealed aluminum pans and heated at a rate of 10 °C/min to a maximum of 270 °C using Exstar 6000 DSC (Seiko Instruments Inc., Japan). An empty aluminum pan was used as reference. All measurements were performed in triplicates. The DSC analysis was carried out on the following samples: voriconazole, Witepsol® W35, PPC, VCZ-SLN, blank SLN and physical mixture (voriconazole and blank SLN). 2.3.3 Investigation of drug incorporation efficiency (IE) Entrapped drug concentration was determined indirectly. Drug IE was evaluated by an ultrafiltration technique with a 100 kDa centrifugal filter device composed of a regenerated cellulose membrane. An aliquot (0.5 ml) of undiluted SLN dispersion was placed into an Amicon ultracentrifugal filter. The sample was centrifuged for 10 minutes at 12000 g. The filtered aqueous phase contained the free API. The concentration of non-embedded drug was analyzed by HPLC (Agilent 1100, Agilent Technologies, USA) equipped with an Agilent C18

column (4.6 mm × 150 mm, 5 μm). 10 µl of the samples were injected. The mobile phase of 80% acetonitrile and 20% of 0.05 M sodium dihydrogen phosphate buffer was run isocratically at room temperature at a flow rate of 0.7 ml/min, and voriconazole was detected by UV detector at 254 nm. The linearity of the calibration curve was checked over the range of 100-1000 μg/ml (Fig.1.). The IE was calculated according to Eq.1.

IE (%) = [(Total drug content- Amount of free drug)/ Total drug content] x 100

(1)

2.3.4 Fourier transform infrared spectroscopy (FTIR) The FTIR spectra of VCZ, PPC, Witepsol® 35, blank SLNs, VCZ loaded SLN and physical mixtures of lipids and drug were recorded using FTIR spectrometer (Alpha Bruker Corp., USA, in the range of 4000–400 cm-1). 2.3.5 Dialysis test In vitro release of voriconazole from VCZ-SLN was investigated using a dialysis tube. 0.5 ml of the samples was placed into a dialysis bag and immersed in 25 ml PBS buffer. The system was maintained at 37 °C with a mixing rate of 100 rpm. 0.5 ml of the sample was withdrawn from the dissolution media at designated time intervals (5, 15, 30, 45, 60, 90, 120, 180, 240, 720, 1440 min) and the sample volume was replaced with fresh medium. Voriconazole concentrations of the sample were measured by the HPLC-UV method described above. 2.3.6 Antifungal study The antifungal activity was investigated by paper disc diffusion method. The antifungal activities of VCZ-SLNs were evaluated against C. glabrata and A. flavus. The fungal strains were maintained on agar slants (malt yeast agar media for C. glabrata, malt extract agar media for A. flavus). The sterile agar medium was inoculated with the fungal suspension. Then, sterile discs (made of Whatman No. 1 filter paper) each about 5 mm diameter impregnated with 2 µl SLN dispersion were placed on the Petri dishes. All the plates were incubated at 40 °C for 5-10 days. The zones of growth inhibition around the disc were measured after the specified period of incubation. Blank SLN was investigated as negative control. The clear zone of inhibition in each Petri dish was analyzed in mm.

3. Results and discussion 3.1 Optimization of the formulation of VCZ-SLN The primary set of experiments was aimed at investigating the influence of the key parameters such as HPH process on nanoparticle formation. Test results were consistent with those in the literature as 3-5 homogenization cycles with HPH at 300-1500 bar were needed to form nanoparticles [26, 27]. Most of the articles of previous authors used homogenization pressure of 600 bar to produce SLN. This does not increase the temperature of the samples too much (approximately 10 °C for 500 bar), therefore 600 bar homogenization pressure was applied during the development of our SLN to avoid the decomposition of VCZ and excipients [28]. It is commonly accepted, that increasing the number of homogenization cycles reduces polydispersity [29]. Low value of PDI, which quantifies distribution width, means that the population of nanoparticles tends to a monodispersity system. Fig. 2 shows the results of the homogenization cycles. It can be seen that the PDI decreased with 36.5% in case of SLN 5, therefore 5 homogenization cycles were preferable to apply during the homogenization process. The parameters of homogenization (pressure, number of cycles) not only influenced the average particle size of the nanoparticles but also the type of solid lipid [30]. Our results indicated that the type of solid lipid also had an impact on the size and size distribution of the prepared nanoparticles. At first, blank SLNs were investigated during the development. However, Khare et al. demonstrated that stearic acid as a lipid material was appropriate to produce SLN with good features and Andrade et al. published that Compritol ® based delivery systems were suitable carrier for VCZ [21, 22]. On the contrary, our outcome showed that these lipids were not fit with HPH method. Because the average particle size of the samples prepared with stearic acid or Compritol® 888 ATO size was more than 0.5 µm even after 5 homogenization cycles and demonstrated high polydispersity value (more than 0.8). The high PDI suggests that populations of SLN from these lipids were heterodisperse [31]. These results pointed out that the total amount of surface active agents was not enough to produce particles in the size range of a few hundred nanometers. Another possible explanation is that lipids with higher melting points cause a higher viscosity in the dispersed phase which in turn increases the average particle size of SLN [32]. At each concentration of Witepsol® W35 the particle size was less than 300 nm (Table 1). Smaller particle size can ensure low irritation, and can increase the compatibility with ocular tissue therefore the selected solid lipid was Witepsol® W35. In summary, 600 bar homogenization pressure and at least 5 homogenizing cycles produced adequate SLN from the selected lipid, Witepsol® W35.

After the determination of the parameters of HPH the effects of the concentration of lipid and VCZ on particle size were investigated. Particle size analysis and examination of the entrapment efficiency were utilized to aid the development process. Fig. 3 shows that increasing the concentrations of VCZ increased the polydispersity. Therefore, it is better to apply a lower concentration of VCZ to avoid the broad size distribution. Table 1 summarizes the effect of the concentration of VCZ and the concentration of lipid on PDI, EE and particles size. In cases of empty SLNs (SLN1, SLN5, SLN9), increasing the lipid content resulted larger particle size and higher value of PDI. Furthermore higher lipid concentration presence of the same VCZ concentration (SLN2, SLN6, SLN10) produced lower encapsulation efficiency. Therefore lower lipid content was preferable to apply during the formulation of VCZ loaded SLN. The encapsulation efficiency was mostly dependent on the concentration of VCZ. While the concentration of VCZ increased twofold from 50 mg to 100 mg, the EE increased by 10%. These results suggest that 50 mg VCZ is ideal to form SLNs due to its low polydispersity and high EE (SLN 2). 3.2 Differential scanning calorimetry DSC analysis was performed to study the physical state of powders. DSC curves of voriconazole, Witepsol® W35, placebo SLNs, its physical mixture and VCZ-SLNs (SLN 2) are shown in Fig. 4. The melting point of voriconazole was observed at 127 °C (Fig. 4.C) while that of Witepsol® W35 was recorded at around 35 °C (Fig. 4.A) due its heterogeneous structure. DSC curve of blank SLNs, which was containing other excipients besides Witepsol® W35 showed a lower melting point than alone. The presence of surfactants and lipid recrystallization during the process of HPH could decrease its melting point from 38 °C to 32 °C (Fig. 4.B). Thermograms of VCZ-SLN (Fig. 4.E) were similar to empty nanoparticles, no peaks of VCZ, only the thermal characteristics of Witepsol® W35 were observed. A possible explanation is that VCZ did not exist in the nanoparticles in a crystalline structure rather encapsulated to nanoparticles. This is underlined by the curve of the physical mixture of VCZ and Witepsol® W35 where features of both are seen (Fig. 4.D). 3.3 Fourier transform infrared spectroscopy FTIR was used to examine the chemical structure changes for VCZ. The analysis was used to detect the possible interactions between phosphatidylcholine, Witepsol® W35 and VCZ in the complex. The spectrum of VCZ (Fig. 5.A) exhibited characteristic peaks, C-O

stretching at 1128 cm-1, O-H streching at 3208 cm-1 aromatic banding vibration at 856 cm-1, 778 cm-1, 667 cm-1 and 663 cm-1, C-C aromatic stretching band at 1598 cm-1 and 1587 cm-1, C-N stretching at 1279 cm-1. In the spectrum of PPC (Fig. 5.B) the characteristic C-H streching band of long fatty acid chain at 2920 cm-1 and 2827 cm-1, C=O streching band at 1734 cm-1, P=O streching band at 1237 cm-1, P-O-C streching band at 1058 cm-1, N+(CH3)3 streching at 974 cm-1 were identified. In the spectrum of Witepsol® W35 (Fig. 5.C) C-H streching band of long fatty acid chain were dominated at 2920 cm-1 and 2850 cm-1, C=O streching band at 1727 cm-1 and 1740 cm-1. The spectrum of the physical mixture (Fig. 5.D) showed a summation of the vibrational frequencies of the individual components. The spectrum of SLN blank (Fig. 5.E) showed a summation of the vibration frequencies of the individual excipients (Fig. 5.C, 5.B). This suggested that there was no recrystallization and interaction between auxiliary materials during the formulation with HPH. In the FTIR spectrum of the VCZ-SLN (Fig. 5.F) there were no appearances or disappearances of any characteristic peaks. Although both the aromatic banding vibration and O-H stretching of VCZ were decreased extremely, they did not disappear. There were some other significant changes, the absorption peaks of C-O of VCZ shifted to a higher wavenumber (from 1128 cm1

to 1147 cm-1). The spectroscopic changes showed that C-O-H bands and aromatic rings of

VCZ interacted with the vehicles and this suggested that the chemical structure of VCZ had not changed after forming SLN. 3.4 Dialysis test Dynamic dialysis curves are presented in Fig. 6. The cumulative release of voriconazole during the initial 60 min was 22.98±0.23%. The rest of VCZ in the VCZ-SLNs (SLN 2) was continuously released over 5 h. The first 5 h release can help the SLNs to reach the therapeutic concentration. After 24 h almost 50% of the incorporated drug was dissolved. The in vitro release study verified that the encapsulated API is able to be liberated from the nanoparticles. 3.5 Antifungal study Antifungal activity of VCZ-SLN was proved by disk diffusion test. Clear zone of inhibition was observed as shown in Fig. 7. Clear zone of inhibition was not observed in case of empty SLN. This suggested that the excipient has no antifungal activity against A. flavus and C. glabra. The diameter of inhibition zones were not dependent on the formulations which proves that SLN 2 and SLN 6 formulations contained the same amount of drug and there was

no significant difference between their inhibition zones (42 ± 1.5 mm; 44 ± 2 mm; p>0.05, n=3). Clear zone of inhibition against A. flavus and C. glabra indicated antimicrobial efficacy of VCZ-SLN formulations.

4. Conclusions Voriconazole, a poorly water soluble triazol with a broad antifungal spectrum, was successfully encapsulated to LNP by HPH. Applying five homogenizing cycles at 600 bar pressure resulted in VCZ-loaded nanoparticles with low PDI. After characterization of SLN we optimized the parameters to form VCZ-SLN. Both DSC analysis and FTIR spectroscopy results indicated the formation of SLN. Dialysis test and antifungal study verified that the embedded VCZ is released from nanoparticles, and the released API could inhibit the reproduction of fungus. The novel SLN drug delivery system, described herein, represents a promising alternative treatment for ophthalmic infections.

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List of Figure Captions

Figure 1. HPLC calibration curve of VCZ Figure 2. Effect of number of homogenization cycles on polydispersity index (Witepsol® W35 =0.5 g) Results are depicted as mean±SD ( n=3) Figure 3. Effect of drug concentration on polydispersity index (Witepsol® W35 =0.25 g; number of homogenization=3) Results are depicted as mean±SD ( n=3) Figure 4. Differential scanning calorimetry thermograms of A: Witepsol® W35, B: Blank SLN; C: VCZ; D: Physical mixture of Blank SLN and VCZ; E: VCZ-SLN (SLN 2) Figure 5. FTIR spectrum of A: VCZ; B: PPC; C: Witepsol® W35; D: Physical mixture; E: blank SLN; F: VCZ-SLN (SLN 2) Figure 6. Release profiles of VCZ loaded SLN (SLN 2) Figure 7. Antifungal effect of VCZ-SLN (SLN 2, SLN 6) on Candida glabra and Aspergillus flavus (n=3)

Tables

Table 1. Effect of Witepsol W35® and drug concentration on mean particle size, PDI and encapsulated efficiency (n=3) Formulations

Witepsol W35 (g)

VCZ

Particle size Polydispersity Encapsuleted efficiency

(mg)

(nm)

(%)

SLN1

0.25

-

139 ± 3.2

0.261 ± 0.01

-

SLN2

0.25

50

182 ± 4.1

0.269 ± 0.01

78.79 ± 3.4

SLN3

0.25

100

215 ± 7.4

0.402 ± 0.02

89.20 ± 4.2

SLN4

0.25

150

212 ± 5.6

0.374 ± 0.02

92.99 ± 6.9

SLN5

0.50

-

179 ± 4.8

0.266 ± 0.01

-

SLN6

0.50

50

216 ± 3.6

0.373 ± 0.01

72.27 ± 5.8

SLN7

0.50

100

255 ± 3.9

0.579 ± 0.03

88.82 ± 2.4

SLN8

0.50

150

281 ± 8.5

0.615 ± 0.03

92.55 ± 1.4

SLN9

0.75

-

182 ± 7.3

0.313 ± 0.01

-

SLN10

0.75

50

189 ± 6.7

0.364 ± 0.01

70.89 ± 5.2

SLN11

0.75

100

202 ± 14.2

0.422 ± 0.02

85.86 ± 7.1

SLN12

0.75

150

218 ± 11.8

0.360 ± 0.02

90.99 ± 5.1