Cross-Linked Fluorescent Supramolecular. Antifungal Drug A Therapeutic Approach for. Onychomycosis

Transcription

1 SUPPORTING INFORMATION Cross-Linked Fluorescent Supramolecular Nanoparticles for Intradermal Controlled Release of Antifungal Drug A Therapeutic Approach for Onychomycosis Fang Wang,,,# Peng Yang,, # Jin-sil Choi, Petar Antovski, Yazhen Zhu, Xiaobin Xu, ϕ,ð Ting-Hao Kuo, Li-En Lin,^ Diane N.H. Kim, Pin-Cheng Huang, Haoxiang Xu,,ᴪ Chin-Fa Lee, Changchun Wang,,* Cheng-Chih Hsu,^,* Kai Chen,,* Paul S. Weiss, ϕ,* Hsian-Rong Tseng,* State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai , China Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging (CIMI), California NanoSystems Institute (CNSI), Institute for Molecular Medicine (IMED), University of California, Los Angeles, Los Angeles, CA , United States 1

2 ϕ Department of Chemistry and Biochemistry, Department of Materials Science and Engineering, California NanoSystems Institute (CNSI), University of California, Los Angeles, Los Angeles, California 90095, United States Ð School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, , Singapore Molecular Imaging Center, Department of Radiology, Keck School of Medicine, University of Southern California, Los Angeles, CA , United States ^Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA 90095, United States Department of Chemistry, Research Center for Sustainable Energy and Nanotechnology, Innovation and Development Center of Sustainable Agriculture, National Chung Hsing University (NCHU), 145 Xingda Road, South Dist., Taichung 402, Taiwan ᴪ Department of Dermatology, Institute of Dermatology, Peking Union Medical College & Chinese Academy of Medical Sciences, 12 Jiangwangmiao Street, Xuanwu Dist., Nanjing , China # These authors equally contributed to this work. *Corresponding Authors: ccwang@fudan.edu.cn (C. C. Wang), ccrhsu@ntu.edu.tw (C.-C. Hsu), chenkai@med.usc.edu (K. Chen), psw@cnsi.ucla.edu (P. S. Weiss) and hrtseng@mednet.ucla.edu (H.-R. Tseng) 2

3 Contents 1. Characterization methods and settings. 2. Preparation of KTZ FSMNPs with various sizes by changing the Ad-PAMAM/CD-PEI ratios (w/w) 3. Preparation of KTZ c-fsmnps with various concentrations of BS3 4. Fluorescence spectra of KTZ c-fsmnps with various Ad-PAMAM/CD-PEI ratios (w/w) and various concentrations of BS3 5. KTZ encapsulation efficiency and capacity of KTZ FSMNPs or KTZ c-fsmnps 6. KTZ release profiles of KTZ FSMNPs or KTZ c-fsmnps 7. Quantification of residual drug concentration from tattoo sites on mouse skin 8. Visualizing KTZ drug in intradermal regions under the tattoo sites by MALDI-MSI 9. References 3

4 1. Characterization methods and settings. Dynamic light scattering (DLS). The hydrodynamic sizes of KTZ FSMNPs and KTZ c- FSMNPs were measured with a Zetasizer Nano instrument (Malvern Instruments Ltd., United Kingdom) equipped with a 10-mW helium-neon laser (λ = nm) and a thermoelectric temperature controller. Measurements were taken at a 90 scattering angle. The hydrodynamic sizes of KTZ FSMNPs and KTZ c-fsmnps were obtained by averaging the values of three or more measurements. Transmission electron microscope (TEM). The morphologies and sizes of KTZ FSMNPs and KTZ c-fsmnps were examined using a TEM. The studies were carried out on a Philips CM 120 electron microscope, operating at an acceleration voltage of 120 kv. TEM samples were prepared by drop-coating 2 L of sample suspension solutions onto carbon-coated copper grids. Excess amounts of solution were removed by filter papers after 45 s. Subsequently, the samples were negatively stained with 2% uranyl acetate for 45 s before TEM studies. UV-Vis absorption. The UV-Vis absorptions of free MPS-PPV, KTZ FSMNPs, and KTZ c- FSMNPs were obtained with a Hitachi UV-Vis system (Hitachi U-4100 spectrophotometer, Hitachi, Japan). The absorptions of free MPS-PPV, KTZ FSMNPs, and KTZ c-fsmnps were obtained from 350 nm to 800 nm with a scan speed of 10 nm /sec with 1nm intervals. Photoluminescence (PL). Steady-state photoluminescences of free MPS-PPV, KTZ FSMNPs, and KTZ c-fsmnps were measured using a custom-made PL system (HORIBA Jobin Yvon 4

5 system, Horiba, Japan). The solutions of MPS-PPV, KTZ FSMNPs, and KTZ c-fsmnps were excited by a Xenon lamp which filtered its wavelength using a monochromator with a wavelength of 490 nm. High-performance liquid chromatography (HPLC). KTZ concentrations were quantified by a HPLC system equipped with a Knauer Smartline pneumatic pump (Knauer, Berlin, Germany), C18 column, K-2600 spectrophotometer, and Gina-star 4 data acquisition software (Raytest, Straubenhardt, Germany). A mixture of acetonitrile and water (containing 0.05% diethylamine) at a volume ratio of 7:3 was used as the mobile phase. The flow rate was set at 1 ml/min. 25 μl of KTZ-containing sample was injected to measure the drug absorption at 227 nm, typically eluted in 4.3 min. The area of the HPLC peak of the KTZ was intergraded for the quantification of KTZ as compared to a calibration curve of free KTZ prepared separately. 2. Preparation of KTZ FSMNPs with various sizes by changing the Ad- PAMAM/CD-PEI ratios (w/w) The particle sizes of KTZ FSMNPs were controlled by altering the ratio of Ad-PAMAM/CD- PEI (w/w). To a solution of Ad-PEG (1.84 mg/ml) in 485-µL of PBS buffer, CD-PEI (0.8 mg/ml) was slowly added under vigorous stirring at room temperature. MPS-PPV (0.12 mg/ml) was then added sequentially and the mixture solution was stirred vigorously for 2 min. Then, a 5-µL aliquot of DMSO containing Ad-PAMAM ( mg/ml) and KTZ (0.4 mg/ml) was added into the mixture solution under vigorous stirring to obtain KTZ FSMNPs. The hydrodynamic sizes of 5

6 KTZ FSMNPs were measured using dynamic light scattering (DLS; Figure S1) and scan electron microscopy (SEM; Figure S2). The hydrodynamic size of KTZ FSMNPs increased from 240 ± 20 to 320 ± ± 30, 440 ± 30, 550 ± 50 and 680 ± 50 nm as the ratio of Ad-PAMAM/CD- PEI changed from 0.25 to 0.5, 1.0, 1.5, 2.0, and 2.5, respectively. Figure S1. Hydrodynamic sizes of KTZ FSMNPs with various sizes by changing the Ad- PAMAM/CD-PEI ratios (w/w): (a) 0.25:1, (b) 0.5:1, (c) 1.0:1, (d) 1.5:1, (e) 2.0:1, and (f) 2.5:1, respectively. 6

7 Figure S2. Scanning electron microscope images of the resulting KTZ FSMNPs with the mixing ratios of the two molecular building blocks (Ad-PAMAM/CD-PEI) (a) 320 ± 30 nm from 0.5/1, (b) 440 ± 30 nm from 1.5/1, (c) 680 ± 50 nm from 2.5/1. 3. Preparation of KTZ c-fsmnps with various concentrations of BS3 In order to fabricate cross-linked KTZ c-fsmnps, the 680-nm sized KTZ FSMNPs were mixed with various concentrations of BS3 (20, 40, 60, 80, and 100 μg/ml) at room temperature with vigorous stirring. After 15 min, Tris buffer (1 ) was added to the reaction solution in order to stop the cross-linking reaction of BS3. The hydrodynamic sizes of the resulting KTZ c-fsmnps were measured by DLS, showing the increasing particle sizes from 2200 ± 180 to 3500 ± 200, 4200 ± 220, and 4800 ± 230nm by altering the feeding concentrations of BS3 from 20, 40, and 60 to 80 µg/ml (Figure S3). 7

8 Figure S3. Hydrodynamic sizes of KTZ c-fsmnps with various concentrations of BS3: (a) 20 µg/ml, (b) 40 µg/ml, (c) 60 µg/ml, and (d) 80 µg/ml. 4. Fluorescence spectra of KTZ c-fsmnps with various Ad-PAMAM/CD- PEI ratios (w/w) and various concentrations of BS3 The fluorescence properties of KTZ c-fsmnps were examined. Under UV light irradiation, the KTZ c-fsmnps displayed dramatically enhanced fluorescence in comparison with the free MPS-PPV aqueous solution, which can be easily distinguished with the naked eye. (Figure S4a) Altering Ad-PAMAM/CD-PEI ratios (w/w) from 0.5:1 to 2.5:1 increased the fluorescent intensity of KTZ c-fsmnps with the highest fluorescent intensity at Ad-PAMAM/CD-PEI=0.25. (Figure 8

9 S4b) We chose this condition (Ad-PAMAM/CD-PEI=0.25) to test the effects of different crosslinker BS3 concentrations. With the increasing concentration of BS3, the highest fluorescent intensity can be obtained at the BS3 concentration of 80 µg/ml. (Figure S4c) Thus, the optimal fluorescent performance of KTZ c-fsmnps were obtained with a specific formulation of the mixing ratio among the molecular building blocks and cross-linker, i.e., Ad-PAMAM/CDPEI ratio (w/w) = 2.5:1 and 80 µg/ml of BS3. Figure S4. (a) Photograph of free MPS-PPV and KTZ c-fsmnps solution with the UV light irradiation (excitation = 365 nm). Fluorescence spectra of KTZ c-fsmnps (b) with various Ad- PAMAM/CD-PEI ratios (w/w), and (c) with various concentrations of BS3 for exploring optimal fluorescent performance at the synthetic condition of Ad-PAMAM/CD-PEI = 2.5 and crosslinking concentration of 80 µg/ml. 9

10 5. KTZ encapsulation efficiency and capacity of KTZ FSMNPs or KTZ c- FSMNPs A collection of 680-nm KTZ FSMNPs or 4800-nm KTZ c-fsmnps with various loading concentrations of KTZ ( mg/ml) was obtained according to the above protocol. Highperformance liquid chromatography (HPLC) was utilized to quantify the KTZ partition in both solution phase and KTZ FSMNPs. Typically, non-encapsulated KTZ was removed from KTZ FSMNPs by centrifugation of KTZ FSMNPs solution at 1300 rpm for 30 min using centrifugal filter devices (3000 NMWL). After recovering the filtrate containing non-encapsulated KTZ, the KTZ partition in the solution was analyzed by HPLC in a system equipped with a Knauer Smartline pneumatic pump, C18 column, K-2600 spectrophotometer, and Gina data acquisition software. A mixture of acetonitrile and water (containing 0.05% diethylamine) at a volume ratio of 7:3 was used as the mobile phase. The flow rate was set at 1 ml/min. 25 μl of KTZ-containing sample was injected to measure the drug absorption at 227 nm, typically eluted in 4.3 min. The area of the HPLC peak of the KTZ was intergraded for the quantification of KTZ as compared to a calibration curve of free KTZ prepared separately. The measurements were performed in triplicate. In order to obtain the KTZ partition in the KTZ FSMNPs directly, the KTZ FSMNPs were dissolved in the methanol for two days to disassemble the KTZ FSMNPs and to release the KTZ completely. The KTZ-containing solutions were filtered through 0.22 μm filters for HPLC analysis at a flow rate of 1 ml/min using the same method. The amount of the KTZ encapsulated 10

11 into KTZ FSMNPs was then calculated by subtracting the free KTZ in the filtrate from the total loading amount of KTZ. KTZ encapsulation efficiency of the resulting KTZ FSMNPs was obtained as the amount of the KTZ encapsulated in the KTZ FSMNPs vector divided by the total loading amount of KTZ. The KTZ encapsulation capacity of the resulting KTZ FSMNPs was obtained as the amount of the KTZ encapsulated in the KTZ FSMNPs vector divided by the total amount of KTZ FSMNPs. 6. KTZ release profiles of KTZ FSMNPs or KTZ c-fsmnps The 680-nm KTZ FSMNPs and 4800-nm KTZ c-fsmnps (10 mg, both the KTZ encapsulation capacities were 9.4 wt%) were each dispersed in 10 ml of 50% human serum (1:1 human serum: 1 PBS, v/v), and the suspension was divided into five equal aliquots. Each of the aliquot sample was then transferred into a dialysis tubing (molecular weight cut off 14,000), which was dialyzed against 18 ml of the 50% human serum, and then incubated at 37 o C under continuous and gentle shaking for 14 days. At selected time intervals, 1 ml of the solution was obtained periodically from the reservoir, and the amount of released KTZ was analyzed by HPLC equipped with an analytical C18 column. The area of the HPLC peak of the released KTZ was intergraded for the quantification of KTZ as compared to a calibration curve of free KTZ prepared separately. 1 ml of fresh buffer medium was added back to the reservoir after each sampling to keep a constant volume. All KTZ drug release data were averaged over three measurements. 11

12 7. Quantification of residual drug concentration from tattoo sites on mouse skin Three different amounts of KTZ c-fsmnps (i.e., 0.2, 1.0, and 2.0 mg) were tattooed at three adjacent locations on the back of the nu/nu mice (n = 3), following the typical tattooing protocol. Then, the tattoo sites were cut down and homogenized on ice. Following the extraction of tissue homogenate with 0.5 ml of methanol, the extracts were vortexed twice for 15 s and centrifuged at rpm for 10 min. The KTZ-containing supernatants were filtrated through 0.22 μm filters for HPLC analysis at a flow rate of 1 ml/min. As a result, residual drug concentrations were examined by quantification of the drug concentrations in solution using HPLC, which showed 6.84 µg, µg and µg of KTZ can be determined under the mouse skins with the tattooing treatment of KTZ c-fsmnps at the concentrations of 10 mg/ml (Figure S5a), 50 mg/ml (Figure S5b), and 100 mg/ml (Figure S5c). a b c Tattoo 20 µg KTZ 6.84 µg under skin Tattoo 100 µg KTZ µg under skin Tattoo 200 µg KTZ µg under skin Figure S5. Quantification of residual drug concentrations from tattoo sites on mouse skin under the treatment of KTZ c-fsmnps at different amounts of (a) 0.2 mg (equivalent to 20 µg of KTZ), (b) 1.0 mg (equivalent to 100 µg of KTZ), and (c) 2.0 mg(equivalent to 200 µg of KTZ) by HPLC analysis. 12

13 We then examined the intradermal retention times of KTZ c-fsmnps at selected time intervals prolonged for 14 days. The residual drug concentrations from mouse skin under the treatment of KTZ c-fsmnps can be quantified by HPLC, showing that the KTZ possessed a finite intradermal retention time up to 14 days. (Figure S6a) In comparison, the non-crosslinked KTZ FSMNPs in the same condition resulted in a residual drug concentration that exhibited a quick drug decay and that could only be detected within 3 days using HPLC. We also applied topical treatment of commercial KTZ topical cream (2%) to evaluate their effect to compare with KTZ FSMNP tattooing treatment. As shown in Figure S6c, although the KTZ cream itself can be quantified using HPLC, it was hard to detect the residual KTZ from topical treatment on mouse skin even after two-day applications. 13

14 a. KTZÌc-FSNPs treatment (i) Day 0 (ii) (iii) Day 1 Tattoo 200 µg KTZ µg under skin µg under skin Day µg under skin (iv) Day 7 (v) Day 10 (vi) 5.09 µg under skin 0.69 µg under skin Day µg under skin b. KTZÌFSNPs treatment (i) Day 0 (ii) Day 1 (iii) Tattoo 200 µg KTZ µg under skin µg under skin Day µg under skin c. KTZ cream (2%) treatment (i) (ii) (iii) Equal to 200 µg KTZ After two-day treatment Day 3 Figure S6. Quantification of residual drug concentration from mouse skin under the tattoo-guided treatments of (a) KTZ c-fsmnps, (b) KTZ FSMNPs, and topical treatment of (c) KTZ cream (2%) by HPLC analysis. 14

15 8. Visualizing KTZ drug in intradermal regions under the tattoo sites by MALDI-MSI Preparation of longitudinally sectioned tattooed skin slices The 4806-nm sized KTZ c-fsmnps with the concentration of 100 mg/ml was tattooed at a region of 5 5 mm 2 on the back of nu/nu mice, following the typical tattooing protocol. The tattoo sites were cut down and frozen at day 0 right after tattoo treatment. To obtain longitudinal section of skins, the frozen skins were sectioned into 12-μm thick slices using Leica CM3050 cryostat with object temperature at -18 C and chamber temperature at -16 C. The sectioning was performed by avoiding skin sections from making contact with optimal cutting temperature (OCT) compounds. The skin slices were subsequently mounted on an ITO-coated microscope glasses (2.5 cm 7 cm) with a conductive tape. Each skin slice had a longitudinal depth of sub-millimeters. After sectioning, the samples were stored under -80 C until further needed. Sample preparation and MALDI-MSI analysis of drug in intradermal skin The matrix application was achieved by sublimation method, which was described elsewhere, S1 followed by a recrystallization step. S2, S3 The sublimation apparatus was purchased from Singlong (Taichung, Taiwan). The apparatus was placed in a sand bath on a hot plate while applying matrix. Sublimation was performed using 150 mg of α-cyano-4-hydroxycinnamic acid (CHCA). The matrix was dissolved in methanol and transferred onto the bottom of the apparatus to create 15

16 homogenized layer of matrix. The matrix sublimation and application were performed at 120 C under vacuum at 0.7 Torr pressure for about 20 min. Amount of the applied matrices were determined by the exposure time. The CHCA-coated sections were rehydrated with 5% acetic acid solution in an oven at 80 C for 3.5 min. Instrumental parameters of MALDI-MSI Skin images were acquired using MALDI-TOF/TOF mass spectrometry (Autoflex Speed MALDI TOF/TOF system, Bruker Daltonics). The instrument was equipped with the third harmonic of Nd:YAG SmartBeamTM-II laser (355 nm). Imaging spectra were recorded and processed by flexcontrol 3.4 and fleximaging 3.0 (Bruker Daltonics). The spectra were acquired in linear mode and positive polarity with pixel-to-pixel resolution of 40 μm using the following parameters: detection gain of 15 (2617 V); laser attenuator offset at 75% of the maximum power; laser operating power at 17% with smart-beam parameter at 2_small; laser repetition rate at 1 khz; acquisition shots accumulated to 1,000 shots per pixel for imaging analysis. The resulting imaging spectra were processed using TopHat baseline subtraction. KTZ within the intradermal regions of the tattooed skin was identified by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The molecular ion of KTZ (chemical formula: C26H29Cl2N4O4; [M+H] + = m/z 531) was imaged in the day-0 tattooed skin slices. The drug distribution of a day-3 skin section was mapped along with a day-0 section, and the results have been showed in Figure S7. 16

17 However, only very weak signals of the residual drug were detected (slightly above the detection limit of the instrument) in the day-3 skin section. The residual drug concentration in the sections of the later time points were below the detection limit. This indicated that the intradermal drug decayed in the first 3 days after tattoo deposition, which agreed with the quantification results using HPLC in Figure S6a. Figure S7. Visualizing KTZ in the intradermal region of the tattooed skin slices by MALDI-MSI (left: day-0 section; right: day-3 section). MALDI-MSI was no longer able to detect KTZ in the skin sections harvested after day-3 due to the limited sensitivity of the technology. 9. References S1. Hankin, J. A.; Barkley, R. M.; Murphy, R. C. Sublimation as a Method of Matrix Application for Mass Spectrometric Imaging. J. Am. Soc. Mass Spectrom. 2007, 18,

18 S2. Yang, J.; Caprioli, R. M. Matrix Sublimation/Recrystallization for Imaging Proteins by Mass Spectrometry at High Spatial Resolution. Anal. Chem. 2011, 83, S3. Gemperline, E.; Rawson, S.; Li, L. Optimization and Comparison of Multiple MALDI Matrix Application Methods for Small Molecule Mass Spectrometric Imaging. Anal. Chem. 2014, 86,