PEGylated Dendrimers as Drug Delivery Vehicles for the. Photosensitizer Silicon Phthalocyanine Pc 4 for Candidal Infections

Transcription

1 Supporting Information For: PEGylated Dendrimers as Drug Delivery Vehicles for the Photosensitizer Silicon Phthalocyanine Pc 4 for Candidal Infections Melanie A. Hutnick 1, Sayeeda Ahsanuddin 2, Linna Guan 2, Minh Lam 2, Elma Baron 2, Jonathan K. Pokorski 1* 1 Department of Macromolecular Science & Engineering, Case Western Reserve University, 2100 Adelbert Road, Cleveland, OH, USA, Department of Dermatology, Case Skin Disease Research Center, Case Western Reserve University/University Hospitals Cleveland Medical Center, Cleveland, OH, USA, * jon.pokorski@case.edu

2 Figure S1. 1 H NMR spectrum of PAMAM G5, methoxypeg5000acetic acid, PEGylated PAMAM G5 nanoparticles. The spectrum was collected on 600 MHz NMR spectrometer in D2O. Integration of the PAMAM a protons to the PEG protons at 3.72 ppm indicate ~17 PEG chains were successfully conjugated to the amine termini of PAMAM G5. A) PEGylated PAMAM G5 nanoparticles B) methoxypeg5000acetic acid C) PAMAM G5. Synthesis of PEGylated and Maleimido Functionalized Nanoparticles: PAMAM in 5% methanol (1.018 ml; 1 µmol) was added to a round bottom flask and solvent was reduced under pressure. The dendrimer was dissolved in a minimal amount of DMSO. HCTU (0.223 g; 0.54 mmol), methoxypeg5000acetic acid (0.2 g; 0.04 mmol), and 6-maleimidohexanoic acid (0.013 g; mmol) were massed into individual scintillation vials and heated to dissolve

3 in a minimal amount of DMSO. The solutions were added to the round bottom flask and the solution turned a clear yellow-orange hue. DIEA was added (184 µl; 1.06 mmol) to the reaction and stirred overnight. The total reaction volume was 10 ml. The reaction mixture was transferred to a slide-a-lyzer and dialyzed at room temperature against 4L of ultrapure water. Solvent was removed under reduced pressure and the pure product was stored at ambient temperature until further use. Figure S2. 1 H NMR spectrum of Maleimido- and PEGylated PAMAM G5 nanoparticles. The spectrum was collected on 600 MHz NMR spectrometer in D2O. Integration of the PAMAM a protons to the PEG protons at 3.72 ppm and the maleimide protons at (d) and

4 (d) ppm indicate ~17 PEG chains and 19 6-maleinmidohexanoic acid groups were successfully conjugated to the amine termini of PAMAM G5. Michael Addition of the Chitin Binding Peptide (CBP) onto the Maleimido Functionalized Nanoparticles: Stock solutions of maleimido-, PEGylated particles (2 mg/ml; g; ~0.051 µmol) and CBP (1 mg/ml; g; 0.78 µmol) were prepared in separate scintillation vials in DMSO. A 0.35 mg (175 μl; µmol) aliquot of maleimido-, PEGylated particles was transferred to a round bottom flask and combined with a 0.5 mg (500 μl; 0.39 µmol) aliquot of the CBP. Triethylamine was added in 100x excess in regards to PAMAM (0.168 μl; mmol). The reaction was stirred overnight at room temperature and was dialyzed against 4L of ultrapure water in a 20,000 MWCO slide-a-lyzer. The solvent was reduced under pressure and pure CBP decorated PAMAM- PEG particles were stored at ambient temperature until further use.

5 Figure S3. 1 H NMR of CBP functionalized nanoparticles. All proton spectra were gathered using a 600 MHz NMR spectrometer in DMSO-d6. A) CBP functionalized PEGylated PAMAM G5 nanoparticle, the overlap at ppm in spectra A and C indicates the presence of CBP on the nanoparticle. B) Maleimido- functionalized PEGylated PAMAM G5 nanoparticles are the precursor to the CBP conjugation. The maleimide group acts as a handle for Michael addition conjugation. C) Chitin Binding Peptide. The peaks at ppm are attributed to the methyl protons of the valine side chains.

6 1.2 Relative Intensity Elution Volume (ml) Figure S4. Size Exclusion Chromatograms. Chromatograms were collected monitoring the absorbance at 220 nm. The traces exhibit a single peak indicating the purity of the compound to be > 95 %. Black = PAMAM, Green = PEG, Blue = PAMAM-PEG, Red = PAMAM-PEG- Maleimide Figure S5. Nanoparticle Characterization. Hydrodynamic radii of the maleimido- functionalized and CBP targeted particles were measured utilizing dynamic light scattering. The radii were A) Rh = 5.4 nm and B) 2.1 nm (Day 1), C) 13.8 nm (6 months), respectively. Pc 4 encapsulated in CBP particles shows aggregation over time. C) Digital photograph of Pc 4 encapsulated in CBP targeted particles. The decrease in hydrodynamic radius may indicate the CBP peptide collapsed onto the PAMAM core.

7 Absorbance (mau) Wavelength (nm) Figure S6. UV-vis spectra to determine the concentration of encapsulated Pc 4. Blue = Pc 4 stock solution; Black = encapsulated Pc 4; Red = PAMAM-CBP. 100 µl aliquot of encapsulated Pc 4 was reduced under pressure and redissolved in 2.5 ml DMF. The absorbance at λmax = 669 nm was measured. Using the Beer-Lambert Law and the molar absorptivity coefficient (ε = 2.4 x 10 5 M -1 cm -1 ) the concentration was determined to be µm. With the original mass of dendrimer used for encapsulation and the final concentration of Pc 4, the number of Pc 4 molecules per dendrimer was determined to be 11 for the untargeted PAMAM and 12.6 for PAMAM-CBP.

8 Figure S7. Cell Uptake Confocal Micrographs of CBP modified dendrimers encapsulated with Pc 4. C. albicans SC5314 was incubated with 2 µm at 37 ºC A) for 2 hours B) and 24 hours, respectively, and visualized by confocal microscopy. Pc 4 was found to penetrate the cell wall and diffuse into the cytoplasmic compartment of the cells.

9 Quantitative Cell Uptake via Flow Cytometry Flow cytometry was conducted to quantify the amount of cell uptake of the photosensitizer by C. albicans. A 100 µl aliquot of cells grown in YNBD were pipetted into 6-well plates and 900 µl of YNBD was added to each well. C. albicans were incubated with samples of free or encapsulated Pc 4 (targeted and untargeted) bringing the final concentration to 2 µm. The foil covered plates were transferred to a rocker in a humid incubated for 2 hours at 37 C. The samples were washed and transferred to 2 ml Eppendorf tubes and centrifuged at 2,500 rpm for 5 minutes at room temperature. The cell pellets were washed twice with 1 ml of 1X PBS ph ml of PBS was used to resuspend the cell pellets and transferred to 6-well plates. The cells were stained with Concanavalin A-488 to appropriately gate the system. After 15 minutes of incubation at 37 C, the cells were washed twice with 1 ml of 1X PBS ph 7.4 resuspended in 1 ml of YNBD to perform flow cytometry.

10 Figure S8. Quantitative Analysis of Cell Uptake by Flow Cytometry. The relative intensity of Pc 4 taken up by C. albicans 9652 R cells after incubating with 2 µm samples at 37 ºC for 2 hours. Plot of the forward scattering intensity versus the fluorescence intensity at 660 nm for A) cells only, 9,978 cells counted B) Pc 4 encapsulated in PAMAM-PEG nanoparticles, 11,389 cells counted C) Pc 4 encapsulated in PAMAM-CBP nanoparticles, 13,491 cells counted D) free Pc 4, 6,939 cells counted E) overlay of plots A-D. F) Histograms depicting the fluorescence intensity of Pc 4 taken up by C. albicans 9652 R cells. Red = Free Pc 4; Black = Pc 4 encapsulated in PAMAM- PEG nanoparticles; Blue = Pc 4 encapsulated in PAMAM-CBP nanoparticles; Green = Concanavalin A- 488 stained cells used for gating the system. According to the flow cytometry data, free Pc 4 was internalized by 99.7% of C. albicans whereas, the untargeted and targeted particles were taken up by 89.0% and 82.7%, respectively.

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12 Figure S9. Cellular Reactive Oxygen Activity Detection Assay. C. albicans 9652R were incubated with 3.5 µm Pc 4 encapsulated in CBP targeted PEGylated-PAMAM nanoparticles and 5 µm 2,7 -dichlorofluorescein diacetate (H2DCFDA). A-C) are micrographs before the cells were irradiated with the red light source. D-F) were imaged after irradiation. Red represents Pc 4 fluorescence, blue indicates DCF fluorescence, and purple is the overlay of these two phenomena. As a control, C. albicans 9652R were incubated solely with 5 µm H2DCFDA to confirm they do not naturally produce ROS. G-I) were imaged prior to irradiation with a red light source. J-L) were imaged after irradiation. The lack of red fluorescence indicates the cell are not naturally fluorescent, while the lack of blue signifies the cells do not produce ROS.

13 Figure S10. Clonogenic Assay. Sabouraud dextrose agar plates were treated with C. albicans that was previously incubated with 3.5 µm Pc 4 encapsulated in CBP-targeted PEGylated-PAMAM nanoparticles for 2 hours. A-C) Plates were not subjected to irradiation while D-F) were irradiated with 1.0 J/cm 2 of nm light. A fungal lawn formed on the plates suggesting the Pc 4 was active, but not as effective as the Pc 4 encapsulated in the PEGylated PAMAM nanoparticles. G) YNDB media plated on SD agar to confirm colony formation was not a result media contamination. The seven plates were incubated at 37 ºC for 24 hours. The following day the plates were inspected for cell growth and digital images were taken.

14 Figure S11. Dose response curve determined via a XTT assay. The blue curve represents the dose dependence of Pc 4 encapsulated in CBP-targeted PEGylated PAMAM nanoparticles incubated with C. albicans 9652 R and irradiated in with 1.0 J/cm 2 of nm light (IC50 = 3.49 x 10-7 M; 0.35 µm) (n = 3). *It is noteworthy that the IC50 dropped by 3 fold with incomplete killing of C. albicans. This result is likely due to the hydrophobic nature of the peptide, making the targeting agent more likely to collapse into the core rather than to be displayed on the corona of the particle.