Article pubs.acs.org/Biomac
PEGylated Dendrimers as Drug Delivery Vehicles for the Photosensitizer Silicon Phthalocyanine Pc 4 for Candidal Infections Melanie A. Hutnick,† Sayeeda Ahsanuddin,‡ Linna Guan,‡ Minh Lam,‡ Elma D. Baron,‡ and Jonathan K. Pokorski*,† †
Department of Macromolecular Science and Engineering, Case Western Reserve University, 2100 Adelbert Road, Cleveland, Ohio 44106, United States ‡ Department of Dermatology, Case Skin Disease Research Center, Case Western Reserve University/University Hospitals Cleveland Medical Center, Cleveland, Ohio 44106, United States S Supporting Information *
ABSTRACT: Fungi account for billions of infections worldwide. The second most prominent causative agent for fungal infections is Candida albicans (C. albicans). As strains of fungi become resistant to antifungal medications, new treatment modalities must be investigated to combat these infections. One approach is to employ photodynamic therapy (PDT). PDT utilizes a photosensitizer, light, and cellular O2 to produce reactive oxygen species (ROS), which then induce oxidative stress resulting in apoptosis. Silicon phthalocyanine Pc 4 is a photosensitizer that has exhibited success in clinical trials for a myriad of skin diseases. The hydrophobic nature of Pc 4, however, poses significant formulation and delivery challenges in the use of this therapy. To mitigate these concerns, a drug delivery vehicle was synthesized to better formulate Pc 4 into a viable PDT agent for treating fungal infections. Utilizing poly(amidoamine) dendrimers as the framework for the vehicle, ∼13% of the amine chain ends were PEGylated to promote water solubility and deter nonspecific adsorption. In vitro studies with C. albicans demonstrate that the potency of Pc 4 was not hindered by the dendrimer vehicle. Encapsulated Pc 4 was able to effectively generate ROS and obliterate fungal pathogens upon photoactivation. The results presented within describe a nanoparticulate delivery vehicle for Pc 4 that readily kills drug-resistant C. albicans and eliminates solvent toxicity, thus, improving formulation characteristics for the hydrophobic photosensitizer.
■
these traditional therapeutics obsolete.14,15 To tackle this issue, photodynamic therapy (PDT) was explored. PDT was discovered over 100 years ago in microbiology and was explored as a treatment for infections prior to the advent of modern-day antimicrobial therapies.16 PDT uses a photosensitizer that is activated in the presence of light to cause cytotoxic reactions. Certain photosensitizers function in the presence of molecular oxygen, while others act directly to damage DNA.17,18 Those that function in the presence of molecular oxygen (3O2) form reactive oxygen species (ROS), most commonly singlet oxygen (1O2) and superoxide (O2−), to cause oxidative stress, which damages cellular DNA and destroys cells via apoptosis. As pathogen resistance to modern-day antibiotics and antifungal medications increases, so has the interest in PDT.16 To date, there are few known reports of bacterial or fungal PDT resistance, and although mechanisms of resistance are yet to be thoroughly explored, PDT is being considered as a viable treatment option for microbial infections.16,19−24
INTRODUCTION Of the nearly 1.5 million species of fungi in the world, 300 species are responsible for >1.7 billion infections worldwide.1−9 Of these, Candida spp. are the second most prevalent pathologic agents.6 Candida albicans (C. albicans) are a species of yeast-like fungi, which exist as part of the normal human gastrointestinal and skin flora. They exist in a symbiotic relationship with other bacteria and fungi; however, when homeostasis is disrupted, the relationship switches from commensalistic to opportunistic and pathogenic.10−12 This often occurs in immunocompromised patients whether they be infants, the infirmed, the acutely ill, or the neutropenic. In humans, C. albicans is commonly responsible for candidiasis, a type of fungal infection that affects skin, nails, and mucous membranes. The polymorphic nature of C. albicans is an important characteristic for its pathogenesis, allowing for the reversible morphogenesis from the blastospore morphology to a hyphal state.10,11 As the unicellular yeast cells transition to the filamentous, pseudohyphae, or true hyphae, the hyphae can invade tissues and keratinized epithelial networks, while the blastospores disperse throughout the tissue.10,13 Candida infections pose a health threat as they can be resistant or tolerant to many antifungal medications including azoles (e.g., fluconazole) and polyenes (e.g., amphotericin B), rendering © XXXX American Chemical Society
Received: September 26, 2016 Revised: January 5, 2017
A
DOI: 10.1021/acs.biomac.6b01436 Biomacromolecules XXXX, XXX, XXX−XXX
Biomacromolecules
■
There are several advantages to incorporating PDT into a treatment plan. It is an outpatient procedure that is less invasive than surgery and can be applied countless times to the targeted region with minimal long-term side effects. PDT and dermatological conditions were revolutionized by the development of topical PDT agents.25 Today, PDT is currently used in conjunction with other therapies to treat actinic keratosis, Bowen’s disease, nonmelanoma skin cancer, and acne vulgaris, among other skin conditions. Porfimer sodium and 5aminolevulinic acid (ALA) are the only FDA approved photosensitizers commercially available. Porfimer sodium is best known for its treatment of small cell lung carcinoma; however, systemic injections cause long-term photosensitivity.26 ALA, by contrast, is a prodrug that must be metabolically converted to the photosensitizer protoporphyrin IX (PpIX). This requires a longer incubation time in the patient to allow the conversion to occur. The photosensitizer silicon phthalocyanine Pc 4, described within, is activated by visible light (λmax = 675 nm) in the far red region, allowing for enhanced tissue penetration. Furthermore, Pc 4’s greater molar absorptivity at a longer wavelength (ε = 2 × 105 M−1 cm−1) than PpIX (λmax = 630 nm; ε ∼ 5 × 103 M−1 cm−1) indicates Pc 4 can absorb a greater number of photons at a deeper tissue penetration.27 Unsurprisingly, Pc 4 has shown promise in several Phase I clinical trials for the treatment of a variety of conditions. The primary concern hindering Pc 4’s progression in clinical use is the hydrophobicity of the drug; its partition coefficient (log P) is 2.01.28 Currently, Pc 4 is solubilized and administered in a potentially toxic organic solvents solution. The solubility of most efficient photosensitizers in nontoxic solvents remains a technological challenge to general acceptance of new PDT agents. This issue is paramount for the future of PDT. Herein, a polymeric nanoparticle that disperses Pc 4 in pure water is developed to combat candidiasis, addressing formulation concerns and improving safety. Dendrimers (“dense stars”) are globular, tree-like branched macromolecules that have been used extensively for formulating and delivering hydrophobic drugs. Dendrimers are monodispersed nanoparticles made by an iterative synthetic strategy, which is advantageous for drug delivery as uniform size promotes reproducible and reliable biological data. Herein, we describe the encapsulation of Pc 4 into PEGylated dendrimers and its efficacy as a PDT agent against C. albicans. Particles were designed to be of small size and charge, shielded by the hydrophilic polymer polyethylene glycol (PEG). Additionally, chitin-targeted dendrimers were synthesized. Chitin, a polysaccharide formed through β(1,4)-linked N-acetylglucosamine units, is an essential component to fungal cell walls.29 Its primary purpose is to provide strength and toughness to the cell wall; however, its absence from mammalian cells provides a unique handle for developing a targeted drug delivery system. A chitin binding peptide, identified via phage display, was conjugated onto PEGylated dendrimers to develop the first chitin targeted drug delivery vehicle. Pc 4 was efficiently encapsulated within the delivery vehicles in pure water or buffer, eliminating potential toxicity concerns of previous formulations. Finally, we demonstrate that both free and dendrimer-encapsulated Pc 4 show equivalent potency in cellkilling assays against C. albicans. This work provides a nontoxic and efficacious formulation alternative for topical treatment against C. albicans that could be easily extended to other dermatologic diseases.
Article
EXPERIMENTAL SECTION
Materials. Methoxypoly(ethylene glycol) acetic acid (methoxyPEG5000 acetic acid, MW 5000 Da, ≥80%) was purchased from SigmaAldrich. Solutions of 5% poly(amidoamine) generation 5.0 dendrimers with ethylenediamine cores (PAMAM G5) and 6-maleimidohexanoic acid (90%) were purchased from Aldrich. O-(H-6-chlorobenzotriazol1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HCTU, 99.7%) was purchased from Chem-Impex International, Inc. Dimethyl sulfoxide (DMSO, > 99.7%) was acquired from Fisher Scientific. N,NDiisopropylethylamine (DIEA, > 98%) was obtained from Acros Organics. Genscript was commissioned to synthesize the Chitin Binding Peptide (CBP Sequence: CEGKGVEAVGDGR, >90%, Piscataway, NJ). XTT Cell Proliferation Assay Kit was purchased from American Type Culture Collection (ATCC, Manassas, VA). H2DCFDA was purchased from Invitrogen (Carlsbad, CA). Pc 4 was generously provided by Dr. Malcolm E. Kenney. C. albicans 9652 R and SC5314 were kindly provided by Dr. Mahmoud A. Ghannoum. Instrumentation. 1H NMR spectra were collected using a 600 MHz Varian Inova NMR spectrometer. The purity of the conjugates was assessed via Size Exclusion Chromatography (SEC) conducted on a GE Healthcare Ä KTAFPLC 900 chromatographer fashioned with a Sephacryl 1000 SF 10/300 size exclusion column. The mobile phase was 50 mM phosphate buffer, 150 mM NaCl (pH 7.4) at a flow rate of 0.4 mL/min. Samples were prepared in the mobile phase buffer at a concentration of 1 mg/mL and filtered with cellulose acetate syringe filters (0.45 μm) prior to analysis. Hydrodynamic radius was measured via dynamic light scattering on a Wyatt DynaPro NanoStar instrument at 25 °C using 10 mm path length plastic cuvettes. UV−vis spectra were collected using a PerkinElmer Lambda 800 spectrometer utilizing a 10 mm path length quartz cuvette. UV absorbance of 96-well plates were analyzed with a spectrophotometer (Spectronic Genesys 5 Analytical Instrument). Cell assays were conducted using an incubator (Forma Scientific) and rocker (Bellco Glass Inc.). Assays that involve irradiation were executed using red light emitting diode (LED) array (EFOS) and measured with a J16 digital photometer (Tektronix). Cell imaging was conducted using glass slides and #1.5 thickness cover glass. Confocal micrographs were obtained with a Leica TCS SPE confocal microscope with immersion oil for 40× or 63× objectives equipped with 488 and 635 nm solid state diode lasers. Methods. Synthesis of PEGylated-PAMAM Dendrimers. PAMAM in 5% methanol solution was added to a round-bottom flask (1.02 mL; 1 μmol) and solvent was removed under reduced pressure. The dendrimer was dissolved in a minimal amount of DMSO. MethoxyPEG5000 acetic acid (0.201 g; 0.04 mmol) and HCTU (0.223 g; 0.54 mmol) were each massed into separate scintillation vials and heated to dissolve in a minimal amount of DMSO. All reagents were combined in a round-bottom flask and the reaction mixture turned a clear yellow solution. DIEA was added (184 μL; 1.06 mmol) and the reaction was stirred overnight. Total reaction volume was 10 mL. The reaction was transferred to a slide-a-lyzer (Life Technologies 3−12 mL MWCO: 20000) and dialyzed against 4 L of ultrapure water (18 MΩ·cm) at room temperature. Solvent was removed under reduced pressure and pure product was stored at ambient temperature until further use. Nuclear magnetic resonance spectroscopy was utilized to study the chemical structure of the functionalized particles. 1 H NMR, (600 MHz, D2O, ppm): δ PEG (−CH2CH2O−) = 3.72 (s); δ PAMAM (−CH2CONH−) “a” = 2.46 (b); δ PAMAM protons = 3.30 (b) 2.5∼2.9 (b). Pc 4 Encapsulation. Stock solutions of Pc 4 (10 mg/mL) were prepared in DMF. Pc 4 stock solution (2.5 mL) was diluted with H2O (2.5 mL) to form a 5 mg/mL H2O/DMF (1:1) solution. Dendrimer stock solutions (0.3 mg/mL) were prepared in a 1:2 DMF/H2O mixture (3 mL). The dendrimer solution was slowly added to the Pc 4 solution. The solution was transferred to a 20000 MWCO slide-a-lyzer and dialyzed against 1 L of ultrapure water (2 h, 4×) at room temperature in a foil-covered beaker. Determining the Concentration of Pc 4 Encapsulation. UV−vis spectroscopy was used to determine drug concentration. To most accurately assess the concentration of encapsulated Pc 4, a 100 μL B
DOI: 10.1021/acs.biomac.6b01436 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
Figure 1. Synthesis and characterization of PEGylated nanoparticles. (A) Scheme for the functionalization of PAMAM G5.0 dendrimers; R = one G5 arm. (B) 1H NMR spectrum of PEGylated-PAMAM particles (D2O, 600 MHz, 512 scans). (C) DLS data confirming the size of the PEGylated nanoparticles in H2O before and after encapsulation with Pc 4. (D) Pc 4 dissolved in DMF (left) and water-solubilized Pc 4 encapsulated in PEGylated-PAMAM nanoparticles (right). total of 10 μL of PBS was used to resuspend the cell pellet for confocal imaging. Cellular Reactive Oxygen Species Detection Assay. Intracellular ROS, was monitored by the conversion of nonfluorescent 2′,7′dichlorodihydrofluorescein diacetate (H2DCFDA) to fluorescent 2′,7′dichlorofluorescein (DCF). A 250 μL aliquot of cells cultured in YNBD broth were pipetted in six-well plates. Additionally, 250 μL of the Pc 4 encapsulated particles were added to the wells. The foilwrapped plate was placed on a rocker for 2 h in a humid, 37 °C incubator. Cells were washed twice with 500 μL of 1× PBS pH 7.4 and returned to the plate. H2DCFDA (5 μM final concentration) was added to the cells and allowed to incubate for 15 min at room temperature. The cells were pelleted and resuspended in a minimal amount of PBS. A total of 5−10 μL of the cell suspension was placed on a glass slide and cover glass (#1.5 thickness). Live cells were imaged with a solid state diode 635 nm laser to confirm Pc 4 was internalized. Upon verification of Pc 4’s uptake, the glass slide was irradiated with a red light emitting diode (LED) array at a dose of 0.2 J/cm2 (fluence rate of 1.0 mW/cm2, λmax(Ex) of ∼670−675 nm) at room temperature. Cells were imaged postirradiation with a solid state diode 488 nm laser to visualize DCF. Control experiments included incubation solely with H2DCFDA with and without irradiation by the red LED array. Clonogenic Assay. To qualitatively assess cell viability upon treatment with the particles and irradiation, a clonogenic assay was performed. Sabouraud dextrose agar plates were prepared by suspending 65.0 g of powder (Becton Dickinson and Co.) in 1 L of Milli-Q water and autoclaving to 121 °C for 15 min. The solution was cooled to ∼50 °C for handling and poured into sterile Petri dishes in a
aliquot of the Pc 4 was evaporated under reduced pressure, dissolved in DMF, and the concentration was determined spectrophotometrically using known molar absorptivity values (ε = 2.4 × 105 M−1 cm−1 at λmax = 669 nm). Organism and Culture Conditions. C. albicans 9652 R and SC5314 isolates were thawed from cryostocks and plated onto a Sabouraud dextrose (SD) agar dish. A loopful of colonies was grown in 10 mL of nutrient rich SD broth in a 50 mL polypropylene tube at 37 °C for 18−24 h, shaking at 150−200 rpm. SD broth was prepared by dissolving 15 g of SD powder in 500 mL of Milli-Q water followed by autoclaving at 121 °C for 25 min, cooling to room temperature and storing at 4 °C.30 For experiments, C. albicans were cultured in a synthetic media yeast nitrogen base dextrose (YNBD). Yeast nitrogen base (3.35 g) and dextrose (4.505 g) were dissolved in 500 mL of sterile Milli-Q water and was autoclaved at 121 °C for 25 min. From the SD culture, a 100 μL aliquot was transferred to 10 mL of YNBD in a sterile 50 mL polypropylene tube in a biological safety cabinet. The tube was placed in an incubator shaker at 37 °C for 18−24 h at 150−200 rpm.30 C. albicans Uptake Studies. 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. Solutions of appropriate concentrations of encapsulated Pc 4 samples in water were added, bringing the final volume to 2 mL. The plates were covered with foil and transferred to a rocker in a humid, 37 °C incubator. Samples were removed from the wells at their indicated time points, transferred to 2 mL Eppendorf tubes, and centrifuged at 2500 rpm for 5 min at room temperature. The cell pellets were washed twice with 1 mL of 1× PBS pH 7.4. A C
DOI: 10.1021/acs.biomac.6b01436 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
to the PEG protons indicate approximately ∼13% functionalization, which correlates well with the targeted ratio of 20% (Figures 1B and S1).34 1H NMR spectra for maleimido- and chitin-targeted particles were collected as well (Figures S2 and S3). The purity was assessed via Size Exclusion Chromatography at 220 nm. Each chromatogram revealed a single peak indicating the nanoparticles were pure and residual reagents were removed by dialysis (Figure S4). Particle size was measured by dynamic light scattering (DLS). The radius of PAMAM G5 in water is reported to be approximately 2.7 nm.32 Once PEGylated, DLS results indicate an increase of the hydrodynamic radius to 7.0 nm. DLS results agree with expected values, since the hydrodynamic radius of linear PEG 5000 is ∼2.3 nm.35 These PEGylated particles were used to encapsulate Pc 4 via dialysis against water. The mechanism of encapsulation is driven by host−guest supramolecular interactions.36 The hydrophobic drug is mixed with the dendrimer solution, allowing the hydrophobic guest to interact with the nonpolar core of the dendrimer host.37 Dendrimer architecture is of paramount importance when designing a delivery vehicle. The internal voids govern the degree of drug loading. The backbone of lower generation dendrimers (G0.5−G3.5) are dynamic, making them inefficient at encapsulating and retaining guest molecules, whereas dendrimers that are too high in generations (G6.5+) undergo de Gennes dense packing, preventing drug molecules from interacting with core.32 Dendrimers between G4−G6 are dynamic enough to allow guest molecules interact with core, while maintaining enough rigidity to retain the drug molecules in the voids.32 For this particular system, the hydrophobic nature of Pc 4 binds to the PAMAM core and drives out the water, causing the core to collapse onto the photosensitizer.36−41 This collapse causes a significant decrease in hydrodynamic radius to 3.4 nm (Figures 1C and S5A,B). Though encapsulation of Pc 4 in water has a profound effect on the dendrimer size, water does not seem to greatly impact the absorbance spectrum. Due to the high degree of symmetry of the phthalocyanine ring (∼D4h), Pc 4 is relatively resistant to solvatochromatic shifts.9 Pc 4 solubilized in DMF and encapsulated Pc 4 solubilized in water are depicted below (Figures 1D and S5C). Additionally, based on the concentration of dendrimer utilized for encapsulation and the final concentration of Pc 4 as determined by UV−vis spectroscopy (Figure S6), there are ∼11 molecules of Pc 4 per dendrimer. Additionally, chitin targeted dendrimers were designed and synthesized utilizing a chitin binding peptide identified via phage display, PAMAM dendrimers, and PEGylation.42 Unfortunately, these particles hindered cell uptake by C. albicans and decreased PDT efficacy. There are few reports of targeted drug delivery vehicles for fungal infections in the literature, even though this is an increasing burden to the medical system. While the results are negative, it is important to consider new ways to target nanoparticulate systems to pathogenic fungi. Therefore, the results of the experiments conducted with the chitin targeted particles are included in the Supporting Information. Experiments conducted with the solely PEGylated dendrimers are described hereafter. Cell Uptake Studies. For PDT to provide any benefits, the cells must first internalize the photosensitizer. It is important that both the blastospore and hyphal morphologies take up the drug because both are implicated in the pathogenesis of candidal infections. Hyphae are able to penetrate the tissue, while the blastospores disperse the pathogen throughout the
biological safety cabinet to prevent contamination. Plates were stored at 4 °C and brought to room temperature prior to use. A 500 μL aliquot of C. albicans 9652R cultured in YNBD was transferred to a foil-wrapped, six-well plate. A total of 500 μL of encapsulated Pc 4 was added (final concentration 3.5 μM) and allowed to incubate for 2 h on a rocker in an incubator at 37 °C. Cells were washed twice with 1× PBS pH 7.4 and transferred back to the six-well plate. One plate was irradiated with the red LED array at a dose 1.0 J/ cm2 (fluence rate of 1.0 mW/cm2), λmax(Ex) of ∼670−675 nm at room temperature, while the other plate was not exposed to light. Cells were plated onto the SD agar in 50, 100, and 200 μL aliquots. SD agar plates were incubated for 24 h at 37 °C. Formation of colonies was assessed visually and captured with a digital photograph. XTT Assay. Cell viability was quantified via an XTT Cell Proliferation Assay Kit (ATCC), a colorimetric test used to determine the metabolic activity of mitochondrial dehydrogenase. Cells were counted utilizing a hemocytometer and trypan blue. The concentration of cells was adjusted to 106 cells/mL with YNBD broth. A total of 100 μL of C. albicans was added to each well of a 96-well plate. The plate was incubated at 37 °C for 30 min. Encapsulated Pc 4 and free Pc 4 were added to the wells in triplicate in the following concentrations: 2, 1, 0.5, 0.25, 0.1, and 0.02 μM. Negative control experiments included the dendrimer (Pc 4 free) and the media (blank). For positive control experiments with free Pc 4, a stock solution of 2 μM Pc 4 in DMF was serially diluted in 1× PBS (pH 7.4) with 10% Fetal Bovine Serum (FBS). Encapsulated Pc 4 was prepared by serial dilution with 1× PBS (pH 7.4). The inoculated plate was incubated for 1 h at 37 °C in the dark. Afterward, the plate was irradiated with a red LED array at a 1.0 J/cm2 dosage (fluence rate of 1.0 mW/cm2), λmax(Ex) of ∼670−675 nm at room temperature. XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxyanilide) was premixed with the activation reagent, PMS (N-methyldipenzopyrazine methyl sulfate), which acts as an intermediate electron carrier to aid in producing its water-soluble orange formazan derivative. A total of 50 μL of activated-XTT solution was added to each well and returned to the incubator. Upon incubation for 4 h, the absorbance was measured with a spectrophotometer at 475 nm and normalized versus background spectra at 660 nm for nonspecific absorbance and 475 nm (blank). Data was analyzed using Origin 2016 software (OriginLab).
■
RESULTS AND DISCUSSION Synthesis and Characterization of PEGylated-PAMAM Particles. Dendrimers can be selected based on both chemical composition and particle size, both of which are critical for drug encapsulation and delivery. Poly(amidoamine) (PAMAM) dendrimers are desirable drug delivery vehicles due to their multivalency, water solubility, and biocompatibility. Additionally, the cationic nature of PAMAM dendrimers have been known to exhibit antimicrobial properties.31 For these studies, PAMAM Generation 5.0 (G5) was chosen because it possesses the essential qualities required to effectively encapsulate hydrophobic drugs.32 In addition to the ideal container properties, there is evidence that suggests PAMAM dendrimers are able to traverse the stratum corneum.33 PAMAM G5 has 128 amine end groups that are easily functionalized. Utilizing HCTU coupling, the carboxylic acid of the methoxyPEG5000acetic acid was conjugated to the terminal primary amines of PAMAM (Figure 1A). PEGylating has been shown to decrease nonspecific adsorption and improve water solubility. After PEGylating, the resultant nanoparticle possesses a core− shell structure that readily sequesters hydrophobic drugs in the core, and solubilizes the system with the hydrophilic PEG shell. 1 H NMR spectroscopy was used to confirm the chemical structure, estimate the degree of functionalization, and approximate the molecular weight of the nanoparticles. The integrations of the PAMAM (−CH2CONH−) protons relative D
DOI: 10.1021/acs.biomac.6b01436 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules tissue. C. albicans 9652 R is an isolate that exhibits resistance to both amphotericin B and fluconazole. Because the strain is resistant to traditional antifungal medications, an alternative approach is needed to treat these infections, which makes this strain an excellent candidate to study photosensitizing agents. To assess the ability of C. albicans 9652 R to uptake free and encapsulated Pc 4, the samples were incubated with cells for 2.5 and 24 h periods. Pc 4’s fluorescent nature allowed for direct visualization of the PDT agent. Confocal micrographs after 2.5 h indicate that both encapsulated and free Pc 4 showed similar uptake, with predominate accumulation in cells exhibiting hyphae morphology (Figure 2A,B). After 24 h of incubation,
targeted particle data is presented in the Supporting Information as a means of comparison. Cellular Reactive Oxygen Species Detection Assay. The effectiveness of PDT as a therapeutic approach is contingent upon ROS production by the photosensitizer. A cellular ROS detection assay was conducted to qualitatively assess the ability to generate ROS following encapsulation of Pc 4. ROS production was monitored by conversion of nonfluorescent 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) to fluorescent 2′,7′-dichlorofluorescein (DCF). Hydrophobic H2DCFDA permeates the cell membrane, where it is de-esterified to the hydrophilic alcohol form dihydrodichlorofluorescein (H2DCF). ROS oxidation of the diol converts H2DCF to the fluorescent DCF form, which can be detected by confocal microscopy. Cells are incubated with encapsulated Pc 4 and H2DCFDA and first imaged to confirm cellular uptake of Pc 4. Pc 4 was effectively internalized by the cells (Figures 3A and S9A). In the preirradiation micrograph there is minimal visible DCF signal (blue) and the background attributed to the excitation of Pc 4 by the confocal laser rather than cellular
Figure 2. Cell uptake confocal micrographs. Uptake of 4.0 μM encapsulated (left) and free Pc 4 (right) for (A, B) 2.5 h and (C, D) 24 h, respectively.
both encapsulated and free Pc 4 show time-dependent uptake but no noticeable delineation between the vehicle delivered Pc 4 and the free drug (Figure 2C,D). This result was further quantified by flow cytometry. Free Pc 4 was internalized by 99.7% of C. albicans cells, whereas the Pc 4 encapsulated in PAMAM−PEG were taken up by 89.0%, respectively (Figure S8). The free Pc 4 showed nearly complete internalization, with a high fluorescent signal. The encapsulated Pc 4 was internalized by nearly all of the cells, however there were two distinct populations. The majority population showed similar internalization efficiency to that of free Pc 4. The minority population showed internalization, but to a much lesser degree. This may be reflected in the cellular morphology of C. albicans (hyphae vs blastospores). Clinically, photosensitizers are often administered to a patient and allowed to incubate for 24−72 h. This delay in the drug-to-light interval or the time in between when the drug is applied and irradiated allows healthy cells to dispose of the photosensitizer to reduce damage, while the abnormal cells readily uptake the drug.43−45 Uptake studies of chitin targeted nanoparticles proved to be less effective than their untargeted counterparts (Figure S7); this trend was consistent throughout all cell-based experiments; hence, the
Figure 3. Cellular reactive oxygen activity detection assay. C. albicans 9652 R were incubated with 3.5 μM Pc 4 encapsulated in PEGylatedPAMAM nanoparticles and 5 μM 2′,7′-dichlorofluorescein diacetate. (A−C) Micrographs before the cells were irradiated with the red light source. (D−F) Micrographs imaged after irradiation. Red represents Pc 4 fluorescence, blue indicates DCF fluorescence, and purple is the overlay of these two phenomena. C. albicans 9652 R were incubated with 3.5 μM free Pc 4 and 5 μM 2′,7′-dichlorofluorescein diacetate. (G−I) Micrographs imaged prior to photoirradiation and (J−L) imaged postphotoirradiation. The relative intensity of blue from (E) to (K) suggests encapsulated Pc 4 is as effective at generating ROS as free Pc 4. E
DOI: 10.1021/acs.biomac.6b01436 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
Figure 4. Clonogenic and XTT Assays. Sabouraud dextrose agar plates were treated with C. albicans 9652 R that were previously incubated with 3.5 μM Pc 4 encapsulated in PEGylated-PAMAM for 2 h. (A−C) Plates were not subjected to irradiation, while (D)−(F) were irradiated with 1.0 J/cm2 of ∼670−675 nm light. The six plates were incubated at 37 °C for 24 h. The following day the plates were inspected for cell growth and images were taken. (G) Dose response curve determined via an XTT assay for free and encapsulated Pc 4. Free Pc 4: IC50 = 1.72 × 10−7 M (red); Encapsulated Pc 4: IC50 = 1.56 × 10−7 M (black; n = 3).
activity (Figure 3B). Next ROS is generated by irradiation of Pc 4 internalized within C. albicans 9652 R, which leads to a much more prominent DCF signal (Figure 3E). Overlaid images further confirm that ROS generation corresponds to Pc 4 localization, as would be expected (Figure 3C,F). Additional control experiments validate that DCF is not generated when the system lacks a photosensitizer and/or light (Figure S9G− L). Cell Viability Assays. To qualitatively determine the effectiveness of the encapsulated Pc 4 to act as a photodynamic therapy agent, a clonogenic assay was conducted. C. albicans 9652 R were incubated with 3.5 μM of encapsulated Pc 4 for 2 h at 37 °C to ensure the particles were sufficiently internalized. Once the cells were washed to ensure there was no excess Pc 4 present, the cells were seeded on SD agar plates in 50, 100, and 200 μL aliquots. One set of samples was subjected to photoirradiation, while the other set remained unexposed to light. After incubating for 24 h, the dark plates formed a fungal lawn on the agar (Figures 4A−C and S10A−C), whereas irradiated plates fully inhibited colony formation (Figures 4D− F and S10D−F). These findings confirm that encapsulated Pc 4 behaves as an effective PDT reagent in concentration ranges that have been effective with free Pc 4 delivered in organic solvent. To quantitatively ascertain the efficacy of encapsulated Pc 4, a cell viability assay was performed (XTT). XTT is a tetrazolium dye, which upon reduction forms a brightly colored, orange formazan derivative that correlates linearly to cell viability. Dose response curves were determined via the XTT assay for free Pc 4 delivered in DMF and dendrimer encapsulated Pc 4 (Figures 4G and S11). Both the dendrimer encapsulated Pc 4 (IC50 = 0.16 μM) and free Pc 4 delivered in DMF (IC50 = 0.17 μM) showed equivalent efficacy. The results indicate the PEGylated dendrimer bears minimal impact on the potency of Pc 4.
eliminate the need for organic solvents when administering the system. These nanoparticles are readily taken up by both the blastospore and hyphal morphologies of C. albicans 9652 R, which are key to eradicating candidal infections. Once internalized by cells and irradiated with a red light source, encapsulated Pc 4 maintains its ability to generate ROS, which is made evident by the DCF assay. The potency of the photosensitizer remains nearly as effective as free Pc 4 as determined qualitatively by the clonogenic assay and quantitatively by the XTT assay. It has previously been shown that PAMAM dendrimers with a hydrodynamic radius of 2.9 nm have effectively traversed the stratum corneum.33 The hydrodynamic radius of the drug encapsulated dendrimers is 3.4 nm, suggesting that these nanoparticles might be capable of skin penetration. Future studies will utilize various targeting agents to more effectively deliver the photosensitizer to the site of interest as well as testing these systems on candidal biofilms.
CONCLUSIONS In conclusion, we have demonstrated the ability to design a nanoparticulate drug delivery system for the photosensitizer silicon phthalocyanine Pc 4 that remains as efficacious as free Pc 4 for elimination of drug-resistant C. albicans. PEGylatedPAMAM dendrimers are effective drug delivery vehicles to solubilize the hydrophobic photosensitizer Pc 4 in water and
ACKNOWLEDGMENTS The authors would like to extend their deepest thanks to Dr. Ghannoum and his staff for C. albicans, bench space, and critical conversations. J.K.P. and M.A.H. acknowledge funding from the National Institute of Health (5P30AR039750), NIAMS Core Center: Skin Diseases Research Center, and the Ohio Department of Development: Center for Innovative Immuno-
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.6b01436. Synthetic procedures, dendrimer characterization, and biological data for targeted particles (PDF).
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Jonathan K. Pokorski: 0000-0001-5869-6942 Notes
The authors declare no competing financial interest.
■
■
F
DOI: 10.1021/acs.biomac.6b01436 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
(32) Tomalia, D. A. Prog. Polym. Sci. 2005, 30 (3−4), 294−324. (33) Yang, Y.; Sunoqrot, S.; Stowell, C.; Ji, J.; Lee, C.-W.; Kim, J. W.; Khan, S. A.; Hong, S. Biomacromolecules 2012, 13 (7), 2154−2162. (34) Kim, T.; Seo, H. J.; Choi, J. S.; Jang, H.-S.; Baek, J.; Kim, K.; Park, J.-S. Biomacromolecules 2004, 5 (6), 2487−2492. (35) Armstrong, J. K.; Wenby, R. B.; Meiselman, H. J.; Fisher, T. C. Biophys. J. 2004, 87 (6), 4259−4270. (36) Morgan, M. T.; Nakanishi, Y.; Kroll, D. J.; Griset, A. P.; Carnahan, M. A.; Wathier, M.; Oberlies, N. H.; Manikumar, G.; Wani, M. C.; Grinstaff, M. W. Cancer Res. 2006, 66 (24), 11913−11921. (37) Medina, S. H.; El-Sayed, M. E. H. Chem. Rev. 2009, 109 (7), 3141−3157. (38) Andrejevic-Blant, S.; Hadjur, C.; Ballini, J. P.; Wagnieres, G.; Fontolliet, C.; van den Bergh, H.; Monnier, P. Br. J. Cancer 1997, 76 (8), 1021. (39) Patri, A. K.; Kukowska-Latallo, J. F.; Baker, J. R., Jr. Adv. Drug Delivery Rev. 2005, 57 (15), 2203−2214. (40) Kojima, C.; Kono, K.; Maruyama, K.; Takagishi, T. Bioconjugate Chem. 2000, 11 (6), 910−917. (41) Morgan, M. T.; Carnahan, M. A.; Immoos, C. E.; Ribeiro, A. A.; Finkelstein, S.; Lee, S. J.; Grinstaff, M. W. J. Am. Chem. Soc. 2003, 125 (50), 15485−15489. (42) Khoushab, F.; Jaruseranee, N.; Tanthanuch, W.; Yamabhai, M. Int. J. Biol. Macromol. 2012, 50 (5), 1267−1274. (43) Li, L.-B.; Luo, R.-C. Lasers Med. Sci. 2009, 24 (4), 597−603. (44) Andrejevic-Blant, S.; Hadjur, C.; Ballini, J. P.; Wagnià , G.; et al. Br. J. Cancer 1997, 76 (8), 1021. (45) Byrne, A. T.; O’Connor, A. E.; Hall, M.; Murtagh, J.; O’Neill, K.; Curran, K. M.; Mongrain, K.; Rousseau, J. A.; Lecomte, R.; McGee, S.; Callanan, J. J.; O’Shea, D. F.; Gallagher, W. M. Br. J. Cancer 2009, 101 (9), 1565−1573.
suppressive Therapeutics (TECH 09-023). M.A.H. acknowledges a Graduate Assistance in Areas of National Need (GAANN) fellowship for supporting this research.
■
REFERENCES
(1) Matee, M.; Scheutz, F.; Moshy, J. Oral Dis. 2000, 6 (2), 106−111. (2) Sobel, J. D. Lancet 2007, 369 (9577), 1961−1971. (3) Corsello, S.; Spinillo, A.; Osnengo, G.; Penna, C.; Guaschino, S.; Beltrame, A.; Blasi, N.; Festa, A. Eur. J. Obstet. Gynecol. Reprod. Biol. 2003, 110 (1), 66−72. (4) Foxman, B.; Muraglia, R.; Dietz, J.-P.; Sobel, J. D.; Wagner, J. J. Low. Genit. Tract Dis. 2013, 17 (3), 340−345. (5) Pfaller, M. A.; Diekema, D. J. Clin. Microbiol. Rev. 2007, 20 (1), 133−163. (6) Brown, G. D.; Denning, D. W.; Gow, N. A. R.; Levitz, S. M.; Netea, M. G.; White, T. C. Sci. Transl. Med. 2012, 4 (165), 165rv13− 165rv13. (7) Types of Fungal Diseases | Fungal Diseases | CDC; http://www. cdc.gov/fungal/diseases/index.html#one (accessed Aug 29, 2016). (8) Thomas, J.; Jacobson, G. A.; Narkowicz, C. K.; Peterson, G. M.; Burnet, H.; Sharpe, C. J. Clin. Pharm. Ther. 2010, 35 (5), 497−519. (9) Havlickova, B.; Czaika, V. A.; Friedrich, M. Mycoses 2008, 51, 2− 15. (10) Tsai, P.-W.; Chen, Y.-T.; Hsu, P.-C.; Lan, C.-Y. BioMedicine 2013, 3 (1), 51−64. (11) Mayer, F. L.; Wilson, D.; Hube, B. Virulence 2013, 4 (2), 119− 128. (12) Netea, M. G.; Joosten, L. A. B.; van der Meer, J. W. M.; Kullberg, B.-J.; van de Veerdonk, F. L. Nat. Rev. Immunol. 2015, 15 (10), 630−642. (13) Filler, S. G.; Sheppard, D. C. PLoS Pathog. 2006, 2 (12), e129. (14) Ghannoum, M. A.; Rice, L. B. Clin. Microbiol. Rev. 1999, 12 (4), 501−517. (15) Kanafani, Z. A.; Perfect, J. R. Clin. Infect. Dis. 2008, 46 (1), 120− 128. (16) Kharkwal, G. B.; Sharma, S. K.; Huang, Y.-Y.; Dai, T.; Hamblin, M. R. Lasers Surg. Med. 2011, 43 (7), 755−767. (17) Sudhamani, C. N.; Bhojya Naik, H. S.; Sangeetha Gowda, K. R.; Giridhar, M.; Girija, D.; Prashanth Kumar, P. N. Spectrochim. Acta, Part A 2015, 138, 780−788. (18) Lam, M.; Oleinick, N. L.; Nieminen, A.-L. J. Biol. Chem. 2001, 276 (50), 47379−47386. (19) Donnelly, R. F.; McCarron, P. A.; Tunney, M. M. Microbiol. Res. 2008, 163 (1), 1−12. (20) Photodynamic Therapy, Methods in Molecular Biology; Gomer, C. J., Ed.; Humana Press: Totowa, NJ, 2010; Vol. 635. (21) Ehrenshaft, M.; Jenns, A. E.; Chung, K. R.; Daub, M. E. Mol. Cell 1998, 1 (4), 603−609. (22) Casas, A.; Di Venosa, G.; Hasan, T.; Batlle, A. Curr. Med. Chem. 2011, 18 (16), 2486−2515. (23) Berg, K. Photochem. Photobiol. Sci. 2015, 14 (8), 1376−1377. (24) Huang, Z. Technol. Cancer Res. Treat. 2005, 4 (3), 283−293. (25) Ozog, D. M.; Rkein, A. M.; Fabi, S. G.; Gold, M. H.; Goldman, M. P.; Lowe, N. J.; Martin, G. M.; Munavalli, G. S. Dermatol. Surg. 2016, 42 (7), 804−827. (26) Panjehpour, M.; Overholt, B. F. Lasers Surg. Med. 2006, 38 (5), 390−395. (27) Baron, E. D.; Malbasa, C. L.; Santo-Domingo, D.; Fu, P.; Miller, J. D.; Hanneman, K. K.; Hsia, A. H.; Oleinick, N. L.; Colussi, V. C.; Cooper, K. D. Lasers Surg. Med. 2010, 42 (10), 888−895. (28) Sholto, A.; Ehrenberg, B. Photochem. Photobiol. Sci. 2008, 7 (3), 344. (29) Lenardon, M. D.; Munro, C. A.; Gow, N. A. Curr. Opin. Microbiol. 2010, 13 (4), 416−423. (30) Chandra, J.; Mukherjee, P. K.; Ghannoum, M. A. Nat. Protoc. 2008, 3 (12), 1909−1924. (31) Engler, A. C.; Wiradharma, N.; Ong, Z. Y.; Coady, D. J.; Hedrick, J. L.; Yang, Y.-Y. Nano Today 2012, 7 (3), 201−222. G
DOI: 10.1021/acs.biomac.6b01436 Biomacromolecules XXXX, XXX, XXX−XXX
