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PEGylated Dendritic Polyglycerol Conjugate Delivers Doxorubicin to the Parasitophorous Vacuole in Leishmania infantum Infections Camino Gutierrez-Corbo, Barbara Dominguez-Asenjo, Laura I. Vossen, Yolanda Pérez-Pertejo, Maria A. Muñoz-Fenández, Rafael Balaña-Fouce, Marcelo Calderón,* and Rosa M. Reguera*

Most drugs against visceral leishmaniasis must be administered parenterally. A controlled drug release at the target site can improve the efficacy and toxicity of antileishmanial drugs in clinical use. Amastigotes live and grow inside the parasitophorous vacuole of host resident macrophages. Therefore, antileishmanial drugs should accumulate in this compartment to kill the parasite and do not produce toxicity to the cell host. PEGylated dendritic polyglycerol conjugates (PG-PEG) can ensure a controlled drug release and the immune activation efficiency of the host. A dendritic PG conjugate with doxorubicin (DOX) attached through a pH-cleavable hydrazone linker (PG-DOX(pH)-PEG), is tested on murine macrophage cell lines and on ex vivo infected BALB/c splenocytes. As a control, a dendritic PG conjugate attached via a non-cleavable linker (PG-DOX(non)-PEG) is used. DOX fluorescence is useful to monitor the fate of the drug inside the infected cells by flow cytometry and confocal microscopy. The results show that PG-DOX(pH)-PEG slowly releases DOX inside the targeted macrophages, protecting the host of toxic drug concentrations. In addition, unlike free DOX, PG-DOX(pH)-PEG is actively internalized through the acidic endocytic pathway and colocalized surrounding the amastigotes. These results prove that PG-DOX(pH)-PEG is a promising candidate for releasing antileishmanial drugs in a controlled manner.

C. Gutierrez-Corbo, B. Dominguez-Asenjo, Dr. Y. Pérez-Pertejo, Dr. R. Balaña-Fouce, Dr. R. M. Reguera Departamento de Ciencias Biomédicas, Facultad de Veterinaria Universidad de León 24071 León, Spain E-mail: [email protected] C. Gutierrez-Corbo, Dr. M. A. Muñoz-Fenández Laboratorio de InmunoBiologia Molecular Hospital General Universitario Gregorio Marañon Spanish HIV HGM BioBank IiSGM and CIBER-BBN, 28007 Madrid, Spain L. I. Vossen, Prof. M. Calderón Institut für Chemie und Biochemie Freie Universität Berlin Takustrasse 3, 14195 Berlin, Germany E-mail: [email protected] The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/mabi.201700098.

DOI: 10.1002/mabi.201700098

Macromol. Biosci. 2017, 1700098

1. Introduction

Leishmaniasis is a complex vector-borne disease caused by heteroxenous protozoan parasites of the genus Leishmania that currently affects ≈12 million people worldwide, primarily in developing countries. These diseases affect a variety of mammals and are transmitted when sandfly (Phlebotominae) species bite mammalian hosts during their blood meal. Human leishmaniasis can course a wide variety of clinical manifestations that range from a self-healing to a fatal form. Although there is a continuum in the clinical spectrum, four clinical forms are widely recognized. Cutaneous leishmaniasis (CL) causes scars in the skin but in most cases it is a self-healing process. Mucocutaneous leishmaniasis is a disfiguring disease that destroys soft tissues at the upper respiratory track causing psychological disturbances and social stigma to patients. However, the most severe and fatal form if untreated is visceral leishmaniasis (VL), responsible for 20.000–30.000 estimated death per year worldwide.[1] Additionally, post-kala-azar dermal leishmaniasis (PKDL) is generally linked to previous episodes of VL and appears 2–3 years post-treatment.[2] The risk of developing PKDL seems to be associated with incomplete sodium stibogluconate treatment.[3] After sandfly feeding, the infective promastigote form can invade the phagocytic host cells at the bite site, where neutrophils[4] play an essential role in parasite pathogenesis acting as Trojan horses for definitive target cells. Cargoes of promastigotes are efficiently and safely transferred into resident macrophages of liver, spleen, and bone marrow, preventing the activation of their defence responses.[5] Promastigotes can also be internalized by macrophages at the inoculation site, ending within intracellular phagolysosome compartment, named parasitophorous vacuole (PV). Parasite visceralization from skin to internal organs is likely mediated by migratory infected myeloid cells.[6] Therefore, the administration of targeted antileishmanial drugs against mononuclear phagocytic system of infected host could be a plausible strategy against VL.

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PV biogenesis is originated from macrophage plasmalemma that progresses to a hybrid compartment enriched with proteins from late endosomes, lysosomes[7] as well as endoplasmic reticulum.[8] Inside macrophage phagolysosomes, promastigotes transiently inhibit acidification of the phagosome in a lipophosphoglycan-mediated process[9] that excludes synaptotagmin V, an essential player in the recruitment dependent on the vesicular proton ATPase that drives to phagosome acidification.[10] Finally, the major determinant in amastigote differentiation process is the increase of temperature over the decrease of extracellular pH.[11] Nowadays, available drugs against leishmaniasis include pentavalent antimonial derivatives (SbV), amphotericin B (AmB) formulations, miltefosine, and paromomycin. All these compounds have serious limitations such as high toxicity and poor selectivity.[12] Most of them must be administered in repeated and high doses by parenteral routes, with long-term schedules that require hospitalization. Therefore, economic burden is not negligible and treatment adherence is a problem particularly in endemic geographical areas with overwhelmed health services.[13,14] Lipid formulations of AmB have improved the safety profile of this drug and are the treatment of choice in Southern Europe.[15] However, the high cost of these medicines precludes this treatment in low-income countries. Additionally to lipid formulations, polymeric nanocarriers are potential candidates for drug delivery. In fact, since the first marketed nanoparticulate approved in 1990, PEGylated and liposomal drug formulations represent more than 40% of drug delivery systems in clinical use.[16] Small molecules are conjugated to nanocarriers with the aim of avoiding passive diffusion into cells and to prevent as far as possible their undesirable effects. Cell membranes are naturally impermeable to complexes larger than 1 kDa. Therefore, carriers are internalized by the endocytic pathway, which by means of intracellular vesicles called endosomes, is able to engulf molecules and extracellular fluid. Once inside the cell, the intracellular fate of the endosomal contents is a key factor of successful drug delivery. Depending upon macromolecule composition and how it interacts with the cell membrane, endosomes will return to the plasma membrane as part of membrane repair mechanism or otherwise they will mature into acidic vesicles called late endosomes. These endosomes may finally fuse with lysosomes, responsible for a complete elimination of the carrier using hydrolytic enzymes.[17] Although the aim of most drug delivery systems is to avoid lysosomal degradation, those infectious diseases such as leishmaniasis where the parasite resides within phagolysosomes (a lysosome-like organelle) would greatly benefit from therapies along the endocytic pathway. A variety of alternative macromolecules have been proposed as drug delivery systems, among which is dendritic polyglycerol (PG). PG has outstanding properties to work as a drug delivery system and has recently attracted attention for its therapeutic relevance in biomedical applications due to many exceptional properties such as structure, easy and controlled kilogram scale preparation, easy tunable functional groups for attaching biologically active molecules, and finally biocompatibility and water solubility.[18–20] In addition, when dendritic PG is coated


with polyethyleneglycol (PEG), it reduces immune activation efficiency, which has been widely used in cancer therapies.[21] Doxorubicin (DOX) is an anthracycline antibiotic with antitumor activity due to multiple mechanisms. DOX poisons eukaryotic DNA topoisomerase II stabilizing cleaving complexes with DNA, preventing the replication and transcription of DNA.[22] In addition, anthracyclines intercalate not only into nuclear DNA but also mitochondrial DNA.[23] At heart, DOX binds with cardiolipin, a major component of the mitochondrial inner membrane, promoting reactive oxygen species (ROS) generation and cardiotoxicity.[24] Interestingly, DOX also causes alterations in cell apoptotic signaling by increasing the levels of Bax (proapoptotic protein) and decreasing Bcl2 (antiapoptotic protein).[25] Herein, we present a promising dendritic PG conjugate as a vehicle for drug delivery in VL. We propose a PEGylated dendritic PG conjugated to DOX via a hydrazone bond. The PEG shell increases the water solubility of the complex and acts as a shield avoiding to be recognized by the immune system.[26] Moreover, the acid-sensitive hydrazone linker[27] is used for drug conjugation and further release the drug in the acidic lysosomal compartments where the parasite resides. As a proof of concept, DOX is also used for intracellular in vivo imaging as a result of its fluorescent nature.[28] In this study, we tested the biocompatibility and uptake in two different macrophage cell lines (RAW 264.7 and J774A.1), antileishmanial activity in an ex vivo infected system, and subcellular location of a pH-cleavable conjugate PG-DOX(pH)-PEG and a noncleavable control PG-DOX(non)-PEG compared to free DOX.

2. Experimental Section 2.1. Chemical Materials All chemicals were of analytical grade and purchased from Fluka (Germany), Aldrich (Germany), or Merck (Germany), and used as received unless otherwise stated. Maleimido-poly(ethylene glycol) (PEG-mal) with MW = 2 kDa was purchased from Rapp Polymer, Germany. The hydrazone derivative of DOX (DOX-EMCH) was prepared as described previously[29] and the noncleavable control DOX-mal was prepared according to published procedures.[30] Dendritic PG (average MW 10 kDa, polydispersity index (PDI) = 1.6, ≈135 OH groups) was prepared according to published procedure.[31] PG amine with 30% of the total hydroxyl groups converted to amino groups (≈95 OH and 40 NH2 groups per PG scaffold) was synthesized according to previously reported methodologies.[32] Briefly, PG amine was prepared by a three-step protocol starting from PG and a conversion of 30% of the OH groups into mesyl (Ms) groups, followed by transformation of the Ms groups into azide (N3) functionalities, and finally reduction of the N3 groups to amine (NH2) groups by using triphenylphosphine as reducing agent. Extensive dialysis was carried out after each reaction step and quantification of the % of NH2 groups was performed using 1H-NMR spectroscopy. Water of Millipore quality (resistivity ≈ 18 MΩ cm−1, pH 5.6 ± 0.2) was prepared using a Millipore water purification system and was used in all experiments and for preparation of all samples. If not otherwise specified, sodium phosphate buffer (10 × 10−3 m) was used for the pH of 7.4, and acidic pH values were reached by a sodium acetate buffer (pH 4.0, 50 × 10−3 m). All measurements were carried out with freshly prepared solutions at 25 °C. pH values were measured with a Scott instruments HandyLab pH meter at 25 °C. Purification by centrifugal filtration was performed using Amicon Ultra Centrifugal Filters (molecular weight

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cutoff, MWCO 3 kDa, Millipore). Size exclusion chromatography (SEC) of PG conjugates was performed with Sephadex G-25 superfine under ambient pressure and temperature. All reactions that involved air or water sensitive compounds were carried out in dried flasks under an argon atmosphere and dried solvents from the solvent purification system MB SPS 800, M. Braun Inertgas-Systeme GmbH, Garching, Germany. Absorption spectra were recorded on a LAMBDA 950 UV– vis–NIR spectrometer (PerkinElmer, USA).

volume (15 µL) was taken out of the mixture, applied onto a 1 mL Sephadex G-25 column and eluted with PB (pH 7.4). The faster band corresponds to the conjugate and the slower band, which increased intensity over time, corresponds to the released drug. The free drug band was collected and freeze-dried. The solid was redissolved in 0.3 mL MilliQ water and the amount of DOX in the solution was directly determined by UV–vis spectroscopy at 495 nm (ε495 = 10680 m−1 cm−1). The spectrometrically determined amount of free DOX was plotted in correlation to the maximal amount of DOX loaded to the conjugate (% released) against time.

2.2. Synthesis of PG-DOX-PEG Conjugates The synthesis of PG-DOX-PEG conjugates was performed according to published methodologies.[27,33,34] In brief, PG amine (10 kDa, 95 OH and 40 NH2 groups), bearing 30% of amino groups, was dissolved in phosphate buffer (PB) (pH 7.4). 2-iminothiolane (1.5 eq. per NH2 group) was added in PB (pH 7.4) and the mixture was stirred for 20 min at room temperature. Afterward, the solution was directly applied onto a Sephadex G-25 superfine column and eluted with MilliQ water to remove excess 2-iminothiolane. The polymeric fraction was collected and to this a solution of DOX-EMCH, or DOX-mal (1.5 eq. per PG) in MeOH was added and stirred for 15 min at room temperature. Afterward, a solution of PEG-mal (50 eq. per PG) in PB (pH 7.4) was added and the mixture stirred for another 2 h. Finally, the reaction mixture was concentrated with Amicon filters (MWCO 3 kDa) and purified by SEC using a Sephadex G-25 superfine column to give the final conjugates PG-DOX(pH)-PEG and PG-DOX(non)-PEG. Conjugate formation was confirmed by appearance of a faster band on the Sephadex G-25 superfine column.

2.3. Physicochemical Characterization of PG-DOX-PEG Conjugates

The animal research described in this manuscript complies with Spanish Act (RD 53/2013) and European Union Legislation (2010/63/UE). The used protocols were approved by the Animal Care Committee of the University of León (Spain), project license number (PI12/00104). Female BALB/c mice (6–8 weeks old) were obtained from Harlan Interfauna Iberica SA (Barcelona, Spain) and housed in specificpathogen-free facilities for this study. The infrared fluorescence-emitting strain iRFP+L. infantum was previously described.[35] Promastigotes were routinely cultured at 26 °C in M199 medium supplemented with 25 × 10−3 m (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) pH 6.9, 10 × 10−3 m glutamine, 7.6 × 10−3 m hemin, 0.1 × 10−3 m adenosine, 0.01 × 10−3 m folic acid, RPMI 1640 vitamin mix (Sigma), 10% (v/v) heat inactivated fetal calf serum (FCS), and antibiotic cocktail (50 U mL−1 penicillin, 50 mg mL−1 streptomycin). Ex vivo splenic infected explants were used to test the antileishmanial activity of free and conjugated DOX in amastigote-infected macrophages.

2.6. Assessment of Cell Toxicity Using Macrophage Cell Lines

2.3.1. Surface Charge Measurements Zeta potential measurements were performed with a Malvern Zetasizer Nano-ZS 90 (Malvern Instruments) with an integrated He–Ne laser (λ = 633 nm). The samples were freshly prepared in Milli-Q water at a concentration of 1 mg mL−1. Prior to the measurements, all samples were filtered through a 0.45 × 10−6 m pore size filter. All measurements were performed at 25 °C using folded capillary cells (DTS 1070).

2.3.2. Hydrodynamic Diameter Measurements Dynamic light scattering (DLS) measurements were performed using a Malvern Zetasizer Nano-ZS 90 (Malvern Instruments) equipped with a He–Ne laser with a wavelength of λ = 633 nm under a scattering angle of 173°. The samples were freshly prepared in Milli-Q water at a concentration of 1 mg mL−1. Prior to the measurements, all samples were filtered through a 0.45 × 10−6 m pore size filter. All measurements were performed at 25 °C using quartz cells.

2.4. Drug Release Profile Determination of PG-DOX-PEG Conjugates 2.4.1. Analysis of DOX Release by SEC and UV–Vis Spectroscopy The drug release of PG-DOX-PEG conjugates was studied by separating the conjugate from the free drug fraction by SEC on a Sephadex G-10 column and collecting the free drug fraction, followed by determination of the concentration of the free drug photometrically by UV–vis spectroscopy. The cleavage study was performed at pH 4.0 and 7.4, respectively. Initially, the conjugate was dissolved in PB pH 7.4 or acetate buffer pH 4 (110 µL) and incubated for 27 h in total. The concentration of the conjugate in buffer was 180 × 10−6 m with a DOX loading of 0.9 wt% (PG-DOX(pH)-PEG) and 1.9 wt% (PG-DOX(non)-PEG), respectively. At specific time points (30 min, 2, 3, 5, and 27 h) a defined


2.5. Mice and Parasites

To determine the toxicity of free DOX and different types of DOXconjugated nanoparticles, we used adherent mouse macrophage cell lines such as RAW 264.7 and J774A.1. They were subcultured every 2–3 d in RPMI supplemented with 10% heat inactivated FCS, 1 × 10−3 m sodium pyruvate, 1× RPMI vitamins, 10 × 10−3 m HEPES, and antibiotic cocktail (100 U mL−1 penicillin, and 100 mg mL−1 streptomycin). Fifty thousand cells per well were seeded in 200 µL of supplemented medium and added to optical 96-well plates. Free DOX, PG-DOX(pH)-PEG, and PG-DOX(non)-PEG conjugates were added at various DOX equivalent concentrations (µm) and incubated at 5% CO2 and 37 °C for 96 h. Cell viability was assessed using Alamar Blue assay according to manufacturer’s recommendations (Invitrogen). Fluorescence readouts were obtained with a Synergy (BioTek) microplate reader and used them to calculate the cytotoxic concentration 50 (CC50) of DOX formulations. Cytotoxicity was also determined in uninfected ex vivo splenic explant, to calculate selectivity index (SI) in primary cell cultures.

2.7. Macrophage Uptake Studies J774A.1 and RAW 264.7 macrophages were used for this study. Aliquots (100 µL) containing J774A.1 cells (2 × 105) were suspended in 0.9 mL of RPMI-1640 medium supplemented with 10% FCS and transferred into 24-well plates (Corning). After 3 h at 37 °C and 5% CO2, to allow cells to adhere well surface, free DOX and PG-DOX-PEG conjugates were added to culture medium (0.5 µm DOX-equivalent). For separation of the internalized and surface-bound nanoparticles, cells were washed 3× with acetate buffer (pH 4.0). DOX-associated fluorescence was measured by flow cytometry (Dako Coulter) at an excitation wavelength of 480 nm and an emission wavelength of 550 nm.[36] At least ten thousand cells were acquired per well. For uptake analysis, side scatter was represented versus Texas-red fluorescence (FL-3) (x and y axes, respectively). Negative control was established with untreated cells as 2% of the gated population. Data were processed with FlowJow software. Positive population expressed as percentage (%) was used for uptake quantification.

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2.8. Flow Cytometric Analysis of Apoptosis/Necrosis Apoptosis and necrosis induced by free DOX and DOX-conjugated to dendritic PG (1.0 µm DOX-equivalent) was measured by annexin-V/ propidium iodide (PI) analysis through flow cytometry. Fraction of annexin-V positive cells was measured with FlowJo software. RAW 264.7 macrophage cultures (105 cells/24-well plate) were treated with free DOX and PG-DOX-PEG conjugates at 1 µm DOX-equivalent amount for 24 h. Cells were washed twice with warm PBS, detached with PBS supplemented with 10 × 10−3 m ethylene diamine tetraacetic acid for 10 min, 37 °C, harvested and resuspended in 100 µL binding buffer (10 × 10−3 m HEPES/NaOH, pH 7.4, 140 × 10−3 m NaCl, 2.5 × 10−3 m CaCl2). Staining was performed with annexin-V and PI (20 µg) following manufacturer’s recommendations. Then, cells were fixed with 1% paraformaldehyde for 10 min at 4 °C and the fixing solution removed by two PBS washes. Cells were centrifuged and resuspended in PBS containing RNAse A (20 µg mL−1) for 30 min at 37 °C in dark before flow cytometry acquisition.

2.9. Infection of RAW 267.4 Macrophage Cultures Freshly L. infantum WT amastigotes isolated from infected BALB/c mice spleens were added to RAW 267.4 macrophage cultures at an infection ratio of 5:1 and incubated at 37 °C and 5% CO2 in complete medium for 6 h. To remove noninternalized parasites, cultures were washed 5× with warm PBS and cultured in complete medium at 37 °C in a 5% CO2 atmosphere. Free DOX or PG-DOX-PEG conjugates (normalized to 1 µm of DOX) were added for 24 h. Noninternalized conjugates were removed by 3× PBS washes and the cells were stained with 1 × 10−6 m cresyl violet for 5 min and 1 µg mL−1 Hoechst 33342 in culture medium. Observation and image acquisition of live macrophages were then

taken by using a Zeiss LSM800 confocal microscope. Cultures were maintained at 37 °C and 5% CO2 within the incubators coupled to the microscopes.

3. Results and Discussion 3.1. Synthesis and Characterization of PG-DOX-PEG Conjugates All PG-DOX-PEG conjugates (Figure 1) were synthesized in a one-pot synthesis as stated in earlier publications.[33,34] The general schematic pathway for the conjugation reaction between DOX prodrugs, PEG-mal (2 kDa) and PG amine bearing 30% amino groups is shown in Scheme 1. The first step comprised the thiolation of the dendritic PG amine (10 kDa, 95 OH, and 40 NH2 groups), previously synthesized using reported procedures.[32,36] The concentration of thiols was determined by an Ellman’s test, as well as the highest thiol concentration after 20 min.[36] Next, DOX, modified with a pH-cleavable hydrazone bond (DOX-EMCH) or a noncleavable amide bond (DOX-mal), is added to the PG thiol solution in methanol where a selective Michael-type addition between the thiols and the maleimide groups takes place. Finally, PEGylation with PEG-mal (2 kDa) renders the final conjugates. The two modified DOX prodrugs (DOX-EMCH and DOX-mal) were synthesized according to scientific literature procedures[29,30] and their structures are depicted in Figure 1.

Figure 1. A) General structures of PG-DOX(pH)-PEG and PG-DOX(non)-PEG conjugates. B) Building blocks for the synthesis of PG-DOX(pH)-PEG and PG-DOX(non)-PEG.


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Scheme 1. Schematic pathway for the synthesis of PG-DOX-PEG conjugates.

3.2. Characterization of the Dendritic Conjugates 3.2.1. Hydrodynamic Diameter and Zeta Potential Measurements of the Conjugates Hydrodynamic diameters of the conjugates were measured by DLS. Conjugates showed Z-average values of 20 and 32 nm with PG-DOX(pH)-PEG having a larger diameter (32 nm) than PG-DOX(non)-PEG (20 nm) (Table 1). The difference in sizes, between the pH sensitive conjugate and the stable one, could be explained with the differences in solubility of DOX-EMCH and DOX-mal due to different sites of linker-conjugation during their synthesis. DOX-EMCH is synthesized by reacting the C-13 keto position with 6-maleimidocaproic acid hydrazide, whereas DOX-mal is synthesized by reacting the daunosamine with maleimidopropionic acid. In the case of DOX-EMCH, the daunosamine group makes the prodrug more hydrophilic. Once attached to highly flexible PG, the positive charged DOXEMCH will be exposed under physiological pH, causing an increase of the hydrodynamic diameter of the conjugate. The more hydrophobic, noncharged, DOX-mal, will stay closely to the core, resulting in a decreased hydrodynamic diameter. Moreover, zeta potential measurements of the conjugates evidence our hypothesis regarding the difference between the charges (+6.0 PG-DOX(pH)-PEG vs +1.2 PG-DOX(non)-PEG). Nevertheless, as a result of PEGylation, overall charge of the conjugates is very close to zero (Table 1). All physicochemical parameters of the conjugates are summarized in Table 1 and size distributions are reported in Figure S1 in the Supporting Information.

in Table 1. The in vitro stability studies of the pH-cleavable conjugate PG-DOX(pH)-PEG and the noncleavable control PGDOX(non)-PEG were performed by SEC and UV–vis spectroscopy assistance. PG-DOX(pH)-PEG showed a minimal release of the drug at pH 7.4 after 27 h (less than 8%) while at acidic pH 4.0 the release was 60%. Moreover, the stability of PGDOX(non)-PEG at pH 4.0 could be demonstrated with less than 14% of DOX release. The collected data are in accordance with our previous publications.[27,30,33,34] These properties should enable the drug to be released in the slightly acidic lysosomal compartment where the parasite resides (Figure 2), preventing its delivery during blood distribution.

3.3. DOX Conjugated to Polyglycerol Carriers Reduces Free DOX Toxicity in an Ex Vivo Physiological Mouse Model of Leishmania Infection

First, we study the biocompatibility of the conjugates within two murine macrophage cell lines. J774A.1 (a monocyte macro phage cell line) and RAW 264.7 (a macrophage cell line) were treated with free DOX, PG-DOX(pH)-PEG, and PGDOX(non)-PEG conjugates. The noncleavable dendritic conjugate PG-DOX(non)-PEG, did not have any effect on both cell lines. Both cultures showed similar CC50 values for free DOX: 1.43 ± 0.14 µm for monocytes (Figure 3A) and 1.87 ± 0.09 µm for macrophages (Figure 3B). The acid-sensitive PG-DOX(pH)-PEG conjugate was less toxic to both; fourfold lower to RAW 264.7 macrophage cell line (CC50 7.42 ± 0.41 µm) and twofold lower to J774A.1 monocyte macrophage cell line (with CC50 2.81 ± 0.24 µm). A similar drug toxicity reduction was reported in J774A.1 macrophages using DOX encapsulated in a biodegrad3.2.2. Drug Loading of the Dendritic Conjugates and Drug Release able polymer methoxypoly-(ethylene glycol)-b-poly(lactic acid).[37] Study However, these tumoral cell lines do not mirror the specific parasite niche. Therefore, as alternative we used a primary cell The DOX concentrations were determined photometrically culture obtained from murine uninfected splenic explants. Free using the molar absorption coefficient of DOX at 495 nm DOX showed high cytotoxicity (CC50 0.28 ± 0.12 µm), whereas (ε = 10 680 m−1 cm−1) after reconstitution of the lyophilized the pH-sensitive PG-DOX(pH)-PEG conjugate was safe beyond 100 µm (Figure 3C). Collectively, our results using different samples in PB (pH 7.4). The obtained loadings are summarized types of phagocytes, strongly suggest a decreased toxicity induced by acid-sensitive Table 1. Physicochemical characterization of PG-DOX-PEG conjugates. hydrazone linker used in PG-DOX(pH)-PEG conjugate compared to free DOX. Thus, PDI Zeta potential DOX loading Z-average Mw (theoretical) the whole process may differ considerably [mV] [wt%] [nm] [kDa] between primary cultures and related tumor PG-DOX(pH)-PEG 32 0.5 55 0.9 +6.0 cell lines, as has been previously pointed by PG-DOX(non)-PEG 20 0.5 56 1.9 +1.2 comparing blood differentiated macrophages


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PG-DOX(pH)-PEG pH 4.0 PG-DOX(non)-PEG pH 4.0 PG-DOX(pH)-PEG pH 7.4

DOX release (%)

60

40

20

0 0

500

1000

1500

t [min]

Figure 2. Representative release profile of PG-DOX(pH)-PEG and PGDOX(non)-PEG incubated at pH 4.0 and 7.4 at 37 °C over 27 h. The DOX release (%) was quantified using a SEC assay with assistance of UV–vis spectroscopy. Mean ± SEM were obtained from triplicates in three independent experiments.

with a monocytic cell line from human leukemia (THP-1), suggesting that the results obtained from the nanoparticle–cell interactions on cell line models may not be applicable to the situation in normal differentiated cells.[38] During in vivo infections, leishmania parasites are located within resident macrophages in the liver, red and white pulp in spleen, and bone-marrow macrophages. The prevailing model supported the hypothesis that all resident tissue macrophages are derived from circulating monocytes. However, recent studies show that tissue-resident populations are derived from embryonic precursors with minimal input from circulating monocytes.[39] In this scenario, nanoparticle internalization studies may differ between tumor cell lines used in in vitro studies and in vivo infections in mice. Next, we investigated the effect of the dendritic conjugates in Leishmania infantum (L. infantum) parasites. Free DOX, and DOX conjugated to PG were added at different DOX equivalent concentrations to the promastigote cultures or the ex vivo infected splenic explants in a 384 optical bottom, black well plate. Vehicle-treated wells (0.4% dimethylsulfoxide) were used as negative controls. After 96 h incubation, we used an Odyssey imaging system (Li-Cor) to obtain fluorescence measures and

Figure 3. Cytotoxicity of polymers against macrophage cell lines. A) RAW 264.7 and B) J774A.1. Free DOX (■), PG-DOX(pH)-PEG (●), PG-DOX(non)PEG (▲). Cell cultures were incubated with different concentrations of polymers or free DOX at 37 °C for 96 h. AlamarBlue was added following manufacture instructions and fluorescence readouts used to express cell viability. CC50 values were estimated by pharmacological approach using SigmaPlot. Mean ± SD error were obtained from duplicates in three independent experiments. X-axis represents µm concentrations of doxorubicin equivalents.


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Figure 4. Curve dose response of free DOX and conjugated DOX polymers to PG against iRFP+L. infantum parasites. A) Effect against promastigotes cultures. Free promastigotes were incubated with different equivalent concentrations of DOX at 26 °C for 96 h. Promastigote viability measured as fluorescence was expressed as % respect untreated control. B) Effects against intracellular amastigotes were incubated with different equivalent concentrations of DOX at 37 °C for 96 h. Free DOX (■), PG-DOX(pH)-PEG (●), PG-DOX(non)-PEG (▲). Mean ± SD error were obtained from duplicates in three independent experiments. EC50 values were estimated by sigmoidal fit using SigmaPlot 10.0. X-axis represents µm concentrations of doxorubicin equivalents.

to calculate EC50 values by using SigmaPlot 10.0 software. Promastigote growth was inhibited by free DOX (EC50 0.29 ± 0.02; EC98 6.77), whereas PG-DOX(pH)-PEG was unable to kill parasites (EC50 > 100 µm) (Figure 4A). These results were further corroborated by flow cytometry showing that DOX was not detected after 24 h PG-DOX(pH)-PEG treatment (see the inset). On the other hand, inhibition of intracellular amastigotes in the ex vivo infected splenic explant showed that pH-sensitive PG-DOX(pH)PEG conjugate was 20-fold less toxic (EC50 2.38 ± 0.19 µm) than free DOX (EC50 0.43 ± 0.05 µm) (Figure 4B). Several DOX-conjugated nanosystems have been used against intracellular leishmania infections, resulting in an almost twofold higher antileishmanial effect than free DOX.[40–43] Despite the fact that our results showed a decrease in the antiparasitic effect after DOX conjugation, the EC50 values obtained are in the micromolar order similar to those obtained by others. This opposite effect can be attributed to either the leishmania species used in the experiments (L. infantum vs Leishmania donovani), or to a higher drug sensitivity in our ex vivo splenic explant[44] than that provided by infected J774A.1 macrophages. Collectively, our results show a SI value for free DOX < 1 (Figure 3C CC50/Figure 4B EC50 (0.28/0.43)), which is not surprising given that J774A.1 is a tumoral cell line that is used as macrophage model for testing leishmania infection. Therefore, the use of an anticancer drug such as DOX might kill the host cells before killing intracellular parasites. On the other hand, PG-DOX(pH)PEG showed an SI > 42 (Figure 3C CC50/Figure 4B EC50 (>100/2.38)) that can be attributed to both, the conjugate and the more physiological assay for antileishmanial testing.

3.4. DOX Uptake Showed Different Patterns in J774A.1 and RAW 267.4 Macrophages Characterized by a Delayed Release When PG-DOX(pH)-PEG Is Used as a Carrier The uptake of PG-DOX(pH)-PEG and PG-DOX(non)-PEG was compared to free DOX in J774A.1 and RAW 264.7


macrophage cell lines. We used DOX fluorescence emission at 550 nm as readout for appraisal of drug uptake. Intracellular DOX fluorescence was quantified in each type of culture after different incubation times of three treatments at 0.5 µm DOX-equivalent concentration. As we were unable to define positive and negative populations (Figure S2, Supporting Information), we quantified results by representing DOX fluorescence versus side scatter and setting a 2% cutoff for DOX positive population in untreated cells (Figure S3, Supporting Information). PG-DOX(non)-PEG was detected at low levels, with similar numbers to untreated cells in both cultures. Free DOX and PG-DOX(pH)-PEG resulted in a time-dependent increase in DOX levels in both J774A.1 and RAW 264.7 macro phages. However, the uptake pattern in each cell type was notably different. In J774A.1 macrophages, PG-DOX(pH)PEG showed a quick increase during the first hours that peaks at 18 h with 62% of cells positive to DOX, whereas free DOX reached 88% (Figure 5A; Figure S3A, Supporting Information). On the contrary, in RAW 264.7 macrophages, PG-DOX(pH)-PEG resulted in a markedly lower DOX level with only 6% DOX-positive macrophages after 3 h, and the maximum percentage (30%) was delayed to 48 h (Figure 5B; Figure S3B, Supporting Information). This lower uptake rate in RAW 264.7 macrophages may justify the lower toxicity (fourfold) for this cell line (see Figure 3B) after 96 h of PGDOX(pH)-PEG treatment compared to J774A.1 macrophages (Figure 3A). A similar delay in DOX uptake was reported in J744.1A macrophages after treatment with a PEG-coated biodegradable poly (lactic acid) (PLA) polymer.[37] This was justified by the release of drug due to conversion of dimeric DOX–DOX to DOX monomer due to the acidic pH of the lysosomal compartment. Our PG-DOX(pH)-PEG conjugate showed a slower and lower uptake in the macrophages compared to free DOX (Figure 5), never the less this can be explained with the different pathways of conjugate versus the free drug (endocytosis vs diffusion).

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Figure 5. Free and conjugated DOX to PG uptake in J774A.1 and RAW 264.7 macrophages at different time points. Once cells were attached to plates, free or conjugated DOX equivalent amounts (0.5 µm) were added to cultures. Macrophages were harvested and the unbound polymers were removed by acidic treatment. Red fluorescence intensity was acquired on logarithmic scale and represented versus scatter side. Gate Dox-positive population was established as 2% in untreated cells. Uptake is expressed as %. Values are means + standard error. A) J774A.1 macrophages. B) RAW 264.7 macrophages.

3.5. PG-DOX-PEG Conjugates Use Endocytosis Pathway to Reach Lysosome, the Natural Niche to Leishmania Parasites during Infection Next, we examined the PG conjugate’s intracellular localization. Confocal laser scanning microscopy was performed to study the localization of conjugates within macrophages. Infected RAW 267.4 macrophages were exposed to 1 µm free DOX or equivalent conjugated DOX for different times and then noninternalized material was removed by extensive warm phosphate buffer saline (PBS) washes. Before image acquisition we used cresyl violet to label the acidic compartments.[45] After 30 min treatment, free DOX was sequestered within vesicular structures that localize in peripheral cell regions with no signs of nuclear localization (Figure 6A). Longer incubation times (2 h) showed a scarce free DOX accumulation inside the nuclei that increases notably over time. Colocalization levels between DOX and cresyl were markedly low (11 ± 10% n = 23 cells) suggesting that free DOX is predominantly trafficked in a nonacidic pathway. It is known that free DOX cellular uptake occurs by a passive diffusion mechanism[46] and during the first 2 h, it accumulates in several cell organelles that include nucleus, lysosomes, mitochondria, and plasma membrane[47] for ending in the nucleus, the final drug location.[48] On the contrary, after 24 h PG-DOX(pH)-PEG treatment DOX rendered high colocalization levels with cresyl, suggesting an acidic endocytic pathway (Figure 6A, bottom line). It is worth noting that cresyl wrapped tightly the amastigotes and yielded a black hollow where the parasite is detected by Hoechst 33342 staining (crosses). DOX delivery by PG-DOX(pH)-PEG appeared inside the entire phagolysosome with an increased fluorescence overlapping the acidic content labeled by cresyl, pointing that DOX colocalizes with cresyl violet inside the phagolysosome. A quantification of the fluorescence level inside nuclei is depicted in Figure 6B. In free DOX treated cells, nuclei showed a 15-fold higher fluorescence level than PG-DOX(pH)-PEG treated cells, confirming that our nanoparticle avoids drug deposition in the nuclei. The accumulation of free DOX in nucleus or mitochondria is a major drawback of this drug as well as of other drugs,


such as the topoisomerase-I poisoning derivatives (camptothecin derivatives and indenoisoquinolines) that have shown to be active against intracellular-leishmania amastigotes but are highly toxic to host cells.[49,50] To confirm whether lysosomes are the organelles where DOX is located, cells were pretransducted with emerald green fluorescent protein (GFP) fused to lysosomal associated membrane protein 1 (LAMP-1) (BacMan 2.0, ThermoFisher) following manufacturer’s instructions. Then, macrophages were exposed to 1.0 µm free DOX or PG-DOX(pH)-PEG treatment for 24 h. Noninternalized drug and conjugates were removed by extensive warm-PBS washing before image acquisition. Free DOX was visualized in different intracellular locations that include lysosomes (Figure 7A), while PG-DOX(pH)-PEG was solely detected inside vesicular structures surrounded by LAMP-1 marker, pointing to these conjugates were sequestered by degradative vesicles of the endolysosomal pathway. A quantification of the fluorescence level inside lysosomes is depicted in Figure 7B. Unlike free DOX that was not detected inside lysosomes, drug delivered by PG-DOX(pH)-PEG conjugate accumulated inside these organelles. These results revealed that the lysosomal compartment is the main depot of internalized PG-DOX(pH)-PEG in macrophages and this is a strategic action to use an acid-cleavable bond to connect DOX.

3.6. PG-DOX(pH)-PEG Prevents Necrosis Promoted by Free DOX in RAW 264.7 Macrophages To confirm microscopy results, we test the necrotic–apoptotic effect induced by free DOX and PG-DOX-PEG conjugates at 1 µm after 24 h treatment in RAW 264.7 macrophages. Untreated cells showed high viability (>96%) that was sharply reduced to less than 60% when cells were treated with free DOX due to an increase in the necrotic population. The number of apoptotic cells remained unaffected. On the contrary, PGDOX-PEG conjugates showed a pattern similar to untreated cells with viabilities of 93% and low level of necrosis (5% for PG-DOX(pH)-PEG) and similar levels for PG-DOX(non)-PEG

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Figure 6. Free DOX and PG-DOX(pH)-PEG conjugates trafficked to spatially distinct intracellular locations within 24 h post-treatment with live RAW 264.7 macrophages. A) Free DOX after different treatment times. Nucleus is stained with Hoechst 34580 (blue), lysosomes are stained with cresyl-violet (red), and DOX is shown in green. Free DOX is not seen inside nuclei within 30 min, but clearly detected after 24 h. The amastigotes nuclei appear in blue (crosses). PG-DOX(pH)-PEG treatment for 24 h (bottom line) showed that DOX strongly accumulates in vesicles containing parasites that colocalize with cresyl-violet. B) DOX fluorescence detected in nuclei of cells treated with free DOX or PG-DOX(pH)-PEG conjugate. A region of interest (ROI) was drawn over nuclei to quantify DOX accumulated in nuclei. Results are represented as mean ± SEM. Macromol. Biosci. 2017, 1700098

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Figure 7. PG-DOX(pH)-PEG conjugates trafficked to lysosomes and colocalize with intracellular parasites inside lysosomes in RAW 264.7 macrophages. Cells were pretransducted with GFP-LAMP-1. Free DOX and PG-DOX(pH)-PEG conjugate were added for 24 h before image acquisition. Nucleus is stained with Hoechst 34580 (blue), LAMP-1 is labeling late endosomes and lysosomes (green) and DOX is shown in orange. DOX fluorescence was detected in lysosomes. B) A line crossing single representative lysosomes was manually traced with a one-pixel width and quantification of the DOX fluorescence in free DOX and PG-DOX(pH)-PEG treated cells is shown.

(Figure 8). These results confirmed our microscopic findings, showing that the acid-sensitive PG-DOX(pH)-PEG conjugate overrides drug nuclei delivery of free DOX and prevents cellular necrosis. Therefore, the reduced viability after free DOX treatment (twofold) correlates well with the reduced cytotoxicity shown by PG-DOX(pH)-PEG in Figure 3 (twofold). Collectively, our results demonstrate that most of the drug accumulates in the nucleus after free DOX treatment, whereas PG-DOX(pH)PEG abrogates this effect. Instead, our pH sensitive conjugate delivers the drug mainly to lysosomes the natural niche of the parasite. Human leishmaniasis is still a disease that lacks of a potential vaccine and its therapeutic arsenal is scarce. Several private initiatives in collaboration with DNDi, academic and industrial


partners are seeking new therapies with better pharmacological profile.[51,52] According to DNDi priority is to develop a safe, effective, oral, and short antileishmanial drug. Despite all efforts conducted during the last decade in drug discovery, no hit molecule did move forward to clinical testing. Due to the low metabolic rates of intracellular amastigotes in mice models, evidenced by low transcription rates and protein turnover,[53] it is likely to suggest that antileishmanial therapy should combine both, good new chemicals and improved controlled release delivery systems for them. Macrophages are both, the primary site of amastigote replication and the major effector cells to combat them.[54] Therefore, the challenge is to effectively target infected resident macro phages. After infection, macrophage polarizes to a M2b-like

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Figure 8. PG-DOX(pH)-PEG conjugates prevent necrosis in RAW 264.7 macrophages. Cells were incubated with 1.0 µm free DOX or conjugated DOX equivalent amounts for 24 h before apoptosis/necrosis evaluation. Dot-plots showing viability (left bottom), early apoptosis (right bottom), late apoptosis (right top), necrosis (left top).

phenotype with a high expression of C-type lectin receptors (CLR). CLRs are a large family of carbohydrate receptors, which are abundantly expressed by antigen-presenting cells including macrophages and dendritic cells. These receptors recognize terminal glycosylated motifs, such as d-mannose, l-fucose, and N-acetyl-d-glucosamine displayed by pathogens. Among them, mannose receptor, and Dectin-1 are coupled to signaling pathways associated with ROS production.[55] Therefore, nanoconjugates designed to target both receptors in infected macrophages could improve both drug delivery and immune response.

Acknowledgements

4. Conclusion

Conflict of Interest

In this study, we could synthesize and characterize a pH-cleavable conjugate where DOX is attached via a hydrazone bond to the PG as well as a stable conjugate (PG-DOX(non)-PEG) as a control. PG-DOX(pH)-PEG was characterized by DLS, exhibiting a nanoscale size of 12 nm. Moreover, the release of the drug at acidic pH for the cleavable and the stable conjugate were investigated and the results are in accordance with previous publications.[30,33,34] We further studied the uptake and antileishmanial activity of the PG-DOX-PEG conjugates on macrophage cell lines (J774A.1 and RAW 264.7). PG-DOX(pH)PEG conjugate showed a reduced toxicity compared to free DOX in a physiological mouse model of leishmanial infection. In addition, PG-DOX(pH)-PEG conjugate is actively accumulating within macrophage lysosomes bearing L. infantum amastigotes. We found a delayed DOX release of PG-DOX(pH)PEG into PV surrounding amastigotes protecting host cells of necrotic cell death. The conjugate undergoes an endocytic pathway reaching the lysosome, which is the natural niche for leishmania parasites. Finally, these results point out that PG conjugates are promising carriers for a controlled drug release in leishmaniasis.

The authors declare no conflict of interest.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.


C.G.C. and B.D.A. contributed equally to this work. This collaborative research was carried out under the auspices of Programa Iberoamericano de Ciencia y Tecnologia para el Desarrollo (CYTED 214RT0482) to M.A.M.F. and R.M.R., Instituto de Salud Carlos III-FEDER (PI12/00104) to R.M.R. M.C. gratefully acknowledges financial support from the Bundesministerium für Bildung und Forschung (BMBF) through the NanoMatFutur award (ThermoNanogele, 13N12561) and the Freie Universität Berlin Focus Area Nanoscale. M.A.M.F. was funded by Instituto de Salud CarlosIII-FEDER PI1/02016, PI12/0014, and RETICPT13/0010/0028.

Keywords dendritic polyglycerol, doxorubicin, drug delivery system, leishmania Received: March 15, 2017 Revised: May 4, 2017 Published online:

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