Journal of Controlled Release 192 (2014) 249–261

Contents lists available at ScienceDirect

Journal of Controlled Release

j ourna l homepage: www.e lsev ie r .com/ locate / jconre l

Microparticle-loaded neonatal porcine Sertoli cells for cell-basedtherapeutic and drug delivery system

S. Giovagnoli a,⁎, F. Mancuso b, S. Vannini a, M. Calvitti b, M. Piroddi a, D. Pietrella a, I. Arato b, G. Falabella b,F. Galli a, M. Moretti a, L.M. Neri d, M. Bodo b, S. Capitani d, D.F. Cameron e, M. Ricci a, G. Luca b,1, R. Calafiore c,1

a Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italyb Department of Experimental Medicine, University of Perugia, Perugia, Italyc Department of Medicine, University of Perugia, Perugia, Italyd Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italye Department of Pathology and Cell Biology, University of South Florida, Tampa, FL, USA

⁎ Corresponding author at: Department of PharmacPerugia, via del Liceo, 1-06123 Perugia, Italy. Tel.: +39 07

E-mail address: stefano.giovagnoli@unipg.it (S. Giovag1 Riccardo Calafiore and Giovanni Luca share senior aut

http://dx.doi.org/10.1016/j.jconrel.2014.08.0010168-3659/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 April 2014Accepted 1 August 2014Available online 8 August 2014

Keywords:Sertoli cellsCell-based delivery systemsStorageCryopreservationAntibacterial activity

Neonatal porcine Sertoli cells (NPSC) are immune privileged cells showing innate phagocytic and antibacterialactivities. NPSC have been shown capable of immunoaltering the body's response and possess lung homingcapacity. These properties encourage investigation of NPSC as functional components of cell-based therapeuticprotocols to treat lung infections and related complications. In this work, for the first time, NPSC were tailoredto carry an antibiotic drug loaded into poly(D,L lactic) acid microparticles (MP).A loading protocol was developed, which afforded 30% drug uptake and high stability over time, with little or noeffects onNPSC viability,morphology, reactive oxygen species production and DNA integrity. FSH receptor integ-rity, and TGFβ (transforming growth factor β) and AMH (anti-Müllerian hormone) expressionswere unchangedafter 1 month of cryopreservation. Protein tyrosine kinase activation due to phagocytosis may have had resultedin changes in inhibin B expression. The activity of MP-loaded or NPSC alone against Pseudomonas aeruginosawasmaintained throughout 1 month of storage.NPSC couple an innate antibacterial activitywith the capacity to embodydrug loadedMP.We showed for thefirsttime that engineered NPSC can be cryopreserved with no loss of their basic properties, thereby possiblyrepresenting a novel approach for cell-based therapeutic and drug delivery system.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

In the past few years, increasing evidence seems to suggest that cellscan be employed as potential and effective therapeutic tools. Cell-mediated drug delivery systems may represent a step forward to amore selective and safer therapeutic strategy for many disorders [1,2].Cells have been engineered to become, themselves, a source of thera-peutic agents or have been loaded with drugs that may be deliveredto targeted sites for local/regional treatments [3–5]. The most recentapplications of living cell-based drug delivery include neurodegenativediseases such as Alzheimer's and Parkinson's disease, cancer, HIV, lungdisorders, and microbial infections [2]. For instance, properly treatedred blood and mesenchymal stem cells have already been used inclinical trials [6–10]. Nevertheless, the actual potential of cells carrying

eutical Sciences, University of5 5585162.noli).horship of this paper.

bioactive molecules, has not, thus far, been fully exploited. A majorhurdle relates to the unclear fate of live cells, once they are introducedin the body, since they may undergo mutations or unwanted pro-liferation [11]. In this regard, non-viable or artificial cells have beenemployed as drug carriers to safely deliver drugs to body targets [12].However, live cells may offer the advantage of coupling cell drugdelivery capabilitywith cell functionality, whichmay result in beneficialtherapeutic effects to the recipient. In this regard, selecting the appro-priate drug loading procedure may greatly help in accomplishing thegoal. To be a good carrier, the cells should be structurally and function-ally stable prior to and following distribution into the body. The carriercells should be able to transport the drug to the desired site byexploiting particular tropism or cell homing properties. It is thereforemandatory for these cells to retain full integrity and viability throughoutthe loading process.

Several different techniques have been employed to load drugs intocells. Osmotic shocks ormicrowaves have been used to induce transientrupture or permeabilization of the cell membranes, while others report-ed on direct injection of agents into the cells [13]. Unfortunately, thesestrategies can induce excessive biological stress and cannot be applied

250 S. Giovagnoli et al. / Journal of Controlled Release 192 (2014) 249–261

to all cell types. Alternatively, the innate phagocytic capacity possessedby some cell types can be used to pick up different materials, includingdrugs for subsequent delivery. Phagocytosis is the primary mechanismof internalization not only for professional scavenger cells, such asmacrophages, but also for sustentacular cells, such as Sertoli cells(SC).

SC form the tight epitheliumof seminiferous tubules of the testis andare entrustedwith nourishing and protecting spermcells on their devel-opmentwithin the tubules [14]. In this role, they effectively phagocytizedebris resulting from this active spermatogenetic process. SC function isregulated by the pituitary follicle-stimulating hormone (FSH), whichis initiated by FSH binding to its specific receptor (FSH-r) located onthe SC membrane [15]. SC produce about 60 different cytokinesand hormones belonging to the transforming growth factor (TGFβ)superfamily. Inhibin B, androgen-binding-protein, and anti-Müllerianhormone (AMH), among others, are secretory products with importantroles inmaintaining SC function [16]. Additionally, other cytokines, suchas TGFβ, interleukins 1 and 6, interleukin 2-inhibitor, and the presenceof several toll-like receptors contribute to the immune competenceassociated with SC [17,18].

Due to these properties, SC have been used to treat diabetes [19] toexpedite and improve the outcome of pancreatic cell transplantation[20,21], to promote neural cell regeneration [22,23], and to serve as im-munomodulators to potentiate success of several other transplantationprocedures [18,24]. SC themselves lack class II major histocompatibilitycomplex antigens [25] thus accounting, at least in part, for the absenceof the host's immune response to either allo- or xenogeneic SC trans-plants. Recently, rat SC pre-loaded with curcumin containing chitosannanoparticles, were injected i.v. into amousemodel of acute pulmonaryinflammation The cells were observed to localize only in depth in thelungs. This drug delivery protocol resulted in an aggressive therapeuticresponse, without immune complications, rendering the animalsdisease-free and demonstrating the ability of the drug-loaded SC toconstitute a safe and effective therapeutic treatment [26]. SC also playan effective role against bacterial infections of the testis [27]. Eventhough generally recognized, this SC function is poorly addressed inthe literature. The antibacterial activity of SC has been ascribed to thesecretion of defensins in rodents and canines [28,29] and to the highmobility group box chromosomal protein 1 in rat and human SC [30].Addressing this property of SC, a bacteriostatic action of rat SC wasrecently postulated with regard to phagocytized Staphylococcus aureus[31]. Among others, we believe that neonatal porcine SC (NPSC) possessa superior potential not only in allogeneic and xenogeneic transplanta-tion procedures but also as therapeutic agents. In fact, NPSCwere able tosurvive long-term xenograft rejection in rodents and dogs [32,33], andthey seem to prevent MAC (membrane attack complex) formation,which makes them resistant to human antibodies and complement-mediated lysis [34]. Moreover, NPSC can be obtained at high yieldfrom pre-pubertal pigs. Therefore, NPSC may represent good andlikely safe candidates for drug carriers, acting as potential therapeu-tic agents. In particular, we theorize that SC may be used in lung in-fections by combining their immunomodulatory and antibacterialproperties with the possibility of targeting the lung. However, nei-ther the NPSC antibacterial activity nor the use of NPSC as adrug carrier has been so far reported in the literature. In this study,we originally focused on the formulation of NPSC within a drug de-livery system by addressing, primarily, cell stability of particle-loaded NPSC and their storage capability. NPSC were loaded with mi-croparticles (MP) bearing an antibiotic drug complex synthesizedpreviously [35,36]. We assessed, for the first time, cryopreservationof NPSC in order to establish the effect of storage in liquid nitrogenon long-term stability of the loaded cells. Cell functionality was eval-uated before and after loading and freeze-thawing steps. Additional-ly, NPSC alone and NPSC loaded with MP containing an antibioticagainst Pseudomonas aeruginosa (P. aeruginosa) were investigatedover time for antibacterial activity.

2. Materials and methods

2.1. Insoluble antibiotic complex preparation

Ofloxacin (Ofx, Sigma–Aldrich, Milan, Italy) was coordinated withpalladium (Pd) by mixing under stirring at room temperature of thedrug suspension in water with a K2PdCl4 (Sigma–Aldrich, Milan, Italy)solution. The Ofx–Pd complex precipitated completely within anhour and it was centrifuged (Optima TL100 Beckman, 4000 rpm,20 °C, 10 min), washed and vacuum dried overnight. The complexstructure and stoichiometry were determined by TGA, ICP-OES and H1

NMR analyses [36].

2.2. Preparation of Ofx–Pd loaded MP

Ofx–Pd loadedMPwere prepared by spray-drying of a suspension inacetonitrile polymer solution by using a Buchi Mini spray-dryer B290(Buchi, Italy). The polymer employed was poly (DL-lactide) (PLA)R203H (MW 20–30 kDa, Boehringer Ingelheim, Germany). The Ofx–Pddispersion in PLA R203H acetonitrile solution underwent sonicationsteps in order to improve homogeneity and to reduce particlesize. The instrumental parameters were set according to previouslypublished experiments [37,38].

2.3. Ofx–Pd quantification methods—spectrophotometric method

The amount of drug complex encapsulated in the MP was deter-mined by UV–vis spectrophotometry by using an Agilent 8453 (Agilent,Germany) spectrophotometer. The analysis was performed at λmax =287 nm upon calibration in the range 2.5–30 μg/mL in 0.1 M pH 7.4phosphate (r2 = 0.9986).

Samples were prepared by extraction of the drug from the MP.Briefly, a weighed MP amount was dispersed in 1 M NaOH solutionand after overnight rotation the obtained solution was neutralizedwith an equal volume of 1 MHCl solution. The samples were submittedto UV–vis analysis after proper dilution in 0.1 M pH 7.4 phosphatebuffer. The measurements were performed in triplicate and the errorwas expressed as S.D.. The amount of Ofx–Pd entrapped in MP wasexpressed as %Drug Content (%DC) according to Eq. (1):

%DC ¼ mg Ofx−Pd=mg dry powderð Þx100: ð1Þ

2.4. Ofx–Pd quantification methods—HPLC method

An isocratic HPLC method was assessed for drug quantificationin cells by using a Portlab STAYER HPLC system equipped with UVdetection, a parallel pump and Triathlon autosampler (Portlab, Milan,Italy). The conditions were as follows: column Crom-Sil 120 Diol 5 μm250 × 4.6 mm (Grace, Milan, Italy) equilibrated at 31 °C; mobilephase: 50 mM pH = 3.85 trimethylammonium formate: acetonitrile35:65 filtered and sonicated for 30 min to eliminate particulates andgas; flow rate 0.8 mL/min. Detection was performed by UV–vis at287 nm. A standard curve was built between 0.5 and 10 μg/mL (r2 =0.99967).

Biological samples were analyzed after proper purification throughvalidated HybridSPE®-Phospholipid cartridges (Sigma–Aldrich, Milan,Italy).

2.5. MP size and morphology

Particle size analysis was performed by an Accusizer C770 particlecounter (PSS, Santa Barbara, CA) equippedwith an autodilution system.MP were dispersed in 1% w/v Tween 80 solution, slightly sonicatedand immediately analyzed. MP size was expressed as volume mean

251S. Giovagnoli et al. / Journal of Controlled Release 192 (2014) 249–261

diameter and number mean diameter and population spread as span(Eq. (2)):

span ¼ d90−d10ð Þ=d50 ð2Þ

where d90, d10 and d50 are the diameters ≤90%, 10% and 50% of thepopulation distribution, respectively.

The Ofx–Pd MP were morphologically characterized by ScanningElectron Microscopy (SEM) using a FEG LEO 1525 microscope (LEOElectron Microscopy Inc., NY). The acceleration potential voltage wasmaintained at 10 keV. Samples were suspended in water and placedonto carbon tape coated aluminum stubs. After complete water evapo-ration the stubs were sputter coated with chromium prior to imagingby a high resolution sputter (Quorum Technologies, East Essex, UK).Coating was performed at 20 mA for 30 s.

2.6. In vitro drug release

The drug release of Ofx–Pd MP in vitrowas carried out in 15 mL Fal-con tubes containing 10 mL of 0.1 M phosphate buffer at pH 7.4. Thetubes were incubated at 37 °C without shaking. At designated timeintervals, samples were collected and the release mediumwas replacedwith fresh buffer to maintain the same total volume throughout thestudy. The Ofx–Pd concentration was determined by UV–vis spectro-photometry at λ = 287 nm. Each release study was performed intriplicate. Drug release was calculated in terms of %w/w of Ofx–Pdreleased.

2.7. NPSC isolation

Neonatal prepubertal “largewhite” pigs, aging 7–15 days, were usedas NPSC donors. NPSCwere isolated according to previously establishedmethods modified in our laboratory [21,39]. The testes were excisedfrom piglets under general anesthesia. On removal of their fibrouscapsules, the testes were finely minced to obtain a homogenate thatunderwent enzymatic digestion with trypsin/DNAase I in Hanks'balanced salt solution (HBSS; Sigma–Aldrich, St. Louis, MO) untilseminiferous tubule separation. After washing, the tissue suspensionwas incubated with a 2 mg/mL HBSS Collagenase P (Roche DiagnosticsS.p.A., Monza, Italy) solution. The tissue pellet was washed twice inHBSS, centrifuged at 118 ×g for 3 min, sieved through a 500-mm stain-less steel mesh and resuspended in glycine to eliminate the residualLeydig and peritubular cells [40]. The residual tubules were collectedand culture maintained (1 × 106 on 6 multiwell plates) in HAMF12(Euroclone, Milan, Italy) supplemented with 0.166 nM retinoicacid (Sigma–Aldrich, St. Louis, MO) and 5 mL/500 mL of insulin-transforming selenium (Becton Dickinson cat. no. 354352) in 95%air/CO2 at 37 °C. After 3 days, NPSC were incubated with 10 mMtris(hydroxymethyl)aminomethane hydrochloride buffer (Tris; Sigma–Aldrich, Milan, Italy) to eliminate any residual germ cells [41]. Twentyfour hours after Tris treatment, NPSC viability was determined utilizingethidium bromide (EB; Sigma–Aldrich, Milan, Italy) and fluoresceindiacetate (Sigma–Aldrich, Milan, Italy) staining and observed byfluorescence microscopy (Nikon, Optiphot-2, Nikon Corporation,Tokyo, Japan).

2.8. Determination of microparticle loading conditions and NPSC uptake

MP loading into the cells was accomplished by exploiting the innatephagocytic capacity of NPSC. Ofx–Pd loadedMPwere properly weighedand dispersed into HAMF12 medium by gentle bath sonication inorder to avoid foaming and MP flotation. NPSC were plated at a densityof 0.5–1 × 105 cells/cm2 and incubated in standard conditions (37 °C,5% CO2). To investigate the NPSC uptake time profile, incubation wasterminated at 1, 2, and 5 h and 1, 2, 3, and 6 days, and supernatantsand pellets were recovered and analyzed by HPLC to determine the

amount of internalized Ofx–Pd. After supernatant recovery, treatedand untreated cells were washed twice in Dulbecco's PhosphateBuffered Saline (D-PBS, Euroclone, Milan, Italy) and detached bytrypsin/ethylenediaminetetraacetic acid (EDTA) (Lonza Sprl, Verviers,Belgium) for 7 min. All pellets were then spun at 260 ×g for 6 min andstored at 4 °C until use. The uptake was quantified after extraction ofthe drug from cell pellets by digestion with a 1 M NaOH solution. Thepellets were sonicated at 60 °C to ensure complete extraction and MPhydrolysis. All samples were kept under overnight rotationand together with the supernatants immediately submitted to HPLCanalysis. Cell count was performed at the same time points of theuptake study by a hemocytometer (Invitrogen, Countess automatedcell counter, Milan, Italy) after trypan blue staining (Sigma–Aldrich,Milan, Italy).

2.9. Fluorescence microscopy analysis of MP-cell interaction

To investigate cell–MP interaction, morphological characterizationof the uptake process was performed by plating NPSC on 4-well NUNCslides (Microtech, Milan, Italy) at the same density and incubationtime periods of the uptake study. Different MP concentrations anddrug contents were tested to address toxic effects and to establish thebest loading conditions. For morphology evaluation, MP were labeledwith fluorescein isothiocianate (FITC) or Nile red (NR) fluorescent tags(Sigma–Aldrich, Milan, Italy), chosen upon cell viability assessment,and incubated from 5 h to 6 days. NPSC alone were used as controlcells. At each time point, cells were washed twice with D-PBS andfixed in 4%w/v paraformaldehyde at pH 7.4, for 10min at room temper-ature. Afterwashingwith D-PBS, 0.1% v/v Triton x-100 solution (Sigma–Aldrich, Milan, Italy) was added for 5 min to allow for membranepermeabilization. NPSC were treated for 10 min with 500 μL of1 mg/mL RNAase solution (Qiagen, Milan, Italy) and exposed to500 μL 4′,6-diamino-2-phenylindole (DAPI) nuclear probe solutionfor 50 s. Cells were incubated with 200 μL 4% w/v trypan blue solutionfor 1 min, which is known to quench the fluorescence of particles thatare not internalized [42]. Washes in D-PBS followed each step. Theframes were then removed and the slides were sealed and rested for24 h prior to fluorescent microscopy observations.

2.9.1. 3D-microscopy observationsImages were recorded using a digital imaging system based on a

fluorescencemicroscope (model Axiovert 200; Carl ZeissMicroImaging,Inc.) equipped with a back-illuminated CCD camera (Roper Scientific),excitation and emission filter wheels (Sutter Instrument Company),and piezoelectric motoring of the z stage (Physik Instrumente; GmbH& Co.). The data was acquired and processed using the MetaFluoranalyzing program (Universal Imaging Corp.). Green emission wascollected in the range 505–535 nm. The red signal was collected astotal emission N570 nm. Red and green signals were collected and proc-essed as described elsewhere [43]. To build 3D images, 10 stackedpictures above and below the equatorial plane of cell nuclei wererecorded for each sample at 1 μm step and slices at different z-axisfocus were processed with ImageJ software to build 3D pictures.

2.10. Cell freezing procedure for storage

NPSCwere seeded in T75flasks and co-incubatedwithOfx–Pd-loadedMP for 5 h. At the end of the incubation time, the supernatant wasremoved and cells were washed three times with HBSS. After theaddition of 7 mL of trypsin/EDTA, the flasks were incubated at 37 °Cfor 7 min. After detachment, fetal bovine serum (FBS, Invitrogen,Milan, Italy) was added and the cell suspension was centrifuged at260 ×g for 4 min (Eppendorf 5804). The pellets were reconstitutedin the cryopreservation medium consisting of 90% FBS:10% DMSO(Sigma–Aldrich,Milan, Italy). The cell suspensionswere then transferred

252 S. Giovagnoli et al. / Journal of Controlled Release 192 (2014) 249–261

in cryovials and frozen at−80 °C following a protocol from our previouswork and then in liquid nitrogen for long-term storage [44].

2.11. Sertoli cell functional characterization

2.11.1. ROS expressionTotal reactive oxygen species (ROS) expression was measured in

NPSC loaded with Ofx–Pd containing MP immediately after treatmentand over time. NPSC were seeded into 96-well black bottom plates(Microtech, Milan, Italy) where intracellular and extracellular ROSwere determined in pellets and supernatants. After supernatant andpellet separation, the pellets were washed with 100 μL D-PBS. Intracel-lular ROSweremeasuredby treating cellswith 1 μMdichlorofluoresceindiacetate (Invitrogen,Milan, Italy) solution in D-PBS at 37 °C for 30min.Calibration was performed also with standard solutions. Fluorescencewas read after washingwith D-PBS. Extracellular ROS were determinedusing the following working solution: Amplex/Red 50 μL, horseradishperoxidase (HRP) 100 μL, citrate buffer pH = 6 4.85 mL (Invitrogen,Milan, Italy). Fifty microliters of working solution was added to thesupernatants and incubated for 15 min at room temperature. Fluores-cence was read by using a plate reader (DTX 880 Multimode Detector,Beckman Coulter).

2.11.2. DNA damage—single-cell microgel (COMET) assayTreated and untreated NPSC were submitted to DNA damage assay

according to a previous protocol [45]. A positive control consisted intreating NPSC with 1 μM 4-nitroquinoline N-oxide (4NQO, Sigma–Aldrich, Milan, Italy) for 1 h at 37 °C. At the end of treatments, thecells werewashed twicewith 5mL ice-cold D-PBS, pH7.4, and detachedwith 300 μl of 0.05% trypsin (Invitrogen, Milan, Italy) in 0.02% Na4EDTA(Carlo Erba Reagenti, Milan, Italy). After 3 min, trypsinization wasstopped by adding 700 μl FBS. Cells were then collected by centrifuga-tion (70 ×g, 8 min, 4 °C). Briefly, cell pellets were gently resuspendedin 300 μl of 0.7% low-melting point agarose (Sigma–Aldrich, Milan,Italy) (in Ca++/Mg++-free PBS, w/v) at 37 °C, layered onto a conven-tional microscope slide pre-coated with 1% normal-melting pointagarose (in Ca++/Mg++-free PBS, w/v), and covered with a coverslip(Knittel-Glaser, Braunschweig, Germany). After brief agarose solidifica-tion at 4 °C, the coverslipwas removed and the slideswere immersed incold, freshly prepared lysing solution (2.5 M NaCl, 100 mM Na4EDTA,Carlo Erba Reagenti, Milan, Italy), 10 mM Tris–HCl and NaOH topH 10, and 1% Triton X-100, added just before use, for at least 60 minat 4 °C. The slides were then placed in a horizontal electrophoresisbox (HU20, Scie-Plas, Cambridge, UK) filled with a freshly preparedsolution (10 mM Na4EDTA, 300 mM NaOH; pH N 13). Prior to electro-phoresis, the slides were left in the alkaline buffer for 20 min to allowDNA unwinding and expression of alkali-labile damage. Electrophoresisruns were then performed in an ice bath for 20 min by applying anelectric field of 34 V (1 V/cm) and adjusting the current to 300 mA(Power Supply PS250, Hybaid, Chesterfield, MO, USA). The microgelswere then neutralized with 0.4 M Tris–HCl buffer (pH 7.5), fixedin 70% ethanol (10 min, Carlo Erba Reagenti, Milan, Italy), allowed toair-dry and stored in slide boxes at room temperature until analysis.All the steps of the COMET assay were conducted in yellow light toprevent the occurrence of additional DNA damage.

Immediately before scoring, the air-dried slides were stained with65 μL of EB (20 μg/mL) and covered with a coverslip. The COMETs ineach microgel were analyzed (blind), at 500× magnification with anepifluorescent microscope (BX41, Olympus, Japan), equipped with ahigh sensitivity black and white CCD camera (PE2020, Pulnix, UK),under a 100 W high-pressure mercury lamp (HSH-1030-L, Ushio,Japan), using appropriate optical filters (excitation filter 510–550 nmand emission filter 590 nm). Images were elaborated by COMET AssayIII software (Perceptive Instruments, UK). A total of 100 randomlyselected COMETs (50 cells/replicate slides) were evaluated for eachexperimental point.

2.11.3. Assessment of SC preparation morphology and purityAnti-Müllerian inhibiting hormone (AMH) immunostaining was

performed according to previously established methods with minorchanges [39]. Briefly, NPSC were fixed for 30 min in 4% paraformalde-hyde in phosphate-buffered saline (PBS, Sigma Aldrich, Milan, Italy).Before immunostaining, the cells were treated with 0.1% Triton X-100PBS solution for 10min at room temperature, blocked with 0.5% bovineserumalbumin (BSA; Sigma–Aldrich, St. Louis,MO) in PBS for 1 h, there-by exposed to the AMH primary antibody (Santa Cruz Biotechnology,goat anti-rat, polyclonal, 1:100 in PBS) over night at +4 °C. The cellswere then washed with PBS 3 times (5 min each), and thereafter ex-posed to the secondary FITC-conjugated horse anti-goat antibody(1:300 in PBS) for 1 h at room temperature. The cells were counter-stained with 1 mg/mL propidium iodide for 10 min, after RNAse treat-ment (10 mg/mL for 10 min.). A negative control for MIS (Müllerianinhibiting substance) immunostaining consisted of 1:1 overnight incu-bation of the primary antibody with an appropriate blocking peptide(sc-6886 P, Santa Cruz Biotechnology) before use. Negative immuno-staining controls by-passed the primary antibody treatment. Cellswere mounted on slides with 0.6% 1,4-diazabicyclo-(2,2,2)-octane(Sigma-Aldrich, Milan, Italy) in 90% glycerol in PBS. Immunofluorescentslides were examined by confocal laser microscopy [Zeiss, axioplan(AX70) Oberkochen, Germany]. To evaluate the percentage of AMHpositive cells, 10 different sections containing at least 500 cells in totalwere counted.

The presence of contaminating peritubular cells was assessed byendogenous alkaline phosphatase activity [46]. The tissue was fixed in4% buffered formaldehyde, rinsed, and incubated for 15–30 min withthe alkaline phosphatase stain prepared by dissolving 10 mg of thesubstrate (naphthol AS-biphosphoric acid) in 40 mL of DMSO and 5mL of distilled water. The solution pH was adjusted by adding 5 mL ofAMP buffer (25 mM 2-amino-2-methyl-1 propanol containing 1.25mM MgC12, pH 8.9, Sigma Aldrich, Milan, Italy). Ten milligrams ofFast-Red B tetrafluoroborate salt (Sigma Aldrich, Milan Italy) wasadded to the substrate solution, the mixture was vortex-mixed inten-sively, and the precipitate was passed through a 0.2-mm nylon filter.The cells were rinsed with AMP buffer and mounted on a glass slidewith Aquamount (Gur, Bath, England). Alkaline phosphatase-positivecells stained in red.

Morphological identification of Leydig cells was accomplished by3-β-hydroxysteroid dehydrogenase activity upon modification ofprevious procedure [47]. Cell pellets, collected from each 1-mL fraction,were incubated for 30 min at 37 °C with 0.3 mL of staining solution[0.07 M PBS pH 7.2 containing 1 mg/mL of nicotinamide, 6 mg/mL ofb-NAD, 1.5 mg/mL of nitroblue tetrazolium, and 100 g/mL of dehydro-epiandrosterone]. The percentage of Leydig cells was estimated bycounting the number of stained versus total cells.

2.11.4. Western blotting analysisAt the time of supernatant collection, control and treated NPSC

monolayers were scraped off to obtain whole cellular extracts. Thecells were suspended in 100 μl of 10 mM Tris base at pH 7.4, 150 mMNaCl, 1 mM EDTA, 1 mM ethyleneglycol bis(aminoethylether)tetraacetic acid, 1% v/v Triton X-100, 0.5% (v/v) Nonidet P-40, 1 MNaF, 0.2MNaO3V, 0.2Mphenylmethanesulfonylfluoride (SigmaChem-ical Co, St. Louis, MO). Themixture was then spun at 1000 g (Mikro 200,Hettich Zentrifugen, Tuttlingen, Germany) for 10 min, the supernatantwas collected and the total protein content determined by the Bradfordmethod [48]. Small sample aliquots were stored at−20 °C for Westernblotting analysis.

Cell extracts were fractioned by 4–12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), 50 μg protein/lane,blotted on nitrocellulosemembrane (Biorad, Hercules, CA) and incubat-ed overnight in buffer containing 10 mM Tris, 0.5 M NaCl, 1% (v/v)Tween 20 (Sigma Chemical Co, St. Louis, MO), 1:100 goat anti-FSHr Ab(Santa Cruz Biotechnology Inc., Santa Cruz, CA), 1:100 goat anti-AMH

253S. Giovagnoli et al. / Journal of Controlled Release 192 (2014) 249–261

Ab (Santa Cruz Biotechnology Inc., Santa Cruz, CA), 1:100 rabbit anti-inhibin α Ab (Santa Cruz Biotechnology Inc., Santa Cruz, CA), 1:100goat anti-inhibin βB Ab (Santa Cruz Biotechnology Inc., Santa Cruz,CA), 1:1000 rabbit anti-actin Ab (Sigma Chemical Co, St. Louis, MO).The Ag–Ab complex was then detected by incubating the membranefor an additional 60 min in buffer containing 1:5000 HRP-conjugatedanti-rabbit IgG secondary Ab (Sigma Chemical Co, St. Louis, MO) and/or1:1000 HRP-conjugated anti-goat IgG secondary Ab (Sigma ChemicalCo, St. Louis, MO). Specific bands were detected by ECL (enhancedchemiluminescence).

2.11.5. Real-Time PCR analysisTotal messenger RNA (mRNA) was isolated, from untreated and

treated NPSC, bymRNA purification kit (Versagene RNA Cell Kit, GentraSystems, Minneapolis, MN) and quantified by reading the opticaldensity at 260 nm. Briefly, 2 μg of total mRNA was subjected to reversetranscription in a final volume of 20 μL. Real-Time PCR was performedusing 16 ng of the cDNA prepared by the RT reaction and SYBRGreen (Stratagene, Amsterdam, the Netherlands). Real-time PCR wasperformed in an Mx3000P cycler (Stratagene, Amsterdam, theNetherlands) using 6-FAM (6-carboxyfluorescein) for detection andROX (6-carboxyl-X-Rhodamine) as reference dye. The mRNA level ofeach sample was normalized against β-actin mRNA and expressed asfold changes versus the level in untreated control cells.

2.11.6. FSH-r functional integrityFSH-r integrity onNPSC surfacewas determined following FSH and/or

testosterone stimulation. Briefly, control NPSC monolayers and MPloaded NPSC were treated for 3 days with 1 mg/mL of FSH (Serono,Rome, Italy); thereafter, 0.2 mg/mL of testosterone enanthate (SIT,Pavia, Italy) was added for 8 h. At the end of the incubation, FSH-rintegrity was evaluated by Western blotting and the total mRNA byReal-Time PCR analysis as reported above.

2.11.7. Inhibin B, AMH and TGFβ assaysSupernatants were collected from monolayers of untreated NPSC

and MP loaded NPSC samples immediately after preparation and after1 month of storage, stored at−20 °C, and submitted to ELISA for the in-hibin B (kit Inhibin B Gen II ELISA, Beckman Coulter,Webster, TX, U.S.A.,intra assay CV= 2.81%; inter assay CV= 4.33%), AMH (kit AMH Gen IIELISA, Beckman Coulter, Webster, TX, U.S.A., intra assay CV = 2.81%;inter assay CV = 4.33%), and TGFβ1, TGFβ2, TGFβ3 secretion assays(Quantikine, R&D Systems, Minneapolis, MN, U.S.A., intra assay CV =2.76%; inter assay CV= 7.46%). Two to four replicates were performedfor each assay. The total mRNAwas determined by Real-Time PCR anal-ysis as reported above on treated and untreated NPSC monolayers be-fore and after storage.

2.12. In vitro antibacterial activity

2.12.1. Minimum inhibitory concentration assay for Ofx–Pd and Ofx–Pdloaded MP

The minimum inhibitory concentration (MIC) of Ofx, Ofx–Pd andOfx–Pd loaded MP was determined by dilution susceptibility testingreference methods of the Clinical and Laboratory Standards Institute.The samples were tested against P. aeruginosa (ATCC 15692). Quantita-tive analyses were performed in triplicate. Microbial inocula were pre-pared by subculturing bacteria into Muller Hinton Broth (MHB) at37 °C for 18 h and then diluted to approximately 104–105 organisms/mL. One hundred microliters of Ofx, Ofx–Pd and Ofx–Pd loaded MPwere serially diluted 1:2 in the appropriate medium MHB and placedin 96-well tissue culture plate. One hundred microliter aliquots ofmicroorganism suspension were added to each well, microplates werethen incubated at 37 °C for 24 h. MIC was expressed as the lowest con-centration, which inhibited growth judged by lack of turbidity of themedium.

2.12.2. NPSC antibacterial activityKilling of P. aeruginosa by NPSC was assessed by using a standard

bacterial killing assay. Briefly, untreated or MP loaded NPSC were cul-tured to 90–95% confluence in tissue culture plates in HAMF12medium.P. aeruginosa (105 bacteria/well) were added and incubated for 1, 6 and24 h. After incubation at 37 °C under 5% CO2, the plates were vigorouslyshaken, monolayers were lysed by adding Triton X100 0–1% in distilledwater, and serial dilutions were prepared in distilled water from eachwell. Plates (triplicate samples) were made by spreading each sampleon tryptic soy agar plate and the colony forming units (CFU) werevisually evaluated after 24 h of incubation at room temperature. Controlcultures consisted of P. aeruginosa incubated without cells.

2.13. Statistical analysis

Data were analyzed for statistical significance by Student's t test at95% and 99% significance levels from at least three replicates.

3. Results and discussion

3.1. MP characterization and determination of NPSC uptake

The use of an insoluble antibiotic complex was useful to restraindrug losses from the MP during the uptake process. This complex wascharacterized thoroughly in a previous work [36]. The spray-dried MPshowed nonsignificant in vitro release (b4.0%), in 0.1 M phosphatebuffer pH 7.4 and 37 °C, over several days (data not shown), which con-firmed the low solubility of the complex in aqueous media. Moreover,MP exhibited good morphological and dimensional features withregular shape and smooth surfaces. The volume mean diameter andthe number mean diameter measured 5.4 and 1.5 μm, respectively,and the span was 1.2 (Fig. 1A and B). To successfully embed Ofx–Pdloaded MP into NPSC, it is crucial that the internalized particles do notalter the overall cell integrity and viability. The first property to beconsidered was size, as large particles are difficult to be internalizedand can easily damage the cell structure. Therefore, Ofx–Pd loadedMP, because they showed both reduced size and homogeneity wereproven good candidates to be engulfed by NPSC. Moreover, polyestersare in clinical use in numerous depot preparations approved by theFood and Drug Administration: Lupron®, Depo-Provera®, Risperdal®and, more recently, Abilify Maintena® are just a few examples, andthey proved to be safe as adjuvants, even though, as yet, their use indrug targeting has not been clinically assessed. The selectedMP showeda high capacity of drug loading (30% w/w) with the Ofx–Pd drug con-tent, which was important to maximize drug loading into the cells.The high loading capacity was the reason for selecting MP in the placeof nanoparticles for the NPSC engulfment process. Additionally, theencapsulation of Ofx–Pd in the PLA polymer was considered an im-portant technical step because it reduced its direct contact with thecells, thereby reducing any possible harmful effects of Ofx–Pd on cellmorphology and viability.

To investigate the effect of particle–cell interaction and feasibility ofMP loading, NPSC were exposed to different MP concentrations anddrug contents. This approach was useful to address the potential cyto-toxic effect of the encapsulated Ofx–Pd complex. Based on microscopicobservations, MP concentrations of 10 to 20 μg/cm2 did not resultin cell crowding with particles, an important feature, since the excessof particulates could cause cell damage and possibly death.

Exposure to blank MP confirmed the non-toxic nature of this MP onNPSC viability as graphically illustrated in Fig. 2B. MP loaded with 30%and 8% w/w Ofx–Pd, however, induced some effects on cell viability.NPSC viability of b60% was observed at 30% w/w Ofx–Pd content,whereas viability at 8% w/w Ofx–Pd content was N85% (Fig. 2B). NPSCuptake reached 35% w/w after 24 h at 8% drug content (p b 0.001),whereas it decreased to less than 15% w/w at 30% w/w drug content(Fig. 2A). When the MP concentration was reduced from 20 to

Fig. 1. Characterization of Ofx–Pd loaded MP: A) Particle size, B) SEM imaging.

Fig. 2. Effects of MP loading in NPSC: A) % uptake at different MP and drug loadings,B) effects of different loadings on NPSC viability (% of control) and C) correlation of cellviability and uptake at 24 h. *p b 0.001, #p b 0.002 (α = 0.05, n = 3). All data wereexpressed as mean ± S.D.

254 S. Giovagnoli et al. / Journal of Controlled Release 192 (2014) 249–261

10 μg/cm2, keeping the Ofx–Pd content on 30% w/w, the uptakereached 25%w/w (Fig. 2A). As illustrated in Fig. 2C, there was a directcorrelation between viability and uptake. As expected, when cellviability increased and a greater number of viable cells increased,drug uptake also increased (Fig. 2C). Therefore, a drug content of30% w/w, albeit providing higher amounts of antibacterial activity,was found too toxic to the cells. A good compromise betweenintracellular high drug concentration and cell viability was achievedwith a MP concentration of 20 μg/cm2 and 8–10% w/w Ofx–Pd drugcontent. The use of this MP concentration and the NPSC uptakecapacity measured resulted in about 0.5 μg Ofx–Pd/105 cells.

3.2. Uptake stability of MP loaded NPSC

According to the parameters established above, NPSC loading wasperformed by incubation with 20 μg/cm2 PLA MP containing about10% w/w Ofx–Pd. The uptake process was monitored over 144 h(Fig. 3A and B) and expressed as the percent of Ofx–Pd accumulatedin cell pellets over the initial amount fed to cells. The uptake amountedto about 35–38% at 48 h, which then leveled off over the 6 day investi-gational period (Fig. 3A). Generally, 3 to 5 h are considered sufficientto complete the uptake process of phagocytic cells. Hence, the time ittook to maximize the uptake by 48 h was likely due to the expandingcell population in vitro (2–3 days doubling time) and not to altereddynamics of the phagocytic process itself. In fact, since NPSC wereplated at low density to prevent confluence, so as to maximize theinteraction with MP, it is likely that part of the MP adhered to thewells cell-free surface (data not shown). The growing NPSC population

engulfed the deposited particles and this process was responsible forthe uptake increase, measured up to day 2 of the beginning. The lowamount of drug found in the supernatants could be ascribed to thelow solubility of Ofx–Pd as well as cell life cycle that requires thereplacement of the incubation medium every 48 h.

The Ofx–Pd intracellular accumulation (Fig. 3B) had a profile similarto the proportion taken up. The maximum amount of intracellularOfx–Pd obtained was consistent with what was calculated previouslyof 0.5–0.6 μg/105 cells.

Fig. 3. NPSC uptake time profile of MP A) in pellets and supernatants and B) Ofx–Pd complex intracellular accumulation. Comparison of NPSC MP uptake over time after freezing:C) incubation after thawing and D) after 1 month storage. *p b 0.003 (α = 0.05, n = 3). All data were expressed as mean ± S.D.

255S. Giovagnoli et al. / Journal of Controlled Release 192 (2014) 249–261

The NPSC were frozen in order to evaluate the long-term stability ofOfx–Pd uptake. The non-frozen and frozen NPSC alone and MP loadedNPSCwere assayed over 6 days of incubation (Fig. 3A and C) to establishif and how long NPSC could maintain the initial uptake rate beforeand after freezing. The uptake was also evaluated over 1 month ofcryopreservation (Fig. 3D). In both cases, the Ofx–Pd uptake was stablewith no significant changes in the thawed cells over 6 days of incubationor after 1 month of cryopreservation.

These results likely demonstrate that NPSC are able to maintainthe initial uptake of Ofx–Pd containing MP over time and upon cryo-preservation, thereby indicating their suitability for good drug deliverycarriers and good candidates for cell-based drug delivery systems.

3.3. Characterization of MP loaded NPSC morphology

The presence of the MP in NPSC was characterized through morpho-logical analysis by fluorescent microscopy. NPSC were loaded with fluo-rescent labeled MP as previously described. Two main effects wereexamined to validate this procedure: a) the effects of labeling on MPproperties and b) the tagging effects on cells. The two selectedfluorescenttags, NR and FITC, showed only negligible effects on MP size distributionwith no changes of the basic content properties or particle morphology(data not shown). These compounds were associated with minor effectson cell viability, although at a 0.5%w/w concentration, viabilitywas prov-en to be N80% in both cases. Eventually, the green FITC fluorescence waspreferred to the red emission of NR due to better contrast.

The images in Fig. 4 depict the FITC labeled MP distributed in theNPSC cytosol at all the studied time points. TheMP fluorescence shiftedfrom green to a pale blue after day 2, due to FITC time related decay. Cellmembranes stained with trypan blue overlapped with DAPI stainednuclei and the MP fluorescence was not quenched, suggesting that MPwere actually internalized within NPSC and not adsorbed on the cell

surface. To prove this hypothesis, stacked images at different focusdistance above and below the equatorial plane of the cells wereacquired at 1 μm step distance and arranged as 3D pictures in Fig. 4A–D.The MP are clearly located in the vicinity of the NPSC nuclei as shown inPanels B and C recorded at −1 and +1 μm, respectively, from the cellequatorial plane. As distance increased, MP disappeared (Panels A andD) indicating that the MP were inside the cells. Panels E–H in Fig. 4showed the same 3D stacked images after 1 month of cryopreservationand further confirmed the presence of the MP inside the NPSC. Theseobservations supported previous results indicating preservation of MPuptake over time, including after cryopreservation (Fig. 3 A–D).

3.4. Effects of loading on NPSC functional integrity

MP were internalized by exploiting the innate NPSC phagocyticcapacity. This procedure is much safer than loading methods based onphysical stimulation or shock. However, either the materials employedor particle hindrance itself can stress the cells, through serious celldamage and death, especially over a long period of time. To understandthe stress level that may have been induced by the incorporation of MPin the NPSC, ROS and DNA damage were evaluated before and afterloading and after cryopreservation.

NPSC, either loaded or unloaded with MP, were followed for 6 daysbefore and after freezing to determine how long cells could go beforeuse. In particular, all post-freezing studies were performed after cellthawing and incubation for 48 h.

Immediately after loading, no particular changes either of intracellu-lar or extracellular ROS were detected (Fig. 5A). The same results wereobtained even after freezing. Significant increase of intracellular ROS(p b 0.001) was observed only at day 6 following incubation eitherbefore or after freezing, while extracellular ROS were unchanged. Theincrease may indicate that MP loaded NPSC should be frozen or

Fig. 4. (Left) Fluorescence images of MP-loaded NPSC, after 5 h, 1 day, 2 days and 6 days, showing DAPI stained nuclei (white arrows), FITC-labeled MP (yellow arrows) and trypan bluestainedmembranes (red). Bars= 10 μm. (Right) 3D projection of stacked digital Z seriesmicroscopy images ofMP-loaded NPSC at 1 day (Panels A–D) and 1 month storage (Panels E–H).For this set of experiments 10optical section stackswere taken above and below the equatorial plane of the cells. The sections shown are at A)−4, B)−1, C)+1, andD)+4 μmand E)−5,F) 1−1, G)+1, and H)+5 μm z-axis focus distance from the equatorial plane. Arrows indicate the internalizedMP. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)

256 S. Giovagnoli et al. / Journal of Controlled Release 192 (2014) 249–261

employed within 3–4 days of MP loading or thawing after storage.Higher intracellular ROS may not be the result of cell damage, but itmay reflect an early sign of a change of the cell interior milieu andintegrity. The observed ROS expression upon storage may supportthis hypothesis since ROS expression, as compared to the control ateach time point was practically unchanged after 7 days and 1 monthof storage (Fig. 5B). On the other hand, the intracellular ROS valuesincreased after 1 month of freezing (p b 0.001) if compared to the notfrozen cells (Fig. 5B). Extracellular ROS were instead lower with thisresult to be ascribed to a dilution effect since NPSC were incubated infresh medium. The increase after 1 month of storage related to ROSintracellular accumulation during storage and it was maintained asthe result of stress due to cryopreservation rather than to the presenceof the particles, since loadedNPSC and controlswere practically identical(Fig. 5B).

Because of the heavy cell manipulations associated with loading andfreezing, the overall stress endured by the cellswas consideredmild andlower than expected.

Fig. 5C and D show the COMET assay results performed, as for theROS study, before and after freezing and upon storage. Comparing thevalues of tail intensity obtained for the MP loaded NPSC and thecontrols, the absence of DNA damage was evident with values always

b1% either in the period following loading and after freezing (Fig. 5C)or upon 1 month of storage (Fig. 5D). The sensitivity of the test wasconfirmed by using 4NQO as a positive control, as shown in Fig. 5C.

However, ROS expression and DNA damage assays alone are notsufficient to exclude specific adverse effects on NPSC functional compe-tence. In this regard, the expression by NPSC of fundamental factorsbelonging to the TGFβ superfamily, such as TGFβ1,2,3 isoforms, inhibinB, AMH, and the FSH-r were evaluated for a specific change. The expres-sion of AMH, inhibin B and TGFβwas determined following 1 month ofstorage.We particularly focused on AMH and inhibin B because they arerecognized as specific functional markers for SC [49] and reduced AMHand inhibin expression has been associated with SC neoplastic muta-tions [50,51]. Therefore, a change in the expression of these factorswould indicate an alteration in the SC activity, function and propensityto cell mutation.

Overall, MP loading did not significantly affect expression of thesefactors, since the expressed proteins and mRNA's were similar in MPloaded cells and controls (Fig. 6C–E). Changes, however, were observedafter 1 month of storage: a) inhibin B decreased about 5-fold in bothsupernatants of controls and loaded NPSC (Fig. 6C), b) the TGFβ2

mRNA expression was 1/3 of the initial values (p b 0.005) with nodifferences between controls and loaded NPSC (Fig. 6D), c) AMH

Fig. 5. Evaluation of intracellular and extracellular ROS expression: A) Not frozen and frozen NPSC were followed up for 6 days, B) NPSC and MP loaded NPSC frozen for 7 and 30 daysanalyzed after thawing and compared to not frozen cells. COMET test of NPSC andMP loadedNPSC before and after storage: C)NPSCwere followed up for over 6 days of incubation beforeand after freezing (positive control with 4NQO is shown) and D) analysis after 7 and 30 days of storage in comparison to not frozen cells. *p b 0.001 (α = 0.05, n = 3). All data wereexpressed as mean ± S.D.

257S. Giovagnoli et al. / Journal of Controlled Release 192 (2014) 249–261

expression in cell pellets doubled in both controls and loaded NPSC(p b 0.005), while the inhibin βB subunit doubled in controls(p b 0.005) and disappeared in the loaded NPSC (Fig. 6E).

These results may indicate that the overall change of NPSCcompetence and function wasminimal even after 1 month of cryopres-ervation. In fact, excluding the change in TGFβ2, total TGFβ and AMHwas not significantly modified. The biggest alterations were observedin the secreted inhibin B (Fig. 6C) and in the βB subunit thatdisappeared (p b 0.005) in the pellets of the loaded NPSC after storage(Fig. 6E). The reason behind the βB subunit disappearance is unclear;however, suppression of theβB subunit is reportedwhen protein kinasepathways are activated in immature SC as a consequence of phagocyto-sis [52]. On the other hand, the inhibin B mRNA expression remainedunaltered suggesting that NPSC maintained their function despite thechanges in inhibin B expression (Fig. 6D). Therefore, MP loading mayhave activated kinase pathways with consequences on the long-termexpression of inhibin B, a hypothesis to be further tested. Additionally,FSH-r integrity andmRNA expressionwere determinedwith orwithoutFSH and testosterone stimulation to assess the MP loading effects(Fig. 6A andB). In general, no difference in FSH-r integritywas observed,whether NPSC were stimulated or not, and no differences wereobserved between MP loaded NPSC and controls (Fig. 6A). The FSH-rmRNA was significantly up-regulated (p b 0.05) in basal NPSC afterloading (Fig. 6B). As expected, following FSH stimulation of NPSC,mRNA increased significantly in the controls, whereas a negativetrend was observed in MP loaded NPSC upon FSH and/or testosteronestimulation.

Two observations remain, so far, unexplained. The first is the highermRNA expression for FSH-r and down-regulation observed upon FSHand testosterone stimulation in the MP loaded cells. The second is theAMH and the inhibin βB-subunit significant increase in the cell pellets

at 1 month of storage (Fig. 6E). As stated above, an explanation couldbe that protein kinase induced-phagocytosis may alter NPSC responseto external stimuli and/or maturation profile. We may speculate that,since SC are known to express all three TAM receptor protein tyrosinekinases (TYRO3, AXL and MER) that are in control of phagocytosis andimmune response [53], the changes observed upon MP loading mayrelate to activation of these enzymes, in turn able to influence SCexpression. The link, however, between protein tyrosine kinase activityand SC expression has not been fully described in the literature andwillbe the matter of further investigation.

3.5. NPSC antibacterial activity against P. aeruginosa

In order to understandwhetherMP loaded NPSC retain antibacterialactivity, even after long-term storage, we investigated the activity ofNPSC loaded with Ofx–Pd containing MP and unloaded NPSC againstP. aeruginosa. This ubiquitous pathogen was chosen because it isthe source of many complications and life-threatening infections inimportant disorders affecting the lungs, such as cystic fibrosis. To ourknowledge this is the first time that NPSC have been investigated aspotential antibacterial agents.

Preliminarily, NPSC loadedwith blankMPwere tested and no activitydifferences in comparison with NPSC alone were observed (data notshown). This ruled out any possible inhibition on NPSC due to thepolymer or particles. In addition, the MIC values against P.aeruginosawere determined to address possible differences between the free andloaded complex and the free drug. The MIC of Ofx–Pd loaded MPamounted to 3.13 μg/mL, about four times that of free Ofx–Pd(0.78 μg/mL) or 9 times that of free Ofx (0.39 μg/mL). This activityreduction was expected due to the high insolubility of the complexthat is slowly dissolved or released and, over the time period investigated,

Fig. 6. A) Evaluation of FSH-r integrity upon loading: by densitometric analysis and B) mRNA expression by Real-Time PCR: basal (without stimulation), +FSH: after FSH stimulation,+FSH + T: after FSH and testosterone stimulation. Evaluation before and after 1 month of storage of: C) AMH and Inhibin B in the supernatants by ELISA assay, D) TGFβ isoforms,AMHand Inhibin B by Real-Time PCR and E) AMH and inhibin B subunits in cell pellets by densitometric analysis. Each experimentwas replicated two to four times and one representativeexperiment is shown in each panel. *p b 0.05, #p b 0.005. All data were expressed as mean ± S.D.

258 S. Giovagnoli et al. / Journal of Controlled Release 192 (2014) 249–261

not as available as the free drug. NPSC were infected with low bacteriaconcentration (about 1:10 bacteria/cells ratio) that was found not toaffect cell viability. Due to the phagocytic capacity of NPSC, analyseswere performed on the supernatants and cell pellet lysates to evaluateextra and intracellular activities. Following this bacterial infection,untreated and MP loaded NPSC showed significant killing capacity(p b 0.0001, p b 0.001), especially considering the exponential growthof control bacteria cultures (Fig. 7A). NPSC showed intracellular andextracellular antibacterial activities over time, with 1 and 1.5 log

reductions in cell lysates and 0.2 and 1.0 in the supernatants for NPSCalone and MP loaded NPSC, respectively, at 24 h. These results indicatethat there is a synergistic effect of cells and Ofx–Pd, which is releasedfrom the MP diffusing outside the cell as well, as shown by the differ-ences observed in the supernatants.

The limited killing score of NPSC alone and MP loaded NPSC wasexplained considering that the actual Ofx–Pd concentration estimatedin the wells (0.5–0.6 μg/mL) was well below the measured MIC(3.13 μg/mL) and this resulted in a partial killing effectiveness.

Fig. 7.NPSC antibacterial activity against P. aeruginosa. A: NPSC alone or loaded with MP containing Ofx–Pd complex were incubatedwith P. aeruginosa for 1, 6 and 24 h. After incubation,supernatants were recovered diluted and plated in Tryptic Soy Agar (TSA);monolayerswere lysed and the resulting cell lysateswere diluted and plated in TSA. B: antimicrobial activity ofNPSC fresh or stored in liquid nitrogen for 30 days. NPSC alone orMP loaded NPSCwere incubatedwith P. aeruginosa for 24 h. The graphs are the result of two independent sets of exper-iments. *p b 0.0001, #p b 0.001 (α = 0.05, n = 6). All data were expressed as mean ± S.D.

259S. Giovagnoli et al. / Journal of Controlled Release 192 (2014) 249–261

Moreover, the bacteria concentration employed, if low enough toprevent cell death, was likely too high looking at the fast growth profileof P. aeruginosa. This protocol will be optimized in future experiments.

The most important result, however, was that NPSC maintained theantibacterial activity even after cryopreservation as demonstrated byoverlapping profiles of supernatant and cell lysate CFU recorded at 0and 30 days of storage (Fig. 7B). This important finding indicates thatMP loaded NPSC may withstand cryostatic storage without functionaldeterioration and therefore fulfill need for banked, bioengineeredcells suitable for use in cell-based therapeutic protocols for clinicalapplication.

4. Conclusions

The unique features of NPSC can be exploited to create easy andefficient cellular tools for novel cell-based drug delivery systems. First,it was shown that NPSC are associated with good loading capacity andstability over time. Secondly, it was demonstrated that Ofx–Pd contain-ingMP loaded NPSC remain stable and functional upon storage by cryo-preservation and that the thawed cells maintain antibacterial activitywhether be loaded or not with MP.

This work, for the first time, focused on the possibility of employingNPSC as candidates for use in cell-based therapeutic systems. Of course,as yet some open questions remain, especially with regard to a betterunderstanding of drug loaded NPSC antibacterial mechanisms andefficacy. In fact, we turned our attention on the main effects of loadingand storage onNPSC, rather than on thorough analysis of the underlyingmechanisms. In fact, the latter is worth to pursue only after showing thecell's retention of viability and functional competence upon, oftentraumatizing, in vitro manipulation. According to our results, NPSCseem to fulfill this first screening step and for this reason experimentsare currently being expanded to determine best storage conditionsover longer periods of time. Because of the virtual lack of appropriatein vitro experimental settings to assess the SC-based immunoregulatoryproperties of our product, we do believe that in vivo animal models aremandatory to begin, in order to thoroughly determine the efficiency ofour system in infectious/inflammatory disorders.

Acknowledgment

This work was supported by a grant fromMr. Gary Harlem (AltucellInc. 3 Astor Court, DixHills, NewYork, NY, USA). The kind support of the

260 S. Giovagnoli et al. / Journal of Controlled Release 192 (2014) 249–261

“Consorzio Interuniversitario per i Trapianti d'Organo”, Rome, Italy isgratefully acknowledged.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jconrel.2014.08.001.

References

[1] F. Pierigè, S. Serafini, L. Rossi, M. Magnani, Cell-based drug delivery, Adv. Drug Deliv.Rev. 60 (2008) 286–295, http://dx.doi.org/10.1016/j.addr.2007.08.029.

[2] E.V. Batrakova, H.E. Gendelman, A.V. Kabanov, Cell-mediated drug delivery, ExpertOpin. Drug Deliv. 8 (4) (2011) 415–433, http://dx.doi.org/10.1517/17425247.2011.559457.

[3] J. Shao, J. DeHaven, D. Lamm, D.N. Weissman, C.J. Malanga, Y. Rojanasakul, J.K. Ma, Acell-baseddrugdelivery system for lung targeting: I. Preparation andpharmacokinetics,Drug Deliv. 8 (2001) 61–69, http://dx.doi.org/10.1080/107175401750176981.

[4] J. Shao, J. DeHaven, D. Lamm, D.N. Weissman, C.J. Malanga, Y. Rojanasakul, J.K. Ma,A cell-based drug delivery system for lung targeting: II. Therapeutic activities onB16-F10 melanoma in mouse lungs, Drug Deliv. 8 (2001) 71–76, http://dx.doi.org/10.1080/107175401750177007.

[5] M.A. Fischbach, J.A. Bluestone, W.A. Lim, Cell-based therapeutics: the next pillarof medicine, Sci. Transl. Med. 5 (2013) 179 ps7, http://dx.doi.org/10.1126/scitranslmed.3005568.

[6] J.W. Yoo, D.J. Irvine, D.E. Discher, S. Mitragotri, Bio-inspired, bioengineered andbiomimetic drug delivery carriers, Nat. Rev. Drug Discov. 10 (2011) 521–535,http://dx.doi.org/10.1038/nrd3499.

[7] S. Biagiotti, M.F. Paoletti, A. Fraternale, L. Rossi, M. Magnani, Drug delivery by redblood cells, IUBMB Life 63 (8) (2011) 621–631, http://dx.doi.org/10.1002/iub.478.

[8] S. Ravilla, B.R. Chandu, S. Nama, B. Nagaveni, Erythrocytes as carrier for drugs,enzymes and peptides, J. Appl. Pharm. Sci. 02 (04) (2012) 166–176, http://dx.doi.org/10.7324/JAPS.2012.2503.

[9] V.R. Muzykantov, Drug delivery by red blood cells: vascular carriers designed byMother Nature, Expert Opin. Drug Deliv. 7 (4) (2010) 403–427, http://dx.doi.org/10.1517/17425241003610633.

[10] I.H. Gamaledin, F.I. Mohamed, K.A. Fars, I.A. Alsarra, Application and safety oferythrocytes as a novel drug delivery system, Asian J. Biochem. 6 (4) (2011)309–321, http://dx.doi.org/10.3923/ajb.2011.309.321.

[11] L. Dawson, A.S. Bateman-House, D. Mueller Agnew, H. Bok, D.W. Brock, A.Chakravarti, M. Greene, P.A. King, S.J. O'Brien, D.H. Sachs, K.E. Schill, A. Siegel, D.Solter, S.M. Suter, C.M. Verfaillie, L.B. Walters, J.D. Gearhart, R.R. Faden, Safetyissues in cell-based intervention trials, Fertil. Steril. 80 (5) (2003) 1077–1085,http://dx.doi.org/10.1016/S0015-0282(03)02218-0.

[12] T.M. Chang, From artificial red blood cells, oxygen carriers, and oxygen therapeuticsto artificial cells, nanomedicine, and beyond, Artif. Cells Blood Substit. Immobil.Biotechnol. 40 (3) (2012) 197–199, http://dx.doi.org/10.3109/10731199.2012.662408.

[13] L. Rossi, S. Serafini, M. Magnani, Red blood cell loading: a selection of procedures,In: M. Magnani, editor, Erythrocyte engineering for drug delivery and targeting.Georgetown, Texas: Eurekah.com, and New York: Kluwer Academic/PlenumPublishers, 2003. p. 1–18.

[14] M.K. Skinner, M.D. Griswold (Eds.), Sertoli Cell Biology, vol.1, Elsevier AcademicPress, San Diego, CA, USA, 2005.

[15] M. Simoni, J.R. Gromoll, E. Nieschlag, The follicle-stimulating hormone receptor:biochemistry, molecular biology, physiology, and pathophysiology, Endocr. Rev. 18(6) (1997) 739–773, http://dx.doi.org/10.1210/edrv.18.6.0320.

[16] Y. Makanji, C.A. Harrison, D.M. Robertson, Feedback regulation by inhibins A and Bof the pituitary secretion of follicle-stimulating hormone, in: G. Litwack (Ed.),Vitamins and Hormones, vol. 85, Academic Press, Burlington, 2011, pp. 299–321.

[17] B. Sun, N. Qi, T. Shang, H. Wu, T. Deng, D. Han, Sertoli cell-initiated testicular innateimmune response through toll-like receptor-3 activation is negatively regulated byTyro3, Axl, and Mer receptors, Endocrinology 151 (6) (2010) 2886–2897, http://dx.doi.org/10.1210/en.2009-1498.

[18] P. Mital, G. Kaur, J.M. Dufour, Immunoprotective Sertoli cells: making allogeneicand xenogeneic transplantation feasible, Reproduction 139 (3) (2010) 495–504,http://dx.doi.org/10.1530/REP-09-0384.

[19] F. Fallarino, G. Luca, M. Calvitti, F. Mancuso, C. Nastruzzi, M.C. Fioretti, U. Grohmann,E. Becchetti, A. Burgevin, R. Kratzer, P. van Endert, L. Boon, P. Puccetti, R. Calafiore,Therapy of experimental type 1 diabetes by isolated Sertoli cell xenografts alone, J.Exp. Med. 206 (11) (2009) 2511–2526, http://dx.doi.org/10.1084/jem.20090134.

[20] G. Luca, R. Calafiore, G. Basta, M. Ricci, M. Calvitti, L. Neri, C. Nastruzzi, E. Becchetti, S.Capitani, P. Brunetti, C. Rossi, Improved function of rat islets upon co-microencapsulation with Sertoli cells in alginate/poly-l-ornithine, AAPSPharmSciTech.2 (2001), http://dx.doi.org/10.1208/pt020315 (article 15).

[21] G.S. Korbutt, J.F. Elliott, R.V. Rajotte, Cotransplantation of allogeneic islets withallogeneic testicular cell aggregates allows long term graft survivalwithout systemicimmunosuppression, Diabetes 46 (1997) 317–322, http://dx.doi.org/10.2337/diab.46.2.317.

[22] A.E. Willing, D.F. Cameron, P.R. Sanberg, Sertoli cell transplants: their use in thetreatment of neurodegenerative disease, Mol. Med. Today 4 (11) (1998) 471–477,http://dx.doi.org/10.1016/S1357-4310(98)01355-0.

[23] P.R. Sanberg, C.V. Borlongan, S. Saporta, D.F. Cameron, Testis-derived Sertoli cellssurvive and provide localized immunoprotection for xenografts in rat brain, Nat.Biotechnol. 14 (1996) 1692–1695, http://dx.doi.org/10.1038/nbt1296-1692.

[24] P.J. Turek, S.B. Malkowicz, J.E. Tomaszewski, A.J. Wein, D. Peehl, The role of theSertoli cell in active immunosuppression in the human testis, Br. J. Urol. 77 (1996)891–895, http://dx.doi.org/10.1046/j.1464-410X.1996.00322.x.

[25] D.F. Emerich, R. Hemendinger, C.R. Halberstadt, The testicular-derived Sertoli cell:cellular immunoscience to enable transplantation, Cell Transplant. 12 (2003)335–349, http://dx.doi.org/10.3727/000000003108746894.

[26] A. Kumar, M. Glaum, N. El-Badri, S. Mohapatra, E. Haller, S. Park, L. Patrick, L.Nattkemper, D. Vo, D.F. Cameron, Initial observation of cells mediated drug deliveryto the deep lung, Cell Transplant. 20 (2011) 609–618, http://dx.doi.org/10.3727/096368910X536491.

[27] K. Nagaosa, C. Nakashima, A. Kishimoto, Y. Nakanishi, Immune response to bacteriain seminiferous epithelium, Reproduction 137 (2009) 879–888, http://dx.doi.org/10.1530/REP-08-0460.

[28] Y. Sang, M.T. Ortega, F. Blecha, O. Prakash, T. Melgarejo, Molecular cloning andcharacterization of three β-defensins from canine testes, Infect. Immun. 73 (5)(2005) 2611–2620, http://dx.doi.org/10.1128/IAI.73.5.2611-2620.2005.

[29] V. Grandjean, S. Vincent, L. Martin, M. Rassoulzadegan, F. Cuzin, Antimicrobialprotection of the mouse testis: synthesis of defensins of the cryptdin family, Biol.Reprod. 57 (1997) 1115–1122, http://dx.doi.org/10.1095/biolreprod57.5.1115.

[30] C.K. Zetterström, M.L. Strand, O. Söder, The high mobility group box chromosomalprotein 1 is expressed in the human and rat testis where it may function as anantibacterial factor, Hum. Reprod. 21 (11) (2006) 2801–2809, http://dx.doi.org/10.1093/humrep/del256.

[31] A. Shiratsuchi, Y. Osada, Y. Nakanishi, Differences in the mode of phagocytosis ofbacteria between macrophages and testicular Sertoli cells, Drug Discov. Ther. 7 (2)(2013) 73–77, http://dx.doi.org/10.5582/ddt.2013.v7.2.73.

[32] J.M. Dufour, R.V. Rajotte, K. Seeberger, T. Kin, G.S. Korbutt, Long-term survival ofneonatal porcine Sertoli cells in non-immunosuppressed rats, Xenotransplantation10 (2003) 577–586, http://dx.doi.org/10.1034/j.1399-3089.2003.00059.x.

[33] P.F. Gores, D.H. Hayes, M.J. Copeland, G.S. Korbutt, C. Halberstadt, S.A.Kirkpatrick, R.V. Rajotte, Long-term survival of intra-testicular porcine isletsin non-immunosuppressed beagles, Transplantation 75 (2003) 613–618,http://dx.doi.org/10.1097/01.TP.0000052376.89400.8D.

[34] J.M. Dufour, M. Hamilton, R.V. Rajotte, G.S. Korbutt, Neonatal porcine Sertoli cellsinhibit human natural antibody-mediated lysis, Biol. Reprod. 72 (2005) 1224–1231,http://dx.doi.org/10.1095/biolreprod.104.038315.

[35] L.M.M. Vieira, M.V. de Almeida, M.C.S. Lourenco, F.A. Bezerra, A.P.S. Fontes,Synthesis and antitubercular activity of palladium and platinum complexes withfluoroquinolones, Eur. J. Med. Chem. 44 (2009) 4107–4411, http://dx.doi.org/10.1016/j.ejmech.2009.05.001.

[36] S. Giovagnoli, M.L. Marenzoni, M. Nocchetti, C. Santi, P. Blasi, A. Schoubben, M. Ricci,Synthesis, characterization and in vitro extracellular and intracellular activity againstMycobacterium tuberculosis infection of new second-line antitubercular drug-palladium complexes, J. Pharm. Pharmacol. 66 (2014) 106–121, http://dx.doi.org/10.1111/jphp.12162.

[37] F. Palazzo, S. Giovagnoli, A. Schoubben, P. Blasi, C. Rossi, M. Ricci, Development of aspray-drying method for the formulation of respirable microparticles containingofloxacin–palladium complex, Int. J. Pharm. 440 (2013) 273–282, http://dx.doi.org/10.1016/j.ijpharm.2012.05.045.

[38] S. Giovagnoli, F. Palazzo, A. Di Michele, A. Schoubben, P. Blasi, M. Ricci, The influenceof feedstock and process variables on the encapsulation of drug suspensions byspray-drying in fast drying regime: the case of novel antitubercular drug–palladiumcomplex containing polymeric microparticles, J. Pharm. Sci. 103 (4) (2014)1255–1268, http://dx.doi.org/10.1002/jps.23902.

[39] G. Luca, M. Calvitti, C. Nastruzzi, L. Bilancetti, E. Becchetti, G. Angeletti, F. Mancuso, R.Calafiore, Encapsulation, in vitro characterization, and in vivo biocompatibility ofSertoli cells in alginate-based microcapsules, Tissue Eng. 13 (3) (2007) 641–648,http://dx.doi.org/10.1089/ten.2006.0137.

[40] J.P. Mather, D.D. Philips, Primary culture of testicular somatic cells, in: D.W. Barnes,D.A. Sirbasku, G.H. Sato (Eds.), Methods for Serum Free Culture of Cells of theEndocrine System, Liss, New York, 1999, pp. 24–45.

[41] M. Galdieri, E. Ziparo, F. Palombi, M.A. Russo, M.J. Stefanini, Pure Sertoli cell cultures:a new model for the study of somatic-germ cell interactions, J. Androl. 2 (1981)249–254, http://dx.doi.org/10.1002/j.1939-4640.1981.tb00625.x.

[42] A.A. Torrano, J. Blechinger, C. Osseforth, C. Argyo, A. Reller, T. Bein, J. Michaelis, C.Bräuchle, A fast analysis method to quantify nanoparticle uptake on a single celllevel, Nanomedicine 8 (11) (2013) 1815–1828, http://dx.doi.org/10.2217/nnm.12.178.

[43] P. Pinton, S. Leo, M.R. Wieckowski, G. Di Benedetto, R. Rizzuto, Long-termmodulationof mitochondrial Ca2+ signals by protein kinase C isozymes, J. Cell Biol. 165 (2004)223–232, http://dx.doi.org/10.1083/jcb.200311061.

[44] G. Luca, C. Lilli, C. Bellucci, F. Mancuso, M. Calvitti, I. Arato, G. Falabella, S. Giovagnoli,M.C. Aglietti, A. Lumare, G. Muzi, R. Calafiore, M. Bodo, Toxicity of cadmium onSertoli cell functional competence: an in vitro study, J. Biol. Regul. Homeost. Agents27 (3) (2013) 805–816.

[45] R.R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y.Miyamae, E. Rojas, J.C. Ryu, Y.F. Sasaki, Single cell gel/COMET assay: guidelinesfor in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen. 35 (3)(2000) 206–221, http://dx.doi.org/10.1002/(SICI)1098-2280(2000)35:3b206::AID-EM8N3.0.CO;2-J).

[46] R.E. Chapin, J.L. Phelps, B.E. Miller, T.J.B. Gray, Alkaline phosphatase histochemistrydiscriminates peritubular cells in primary rat testicular cell culture, J. Androl. 8(1987) 155–161, http://dx.doi.org/10.1002/j.1939-4640.1987.tb02427.x.

261S. Giovagnoli et al. / Journal of Controlled Release 192 (2014) 249–261

[47] E. Steinberger, A. Steinberger, O. Vilar, Cytochemical study of delta-5-3-beta-hydroxysteroid dehydrogenase in testicular cells grown in vitro, Endocrinology 79(1966) 406–410, http://dx.doi.org/10.1210/endo-79-2-406.

[48] M.M. Bradford, A rapid and sensitive for the quantitation of microgram quantititesof protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976)248–254, http://dx.doi.org/10.1016/0003-2697(76)90527-3.

[49] R.P. Grinspon, N. Loreti, D. Braslavsky, P. Bedecarrás, V. Ambao, S. Gottlieb, I. Bergadá,S.M. Campo, R.A. Rey, Sertoli cell markers in the diagnosis of paediatric malehypogonadism, J. Pediatr. Endocrinol. Metab. 25 (1–2) (2012) 3–11, http://dx.doi.org/10.1515/jpem-2011-0453.

[50] R. Rey, J.C. Sabourin, M. Venara, W.Q. Long, F. Jaubert, W.P. Zeller, P. Duvillard, H.Chemes, J.M. Bidart, Anti-Mullerian hormone is a specific marker of sertoli- andgranulosa-cell origin in gonadal tumors, Hum. Pathol. 31 (10) (2000) 1202–1208,http://dx.doi.org/10.1053/hupa.2000.18498.

[51] P. Lopez, F. Vidal, M. Rassoulzadegan, F. Cuzin, A role of inhibin as a tumor suppres-sor in Sertoli cells: down-regulation upon aging and repression by a viral oncogene,Oncogene 18 (1999) 7303–7309, http://dx.doi.org/10.1038/sj.onc.1203143.

[52] V. Eede, J.A. Muir, A.E. O'Connor, W.R. Winnall, A.E. Drummond, M.P. Hedger,Differential regulation of activin and inhibin production by interleukin 1 (IL1),transforming growth factor β1 (TGFβ1) and protein kinase C (PKC) in the Sertolicell and granulosa cell, Reprod. Fertil. Dev. 20 (2008) 6-6, http://dx.doi.org/10.1071/SRB08Abs206 (supplement).

[53] Q. Lu, Q. Li, Q. Lu, Regulation of phagocytosis by TAM receptors and their ligands,Front. Biol. 5 (3) (2010) 227–237, http://dx.doi.org/10.1007/s11515-010-0034-5.