Accepted Manuscript Title: Preparation, characterization and in vitro antiviral activity evaluation of foscarnet-chitosan nanoparticles Author: E. Russo N. Gaglianone S. Baldassari B. Parodi S. Cafaggi C. Zibana M. Donalisio V. Cagno D. Lembo G. Caviglioli PII: DOI: Reference:
S0927-7765(14)00169-6 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.03.037 COLSUB 6358
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
22-11-2013 27-2-2014 23-3-2014
Please cite this article as: E. Russo, N. Gaglianone, S. Baldassari, B. Parodi, S. Cafaggi, C. Zibana, M. Donalisio, V. Cagno, D. Lembo, G. Caviglioli, Preparation, characterization and in vitro antiviral activity evaluation of foscarnet-chitosan nanoparticles, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.03.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PREPARATION, CHARACTERIZATION AND IN VITRO ANTIVIRAL ACTIVITY EVALUATION OF FOSCARNET-CHITOSAN NANOPARTICLES
ip t
E. Russoa*, N. Gaglianonea, S. Baldassaria, B. Parodia, S. Cafaggia, C. Zibanaa, M. Donalisiob, V.
Department of Pharmacy, University of Genova, Viale Cembrano 4, 16148 Genova , Italy;
b
Department of Clinical and Biological Sciences, University of Turin, Ospedale San Luigi Gonzaga
us
a
cr
Cagnob , D. Lembob , G. Cavigliolia.
an
Regione Gonzole 10, 10043 Orbassano (TO) Italy.
M
Corresponding author: Eleonora Russo E-mail: [email protected]
Ac ce p
te
d
Tel/telefax: +390103532601/ +390103532684
1 Page 1 of 35
1. Introduction Foscarnet is an antiviral agent which inhibits the activity of herpesvirus DNA polymerase [1], including herpes simplex virus types 1 and 2 [2-4], varicella zoster and cytomegalovirus (CMV).
ip t
The drug is also active against the human immunodeficiency virus (HIV), and is able to inhibit HIV-1 reverse transcriptase. Foscarnet is the trisodium salt of phosphonoformic acid, a
cr
pyrophosphonate analogue with an extremely simple chemical structure. Due to the instability of its acid form, it is formulated as a hexahydrate trisodium salt, with a molecular weight of 300.1 and
us
solubility in water of 5% w/w (pH 7) [5]. Foscarnet possesses three acidic hydroxyl groups, with pKa values of 0.49, 3.41 and 7.27; thus, the polyanion charge depends on the pH of the solution.
an
Due to its low oral bioavailability, it is usually administered via intravenous infusion (marketed
M
by Clinigen as foscarnet sodium under the trade name Foscavir). A major problem associated with the administration of foscarnet, however, is its marked toxicity. The major adverse effect of
d
foscarnet is renal dysfunction, which appears to be less severe and less prevalent with
te
intermittent dosing than with continuous infusion. Another frequent side effect is related to fluctuations in serum calcium levels, with both increased and decreased serum calcium levels.
Ac ce p
Acute hypocalcaemia during the infusion of foscarnet may lead to muscular spasms, paresthesias, tremors, arrhythmias and seizures, which are reversed if the rate of infusion or the drug dosage is reduced [6]. In this regard, nanocarriers may reduce the toxicity associated with the antiviral drug enhance the therapeutic potential and also delay the onset of resistance [7]. Therefore, a suitable drug delivery system could decrease the incidence of adverse effects and modulate the drug’s delivery rate.
The encapsulation of foscarnet into chitosan nanoparticles could be an interesting approach to reduce the toxic effects of this drug and extend its activity [8]. Chitosan, a deacetylated derivative of chitin, is an effective vehicle for drug delivery, and is particularly able to enhance the absorption of hydrophobic macromolecular drugs. Chitosan has also been found to be capable of improving the
2 Page 2 of 35
effectiveness of drug delivery by holding the therapeutic material in closer proximity to its site of action owing to its mucoadhesive cationic nature [9]. Chitosan nanoparticles prepared by ionotropic gelation have been extensively studied in the past decade as delivery media for drugs with low
ip t
molecular weight; the most commonly used compound for ionotropic gelation is sodium tripolyphosphate (TPP), which is a non-toxic inorganic polyanion. The molar ratio between chitosan
cr
and TPP has been shown to be fundamental for the formation of nanoparticles and especially to the achievement of good drug release characteristics [10]. Based on the above considerations, we
us
hypothesized that foscarnet could induce the ionotropic gelation of chitosan via the interaction of the amino groups of the polymer with both the phosphonic and carboxylic groups present in the
an
foscarnet molecule, in a similar way to the interaction seen between chitosan and TPP. This could
M
allow nanoparticles to be obtained in which foscarnet could become a structural component of the system. The interaction of phosphate group-containing drugs with chitosan to obtain
d
nanocarriers for nucleotide delivery has been recently proposed by Giacalone et al. [11].
te
Obviously, in view of this aim, there was a need to completely investigate such an interaction, as it was unlikely that the same component amounts that are suitable for chitosan and TPP could operate
Ac ce p
for chitosan and foscarnet. The investigation was carried out by studying the experimental conditions for this interaction through a statistical experimental design. We also investigated the effect of glutaraldehyde on the stability of the obtained nanoparticles. Glutaraldehyde is a widely used cross-linking agent, which could be useful for improving the stability of nanoparticles of this type in a physiological environment at high pH [12]. In this paper, we also describe the properties and the in vitro antiviral activity of foscarnet-chitosan nanoparticles, in which the drug directly participates in the formation of the nanoparticulate system.
2. Materials and methods 2.1. Materials
3 Page 3 of 35
Chitosan of medium molecular weight (viscosity 200-800 cP, 1% w/v in 1% v/v acetic acid), with a degree of deacetylation of 85%, sodium phosphonoformate tribasic hexahydrate (foscarnet) and glutaraldehyde solution (25% w/w in water) were all purchased from Sigma (Milan, Italy).
University of Torino, Italy. All the other chemicals were of reagent grade.
us
Water was purified using a Milli-Q Plus system (Millipore, USA).
cr
ip t
Fluorescein-5-isothiocyanate-Chitosan (FITC-Chitosan) was supplied by Prof. R. Cavalli, from the
an
2.2. Preparation of nanoparticles 2.2.1. Foscarnet-chitosan nanoparticles
induced by foscarnet in an acidic solution.
M
Nanoparticles with a positive surface charge were prepared by the ionotropic gelation of chitosan,
d
Different volumes of a foscarnet aqueous solution (C= 2 mg/mL), containing appropriate amounts
te
of the drug, were added to different volumes of a chitosan solution (C= 5 mg/mL in 1% v/v acetic acid), containing appropriate amounts of the polymer (Table 1). Addition was performed at a
Ac ce p
controlled flow rate of 0.5 mL/min, using a peristaltic pump with the terminal Teflon tube tip immersed into the liquid, in order to avoid dropping and consequent local concentration of the reagent, under magnetic stirring (400 rpm). The samples were then diluted with water, when necessary, to the final volume (7.0 mL) and maintained under magnetic stirring at room temperature for 30 min, final pH = 5. Afterwards, samples were refrigerated until use for experimental design. Samples for subsequent studies were centrifuged as indicated in section 2.4.1 and re-dispersed in water or in phosphate buffer saline at pH 7.4 (10 mM phosphate buffer, 150 mM sodium chloride) (PBS).
2.2.2. Fluorescent foscarnet-chitosan nanoparticles
4 Page 4 of 35
Fluorescent nanoparticles were prepared following the procedure described in section 2.2.1, using FITC-chitosan instead of chitosan.
ip t
2.2.3. Cross-linking of foscarnet- chitosan nanoparticles by glutaraldehyde Cross-linked nanoparticles were obtained following the procedure described in section 2.2.1, by
cr
introducing a fixed volume (corresponding to 1.13 mg glutaraldehyde) of a glutaraldehyde aqueous
us
solution (25% w/w) along with the foscarnet solution.
2.3. Statistical experimental design
an
A Doehlert design for two variables was used to study the influences of different amounts of
M
chitosan and foscarnet in the sample on the size and zeta potential of nanoparticles using the response surface methodology. Design of experiments and data analysis was performed using
te
d
Modde software, Version 6.0 (Umetrics, Sweden). Statistical significance was set at p< 0.05.
2.4. Characterization of nanoparticles
Ac ce p
2.4.1. Determination of nanoparticle size and zeta potential Particle size (average apparent diameter, D) was determined by dynamic light scattering using a photon correlation spectroscopy (PCS) assembly (Zetasizer 3000 HS, Malvern Instruments, UK). Determinations were carried out at 25°C, at a fixed angle of 90°. Measurements of particle diameter were made, when not otherwise specified, on the prepared sample without preliminary separation of nanoparticles and subsequent reconstitution of the sample. Results are reported as the mean of three measurements ± SD (standard deviation). Zeta potential (Z) was measured by laser Doppler anemometry on the same instrument. Measurements were performed at 25°C, after the separation of nanoparticles by centrifugation
5 Page 5 of 35
(5000 rpm, 2117 x g, for 3 h at T=10°C) and re-dispersion of the obtained pellet in water, without sample dilution or any salt addition. Results are reported as the mean of ten measurements ± SD.
ip t
2.4.2. Determination of nanoparticle drug loading The amount of foscarnet present in nanoparticles was measured by a direct procedure, based on
cr
phosphorus content determination (APHA 1999. Standard Methods for the Examination of Water and Wastewater: 4500-P Phosphorus): nanoparticles were centrifuged as indicated in section 2.4.1,
us
then re-dispersed in water and an accurately measured aliquot (100 µl) of the suspension was evaporated and digested in a mixture of concentrated nitric and sulphuric acid (3 mL, 2:1 ratio) at a
an
temperature of about 80°C for 24 h, in order to eliminate the organic substance.
M
Nitric acid was then evaporated and the remaining sulphuric acid was quantitatively transferred into a 100 mL volumetric flask. The sample was then diluted with 20 mL of distilled water, and, in the
d
presence of 2 drops of phenolphthalein indicator solution, 1N NaOH was added until a weak pink
te
stain appeared. After dilution with water to the final volume and agitation, 20 mL of the resulting solution were withdrawn and mixed with 4 mL of molybdate reagent (Carlo Erba, Italy) and 0.5 mL
Ac ce p
of stannous chloride reagent (Sigma Aldrich, Italy). In the presence of phosphorus, the solution showed a blue colour, whose intensity was measured photometrically (HP 8452 array spectrophotometer, Hewlett-Packard, USA, managed by HP 89532 software) at λ=690 nm 10 minutes after the colour onset, on the basis of a calibration curve constructed using a proper volume of a foscarnet standard solution which had undergone the same treatment of the sample (concentration range 50 μg/mL–300 μg/mL, R2 >0.99). Drug loading was calculated using the following formula: Drug loading (% w/w) = drug content / weighed amount of lyophilized nanoparticles x 100 Results are reported as the mean of 3 measurements ± SD.
6 Page 6 of 35
2.4.3. Nanoparticle yield determination Each nanoparticle sample was centrifuged as described in section 2.4.1 and the residue was lyophilized. Nanoparticle yield was calculated using the following formula:
ip t
Nanoparticle Yield (% w/w) = weight of the lyophilized nanoparticles / total material weight x 100
cr
Results are reported as the mean of 3 measurements ± SD.
2.5. Drug release study
us
The release of foscarnet from nanoparticles was evaluated by a dialysis method. Nanoparticles were recovered from the initial suspension (V= 110 mL) by centrifugation and re-dispersed in water (3.0
an
mL). Salts were added to the final suspension in order to obtain the composition of PBS used as the
M
receptor phase. Dialysis bags (Spectra/Por-7; MWCO: 1000, Spectrum Laboratories, Inc., Laguna Hills, CA) containing 3.0 mL of the suspension in PBS were then immersed in a thermostated
d
beaker containing 60 mL PBS at 37.0°C±0.1°C and maintained under agitation with a stirring bar
te
rotating at 300 rpm. At predefined intervals, aliquots (V=2.0 mL) of receiving buffer were withdrawn and replaced with an equal volume of fresh medium. Foscarnet spectrophotometric
Ac ce p
determination was then carried out by measuring the absorbance of the withdrawn sample at λ= 233 nm on the basis of a calibration curve obtained from foscarnet standard solutions in PBS (concentration range 20 μg/mL–200 μg/mL, R2 >0.99). The study was carried out at least in triplicate.
Following the same method, free foscarnet diffusion from the dialysis bag into PBS was also investigated. At this aim, a foscarnet amount equivalent to that contained in nanoparticle samples was dissolved in PBS and transferred to the dialysis bag.
2.6. Stability studies
7 Page 7 of 35
The average mean diameter, D, of nanoparticles was used as an indicator to evaluate their stability. After centrifugation (see section 2.4.1), a nanoparticle sample was re-dispersed in PBS and maintained at 37°C in the cuvette of the photon correlation spectrometer. D was measured every ten
ip t
minutes for at least 4 h and then at 24 and 48 h. Attention was paid to the appearance of the sediment in the cuvette and the constancy of kcps was checked during the experiments.
cr
The studies were carried out in triplicate.
us
2.7. Biological studies 2.7.1. Cells and Viruses
an
Low-passage-number human embryonic lung fibroblasts (HELFs) were grown as monolayers in
M
minimum essential medium (MEM) (SIGMA) supplemented with 10% fetal calf serum (FCS) (SIGMA), 1 mM sodium pyruvate and antibiotics.
d
A bacterial artificial chromosome (BAC)-derived human cytomegalovirus (HCMV) strain
te
Towne incorporating the green fluorescence protein (GFP) sequence [13] and HCMV strain AD169 (ATCC VR-538) were propagated on HELF. The GFP marker facilitates the identification of
Ac ce p
infected cells. HCMV titers were determined on HELF cells using the median tissue culture infective dose (TCID50) method [14].
2.7.2. Antiviral assay
To assay the antiviral activities of foscarnet and foscarnet-chitosan nanoparticles, we performed an infectivity inhibition assay where cells were infected with GFP-expressing HCMV at a multiplicity of infection (MOI) of 0.1 for 2 hours at 37°C and then exposed to the drug. Three days postinfection, infected cells were examined by an inverted Zeiss LSM510 fluorescence microscopy. AD-169 HCMV-infected monolayers (MOI 0.004 pfu/cell) were washed and overlaid with 1.2% methylcellulose medium in order to visualize viral plaques in the presence of serial dilutions of
8 Page 8 of 35
the test formulations. After five days of incubation, cells were fixed with cold methanol and acetone for 1 min and subjected to HCMV-specific immunostaining using an anti-HCMV IEA monoclonal antibody (11-003; Argene, Verniolle, France) and an UltraTech HRP streptavidin-biotin detection
ip t
system (Beckman Coulter, Marseille, France). Immunostained plaques were counted, and the percent inhibition of virus infectivity was determined as previously described [15]. IC50 and 95% CI
cr
were determined using Prism software (GraphPad Software, San Diego, California, U.S.A.) to fit a variable slope-sigmoidal dose-response curve. All data were generated from duplicate wells in at
an
us
least three independent experiments.
2.7.3. Cell viability assay
M
HELF cell viability was measured by the MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Cells were seeded at a density of
d
5.5 x 103/well in 96-well plates. After 24 h, they were either incubated with different concentrations
te
of free foscarnet or foscarnet in nanoparticles in triplicate, or left untreated. After the 96 h treatment, cell viability was determined by the CellTiter 96 Proliferation Assay Kit (Promega,
Ac ce p
Madison, WI, USA) according to the manufacturer’s instructions [16]. The effect on cell viability was expressed as a percentage, by comparing the absorbance of treated cells with those of cells incubated with culture medium alone.
2.7.4. Cytotoxicity assay
The cytotoxic effects of nanoparticles on HELF cells, seeded and treated as described before for the cell viability assay, were measured by analyzing the release of lactate dehydrogenase in the culture medium of treated cultures according to the manufacturer’s protocol for the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA). The 50% cytotoxic concentration (CC50) values and 95% CIs were determined using Prism software.
9 Page 9 of 35
2.7.5. Evaluation of cellular internalization by confocal laser scanning microscopy HELF cells were seeded at a density of 2 x 104/well in 24-well plates on glass coverslips. Subsequently, the cultures were incubated with 1 or 5 µg/mL of the labelled fluorescent
cr
Zeiss LSM510 fluorescence microscope, as previously described [17,18].
ip t
nanoparticle suspension for different time points (1, 3 and 24 hours) and analysed on an inverted
2.8. Statistical analysis
us
Student’s test was used to compare paired or unpaired data and one-way analysis of variance (ANOVA) and Bonferroni post hoc test were used for multiple comparisons. Statistical significance
M
an
was set at p
S0927-7765(14)00169-6 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.03.037 COLSUB 6358
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
22-11-2013 27-2-2014 23-3-2014
Please cite this article as: E. Russo, N. Gaglianone, S. Baldassari, B. Parodi, S. Cafaggi, C. Zibana, M. Donalisio, V. Cagno, D. Lembo, G. Caviglioli, Preparation, characterization and in vitro antiviral activity evaluation of foscarnet-chitosan nanoparticles, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.03.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PREPARATION, CHARACTERIZATION AND IN VITRO ANTIVIRAL ACTIVITY EVALUATION OF FOSCARNET-CHITOSAN NANOPARTICLES
ip t
E. Russoa*, N. Gaglianonea, S. Baldassaria, B. Parodia, S. Cafaggia, C. Zibanaa, M. Donalisiob, V.
Department of Pharmacy, University of Genova, Viale Cembrano 4, 16148 Genova , Italy;
b
Department of Clinical and Biological Sciences, University of Turin, Ospedale San Luigi Gonzaga
us
a
cr
Cagnob , D. Lembob , G. Cavigliolia.
an
Regione Gonzole 10, 10043 Orbassano (TO) Italy.
M
Corresponding author: Eleonora Russo E-mail: [email protected]
Ac ce p
te
d
Tel/telefax: +390103532601/ +390103532684
1 Page 1 of 35
1. Introduction Foscarnet is an antiviral agent which inhibits the activity of herpesvirus DNA polymerase [1], including herpes simplex virus types 1 and 2 [2-4], varicella zoster and cytomegalovirus (CMV).
ip t
The drug is also active against the human immunodeficiency virus (HIV), and is able to inhibit HIV-1 reverse transcriptase. Foscarnet is the trisodium salt of phosphonoformic acid, a
cr
pyrophosphonate analogue with an extremely simple chemical structure. Due to the instability of its acid form, it is formulated as a hexahydrate trisodium salt, with a molecular weight of 300.1 and
us
solubility in water of 5% w/w (pH 7) [5]. Foscarnet possesses three acidic hydroxyl groups, with pKa values of 0.49, 3.41 and 7.27; thus, the polyanion charge depends on the pH of the solution.
an
Due to its low oral bioavailability, it is usually administered via intravenous infusion (marketed
M
by Clinigen as foscarnet sodium under the trade name Foscavir). A major problem associated with the administration of foscarnet, however, is its marked toxicity. The major adverse effect of
d
foscarnet is renal dysfunction, which appears to be less severe and less prevalent with
te
intermittent dosing than with continuous infusion. Another frequent side effect is related to fluctuations in serum calcium levels, with both increased and decreased serum calcium levels.
Ac ce p
Acute hypocalcaemia during the infusion of foscarnet may lead to muscular spasms, paresthesias, tremors, arrhythmias and seizures, which are reversed if the rate of infusion or the drug dosage is reduced [6]. In this regard, nanocarriers may reduce the toxicity associated with the antiviral drug enhance the therapeutic potential and also delay the onset of resistance [7]. Therefore, a suitable drug delivery system could decrease the incidence of adverse effects and modulate the drug’s delivery rate.
The encapsulation of foscarnet into chitosan nanoparticles could be an interesting approach to reduce the toxic effects of this drug and extend its activity [8]. Chitosan, a deacetylated derivative of chitin, is an effective vehicle for drug delivery, and is particularly able to enhance the absorption of hydrophobic macromolecular drugs. Chitosan has also been found to be capable of improving the
2 Page 2 of 35
effectiveness of drug delivery by holding the therapeutic material in closer proximity to its site of action owing to its mucoadhesive cationic nature [9]. Chitosan nanoparticles prepared by ionotropic gelation have been extensively studied in the past decade as delivery media for drugs with low
ip t
molecular weight; the most commonly used compound for ionotropic gelation is sodium tripolyphosphate (TPP), which is a non-toxic inorganic polyanion. The molar ratio between chitosan
cr
and TPP has been shown to be fundamental for the formation of nanoparticles and especially to the achievement of good drug release characteristics [10]. Based on the above considerations, we
us
hypothesized that foscarnet could induce the ionotropic gelation of chitosan via the interaction of the amino groups of the polymer with both the phosphonic and carboxylic groups present in the
an
foscarnet molecule, in a similar way to the interaction seen between chitosan and TPP. This could
M
allow nanoparticles to be obtained in which foscarnet could become a structural component of the system. The interaction of phosphate group-containing drugs with chitosan to obtain
d
nanocarriers for nucleotide delivery has been recently proposed by Giacalone et al. [11].
te
Obviously, in view of this aim, there was a need to completely investigate such an interaction, as it was unlikely that the same component amounts that are suitable for chitosan and TPP could operate
Ac ce p
for chitosan and foscarnet. The investigation was carried out by studying the experimental conditions for this interaction through a statistical experimental design. We also investigated the effect of glutaraldehyde on the stability of the obtained nanoparticles. Glutaraldehyde is a widely used cross-linking agent, which could be useful for improving the stability of nanoparticles of this type in a physiological environment at high pH [12]. In this paper, we also describe the properties and the in vitro antiviral activity of foscarnet-chitosan nanoparticles, in which the drug directly participates in the formation of the nanoparticulate system.
2. Materials and methods 2.1. Materials
3 Page 3 of 35
Chitosan of medium molecular weight (viscosity 200-800 cP, 1% w/v in 1% v/v acetic acid), with a degree of deacetylation of 85%, sodium phosphonoformate tribasic hexahydrate (foscarnet) and glutaraldehyde solution (25% w/w in water) were all purchased from Sigma (Milan, Italy).
University of Torino, Italy. All the other chemicals were of reagent grade.
us
Water was purified using a Milli-Q Plus system (Millipore, USA).
cr
ip t
Fluorescein-5-isothiocyanate-Chitosan (FITC-Chitosan) was supplied by Prof. R. Cavalli, from the
an
2.2. Preparation of nanoparticles 2.2.1. Foscarnet-chitosan nanoparticles
induced by foscarnet in an acidic solution.
M
Nanoparticles with a positive surface charge were prepared by the ionotropic gelation of chitosan,
d
Different volumes of a foscarnet aqueous solution (C= 2 mg/mL), containing appropriate amounts
te
of the drug, were added to different volumes of a chitosan solution (C= 5 mg/mL in 1% v/v acetic acid), containing appropriate amounts of the polymer (Table 1). Addition was performed at a
Ac ce p
controlled flow rate of 0.5 mL/min, using a peristaltic pump with the terminal Teflon tube tip immersed into the liquid, in order to avoid dropping and consequent local concentration of the reagent, under magnetic stirring (400 rpm). The samples were then diluted with water, when necessary, to the final volume (7.0 mL) and maintained under magnetic stirring at room temperature for 30 min, final pH = 5. Afterwards, samples were refrigerated until use for experimental design. Samples for subsequent studies were centrifuged as indicated in section 2.4.1 and re-dispersed in water or in phosphate buffer saline at pH 7.4 (10 mM phosphate buffer, 150 mM sodium chloride) (PBS).
2.2.2. Fluorescent foscarnet-chitosan nanoparticles
4 Page 4 of 35
Fluorescent nanoparticles were prepared following the procedure described in section 2.2.1, using FITC-chitosan instead of chitosan.
ip t
2.2.3. Cross-linking of foscarnet- chitosan nanoparticles by glutaraldehyde Cross-linked nanoparticles were obtained following the procedure described in section 2.2.1, by
cr
introducing a fixed volume (corresponding to 1.13 mg glutaraldehyde) of a glutaraldehyde aqueous
us
solution (25% w/w) along with the foscarnet solution.
2.3. Statistical experimental design
an
A Doehlert design for two variables was used to study the influences of different amounts of
M
chitosan and foscarnet in the sample on the size and zeta potential of nanoparticles using the response surface methodology. Design of experiments and data analysis was performed using
te
d
Modde software, Version 6.0 (Umetrics, Sweden). Statistical significance was set at p< 0.05.
2.4. Characterization of nanoparticles
Ac ce p
2.4.1. Determination of nanoparticle size and zeta potential Particle size (average apparent diameter, D) was determined by dynamic light scattering using a photon correlation spectroscopy (PCS) assembly (Zetasizer 3000 HS, Malvern Instruments, UK). Determinations were carried out at 25°C, at a fixed angle of 90°. Measurements of particle diameter were made, when not otherwise specified, on the prepared sample without preliminary separation of nanoparticles and subsequent reconstitution of the sample. Results are reported as the mean of three measurements ± SD (standard deviation). Zeta potential (Z) was measured by laser Doppler anemometry on the same instrument. Measurements were performed at 25°C, after the separation of nanoparticles by centrifugation
5 Page 5 of 35
(5000 rpm, 2117 x g, for 3 h at T=10°C) and re-dispersion of the obtained pellet in water, without sample dilution or any salt addition. Results are reported as the mean of ten measurements ± SD.
ip t
2.4.2. Determination of nanoparticle drug loading The amount of foscarnet present in nanoparticles was measured by a direct procedure, based on
cr
phosphorus content determination (APHA 1999. Standard Methods for the Examination of Water and Wastewater: 4500-P Phosphorus): nanoparticles were centrifuged as indicated in section 2.4.1,
us
then re-dispersed in water and an accurately measured aliquot (100 µl) of the suspension was evaporated and digested in a mixture of concentrated nitric and sulphuric acid (3 mL, 2:1 ratio) at a
an
temperature of about 80°C for 24 h, in order to eliminate the organic substance.
M
Nitric acid was then evaporated and the remaining sulphuric acid was quantitatively transferred into a 100 mL volumetric flask. The sample was then diluted with 20 mL of distilled water, and, in the
d
presence of 2 drops of phenolphthalein indicator solution, 1N NaOH was added until a weak pink
te
stain appeared. After dilution with water to the final volume and agitation, 20 mL of the resulting solution were withdrawn and mixed with 4 mL of molybdate reagent (Carlo Erba, Italy) and 0.5 mL
Ac ce p
of stannous chloride reagent (Sigma Aldrich, Italy). In the presence of phosphorus, the solution showed a blue colour, whose intensity was measured photometrically (HP 8452 array spectrophotometer, Hewlett-Packard, USA, managed by HP 89532 software) at λ=690 nm 10 minutes after the colour onset, on the basis of a calibration curve constructed using a proper volume of a foscarnet standard solution which had undergone the same treatment of the sample (concentration range 50 μg/mL–300 μg/mL, R2 >0.99). Drug loading was calculated using the following formula: Drug loading (% w/w) = drug content / weighed amount of lyophilized nanoparticles x 100 Results are reported as the mean of 3 measurements ± SD.
6 Page 6 of 35
2.4.3. Nanoparticle yield determination Each nanoparticle sample was centrifuged as described in section 2.4.1 and the residue was lyophilized. Nanoparticle yield was calculated using the following formula:
ip t
Nanoparticle Yield (% w/w) = weight of the lyophilized nanoparticles / total material weight x 100
cr
Results are reported as the mean of 3 measurements ± SD.
2.5. Drug release study
us
The release of foscarnet from nanoparticles was evaluated by a dialysis method. Nanoparticles were recovered from the initial suspension (V= 110 mL) by centrifugation and re-dispersed in water (3.0
an
mL). Salts were added to the final suspension in order to obtain the composition of PBS used as the
M
receptor phase. Dialysis bags (Spectra/Por-7; MWCO: 1000, Spectrum Laboratories, Inc., Laguna Hills, CA) containing 3.0 mL of the suspension in PBS were then immersed in a thermostated
d
beaker containing 60 mL PBS at 37.0°C±0.1°C and maintained under agitation with a stirring bar
te
rotating at 300 rpm. At predefined intervals, aliquots (V=2.0 mL) of receiving buffer were withdrawn and replaced with an equal volume of fresh medium. Foscarnet spectrophotometric
Ac ce p
determination was then carried out by measuring the absorbance of the withdrawn sample at λ= 233 nm on the basis of a calibration curve obtained from foscarnet standard solutions in PBS (concentration range 20 μg/mL–200 μg/mL, R2 >0.99). The study was carried out at least in triplicate.
Following the same method, free foscarnet diffusion from the dialysis bag into PBS was also investigated. At this aim, a foscarnet amount equivalent to that contained in nanoparticle samples was dissolved in PBS and transferred to the dialysis bag.
2.6. Stability studies
7 Page 7 of 35
The average mean diameter, D, of nanoparticles was used as an indicator to evaluate their stability. After centrifugation (see section 2.4.1), a nanoparticle sample was re-dispersed in PBS and maintained at 37°C in the cuvette of the photon correlation spectrometer. D was measured every ten
ip t
minutes for at least 4 h and then at 24 and 48 h. Attention was paid to the appearance of the sediment in the cuvette and the constancy of kcps was checked during the experiments.
cr
The studies were carried out in triplicate.
us
2.7. Biological studies 2.7.1. Cells and Viruses
an
Low-passage-number human embryonic lung fibroblasts (HELFs) were grown as monolayers in
M
minimum essential medium (MEM) (SIGMA) supplemented with 10% fetal calf serum (FCS) (SIGMA), 1 mM sodium pyruvate and antibiotics.
d
A bacterial artificial chromosome (BAC)-derived human cytomegalovirus (HCMV) strain
te
Towne incorporating the green fluorescence protein (GFP) sequence [13] and HCMV strain AD169 (ATCC VR-538) were propagated on HELF. The GFP marker facilitates the identification of
Ac ce p
infected cells. HCMV titers were determined on HELF cells using the median tissue culture infective dose (TCID50) method [14].
2.7.2. Antiviral assay
To assay the antiviral activities of foscarnet and foscarnet-chitosan nanoparticles, we performed an infectivity inhibition assay where cells were infected with GFP-expressing HCMV at a multiplicity of infection (MOI) of 0.1 for 2 hours at 37°C and then exposed to the drug. Three days postinfection, infected cells were examined by an inverted Zeiss LSM510 fluorescence microscopy. AD-169 HCMV-infected monolayers (MOI 0.004 pfu/cell) were washed and overlaid with 1.2% methylcellulose medium in order to visualize viral plaques in the presence of serial dilutions of
8 Page 8 of 35
the test formulations. After five days of incubation, cells were fixed with cold methanol and acetone for 1 min and subjected to HCMV-specific immunostaining using an anti-HCMV IEA monoclonal antibody (11-003; Argene, Verniolle, France) and an UltraTech HRP streptavidin-biotin detection
ip t
system (Beckman Coulter, Marseille, France). Immunostained plaques were counted, and the percent inhibition of virus infectivity was determined as previously described [15]. IC50 and 95% CI
cr
were determined using Prism software (GraphPad Software, San Diego, California, U.S.A.) to fit a variable slope-sigmoidal dose-response curve. All data were generated from duplicate wells in at
an
us
least three independent experiments.
2.7.3. Cell viability assay
M
HELF cell viability was measured by the MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Cells were seeded at a density of
d
5.5 x 103/well in 96-well plates. After 24 h, they were either incubated with different concentrations
te
of free foscarnet or foscarnet in nanoparticles in triplicate, or left untreated. After the 96 h treatment, cell viability was determined by the CellTiter 96 Proliferation Assay Kit (Promega,
Ac ce p
Madison, WI, USA) according to the manufacturer’s instructions [16]. The effect on cell viability was expressed as a percentage, by comparing the absorbance of treated cells with those of cells incubated with culture medium alone.
2.7.4. Cytotoxicity assay
The cytotoxic effects of nanoparticles on HELF cells, seeded and treated as described before for the cell viability assay, were measured by analyzing the release of lactate dehydrogenase in the culture medium of treated cultures according to the manufacturer’s protocol for the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA). The 50% cytotoxic concentration (CC50) values and 95% CIs were determined using Prism software.
9 Page 9 of 35
2.7.5. Evaluation of cellular internalization by confocal laser scanning microscopy HELF cells were seeded at a density of 2 x 104/well in 24-well plates on glass coverslips. Subsequently, the cultures were incubated with 1 or 5 µg/mL of the labelled fluorescent
cr
Zeiss LSM510 fluorescence microscope, as previously described [17,18].
ip t
nanoparticle suspension for different time points (1, 3 and 24 hours) and analysed on an inverted
2.8. Statistical analysis
us
Student’s test was used to compare paired or unpaired data and one-way analysis of variance (ANOVA) and Bonferroni post hoc test were used for multiple comparisons. Statistical significance
M
an
was set at p
