Negatively charged gold nanoparticles as a dexamethasone carrier: stability in biological media and bioactivity assessment in vitro

Abstract

Gold nanoparticles (AuNPs) have been extensively used in biological applications because of their high biocompatibility, ease of characterization and the extensive knowledge of their surface chemistry. These features make AuNPs readily exploitable for drug delivery and novel diagnostic and therapeutic approaches. In a previous work, we showed that small size (5–10 nm) AuNPs functionalized by sodium 3-mercapto-1-propanesulfonate (3MPS) can be efficiently loaded with the glucocorticoid drug dexamethasone (DXM) and are stable in water and PBS. In the present study, we further analysed the stability and the drug release kinetics of DXM-loaded AuNPs functionalized by sodium 3-mercaptopropane sulfonate (AuNP-3MPS/DXM) and their unconjugated counterparts (AuNP-3MPS) in different biological media. Moreover, we evaluated AuNP-3MPS cyto-compatibility on two mammalian cell lines and tested their specific activity as drug carriers on DXM-sensitive murine and human tumor cells. The colloidal stability of AuNP-3MPS/DXM was significantly increased in all tested culture media, compared with the unconjugated AuNP-3MPS and both AuNP-3MPS formulations which proved non-toxic to biological systems in vitro. Most importantly, we showed that AuNP-3MPS/DXM continuously release bioactive DXM molecules that efficiently induce cell proliferation arrest and apoptotic cell death on a human lymphoma cell line and upregulation of the DXM-inducible programmed cell death-1 (PD-1) molecule on activated mouse T lymphocytes. These data confirm that the AuNP-3MPS/DXM conjugate is a promising system for drug delivery and open interesting perspectives for future in vivo applications.

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1. Introduction

Recent years have witnessed an unprecedented growth of research interest in the field of nanotechnology due to its wide application in the areas of sensors, biotechnology and biomedicine.^1–6 Among others, gold nanoparticles (AuNPs) are nanostructures for which many medical applications have been already reported.^7–9 Due to their unique chemical and physical properties, superior biocompatibility, and well-established strategies for surface modification,^10–14 AuNPs are applied for use as imaging/diagnostic tools, radiosensitizer agents, drug and gene delivery systems, photo-thermal therapy, biosensors, and tissue engineering.^8,15–20 In all these biomedical applications, shape (spheres, cages, rods, nanobottles, disks),^21–26 dimension (2–500 nm),^27,28 and surface functionalization (natural, positive, negative, bioconjugation)^29–32 play a key role on AuNPs stability, biodistribution, escape from RES system, kinetic release and cellular uptake (phagocytosis, diffusion, endocytosis).^33–36 Moreover, functionalized AuNPs are used as promising drug delivery systems for hydrophobic drugs, such as steroids or anticancer drugs.^37–39 In this field several studies on HeLa cells have been performed, demonstrating that the number of nanoparticles per volume unit is the relevant parameter for the stability and biocompatibility of biological systems.^40,41

Dexamethasone (DXM) is a long-acting synthetic glucocorticoid steroid with anti-inflammatory properties. It is extensively used to treat diseases caused by over-reactivity of the immune system⁴² and to decrease swelling (edema) associated with tumors of the spine and brain undergoing radiation therapy.⁴³ Due to its pro-apoptotic effects, DXM is also used to treat a variety of haematological cancers, including lymphomas^44,45 and childhood leukemias.⁴⁶ However, due to the immunosuppressive activity of glucocorticoid steroid and to the wide expression of its receptor on mammalian cells, strategies aimed at achieving the controlled release of the minimum effective drug dose are highly desirable.

In a previous study we showed that the hydrophobic drug DXM can be efficiently loaded onto negatively-charged AuNPs functionalized by sodium 3-mercapto-1-propanesulfonate (3MPS) and slowly released in aqueous media.⁴⁷

In the present study we assessed the stability of empty and DXM-loaded AuNP-3MPS in different biological media, including mammalian sera, and evaluated their toxic effect of two mammalian cell lines. Furthermore we tested the biological activity of AuNP-3MPS/DXM vs. unconjugated DXM by analysing the upregulation of programmed cell death-1 (PD-1) molecule on the surface of activated T lymphocytes and the pro-apoptotic activity on a human lymphoma cell line. We concluded that AuNP-3MPS are non-toxic to mammalian cells and are biologically active in vitro. Moreover, due to the slow release rate of DXM molecules from AuNP-3MPS, this drug formulation displays prolonged efficacy, as compared to soluble DXM, even at suboptimal drug concentrations and represents a promising delivery system to be further evaluated in vivo.

2. Experimental materials and methods

2.1. Materials and methods

Sodium 3-mercapto-1-propanesulfonate (HS(CH₂)₃SO₃Na, 3MPS), tetrachloroauric(III) acid trihydrate (HAuCl₄·3H₂O), sodium borohydride (NaBH₄) and dexamethasone (DXM) were used as received (Aldrich reagent grade). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), RPMI 1640 and human serum (HS) were purchased from Euroclone. Fetal bovine serum (FBS) was purchased from Sigma-Aldrich. L-Glutamine and penicillin/streptomycin were purchased from Lonza. UV-vis spectra were acquired in H₂O solution by using quartz cells with a Varian Cary 100 Scan UV-vis spectrophotometer. Size distribution of AuNPs in aqueous solution has been investigated by means of Dynamic Light Scattering (DLS) technique by using a Zetasizer Nanoseries Malvern instrument, at opportune temperature (25.0 ± 0.2 °C and 37.0 ± 0.2 °C). Correlation data have been acquired and fitted in analogy to our previous work.^48–50 FE-SEM images have been acquired with the Auriga Zeiss instrument (resolution 1 nm, applied voltage 6–12 kV) on freshly prepared films drop casted from water solution on a metallic sample holder. A Mini Spin-Eppendorf centrifuge was used for purification of AuNPs samples (13 000 rpm, 20 min, 5 times with deionized water). pH measurements were done with a CyberScan pH 600 (Eutech Instruments). Deionized water was obtained from Zeener Power I Scholar-UV (18.2 MΩ).

2.2. Preparation of AuNP-3MPS and AuNP-3MPS/DXM

The AuNPs stabilized with 3MPS were synthesized as previously reported.^47,51 Briefly, starting from 200 mg (5 × 10⁻⁴ mol) of HAuCl₄·3H₂O in 20 mL of deionized water, a solution of 3MPS in 20 mL of deionized water was added under vigorous stirring (Au/S = 1/4 molar ratios). After 3 hours from addition of a water solution of NaBH₄ (Au/NaBH₄ molar ratio = 1/10) a solid black product was purified by centrifugation (13 000 rpm, 20 min, 5 times with deionized water). Au-3MPS main characterizations: UV (λ_max [nm], H₂O) 525 nm; DLS (〈2R_H〉 [nm], H₂O): 14 ± 2 nm; Z potential: −38 ± 3 mV; FESEM [nm] 7–10 nm.

The AuNP-3MPS/DXM were prepared following a previous report:⁴⁷ in the typical loading protocol, AuNPs and DXM are mixed in water (Au/DXM = 5/1 in w/w) under vigorous stirring (room temperature, 4 h) and then the suspension is centrifuged (13 000 rpm, 1 hour) to obtain AuNP-3MPS/DXM (loading efficiency 80%) as a solid residue.

2.3. Stability and release studies

The following media were used for the stability studies: H₂O, HEPES, HS, RPMI-1640, RPMI-1640 supplemented with 10% FBS, L-glutamine (2 mM), penicillin/streptomycin (100 U mL⁻¹) and β-mercaptoethanol (50 μM), from now on referred to as “complete RPMI”. AuNP-3MPS and AuNP-3MPS/DXM were dispersed in each media at the concentration of 0.5 mg mL⁻¹ and the size of AuNP-3MPS nanoparticles was measured up to 15 days at two different temperatures (25 °C and 37 °C). The release assays were performed in complete RPMI using 2 mg of AuNP-3MPS/DXM nanoparticles in 20 mL of cell culture media. The DXM released was detected by UV-vis measurement during 14 days and using a calibration curve.^47,51

2.4. Cell lines and in vitro assays

HeLa cell line (human cervix carcinoma, American Type Culture Collection) was cultured in Dulbecco's Modified Eagle's Medium (DMEM, Euroclone) supplemented with 10% FBS, L-glutamine (2 mM) and penicillin/streptomycin (100 U mL⁻¹). EG.7-OVA cell line (murine thymoma, American Type Culture Collection) was cultured in complete RPMI-1640 supplemented with 0.4 mg mL⁻¹ of Geneticin (G-418 sulfate, Gibco). Karpas-422 cell line (human B cell non-Hodgkin lymphoma, Interlab Cell Line Collection) was cultured in complete RPMI. All cell lines were grown in a humidified chamber at 37 °C and 5% CO₂ and serially passaged twice a week.

For all in vitro assays, AuNPs were resuspended in the culture medium right before use and added to cell cultures immediately. Cell lines grown in their media were used as viability controls. For MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) HeLa cells were seeded into 96-well plates (2.0 × 10⁴ cells per well) and left to adhere for 1 h at 37 °C; EG.7-OVA cells were seeded at 1.0 × 10⁴ cells per well. Scalar dilutions of AuNP-3MPS or AuNP-3MPS/DXM were added and cells were incubated for 48 hours at 37 °C in a humidified incubator with 5% CO₂. Supernatants were carefully removed and replaced by fresh medium and 15 μl per well of MTT stock solution (Promega) before incubating 4 h at 37 °C and 5% CO₂. Then 100 μl per well of stop/solubilising buffer (Promega) were added for 1 additional hour at 37 °C. Optical density (OD) for each well was calculated by subtracting OD_{(570 nm)} − OD_{(630 nm)}. Specific absorbance in culture media is directly related to the metabolic activity and thus on the number of viable cells in each sample. Samples consisted of either triplicates or sextuplicates.

For viable cell count analysis, Karpas-422 were seeded at 2.0 × 10⁵ cells per well in 24-well plates and treated with either AuNP-3MPS or AuNP-3MPS/DXM for 10 days. At each time point, 50 μl of cell suspension were diluted in 50 μl of trypan blue (Sigma) and counted in a glass hemocytometer (Neubauer Chamber) under an optical microscope (PrimoVert, ZEISS). For apoptosis detection, 10⁵ Karpas-422 cells were stained with Annexin V-FITC and propidium iodide (PI) according to the manufacturer's instructions (BD Pharmingen™) and immediately analyzed by flow cytometry (FacsCalibur, BD).

Splenocytes from C57Bl/6 mice (Charles River Laboratories) were enriched of lymphocytes by density gradient centrifugation (Lympholyte-M, Cederlane) before stimulation with the mitogen concanavalin-A (5 μg mL⁻¹) in the presence of equal concentrations of DXM or AuNP-3MPS/DXM. Empty AuNP-3MPS were used as controls. After three days of culture at 37 °C and 5% CO₂ cells were stained with anti-CD3/PE-Cy7 (BD, clone 145-2C11) and anti-PD1/APC (e-Bioscience, clone J43) antibodies and with the vital dye LIVE/DEAD® Fixable Dead Cell Stain (Invitrogen). Analysis was performed by flow cytometry (Gallios, Beckman Coulter).

2.5. Statistical analysis

The statistical significance of differences was evaluated by Student's t-test and Wilcoxon signed-rank test. Values of p < 0.05 were considered significant.

3. Results and discussion

3.1. Stability of AuNP-3MPS and AuNP-3MPS/DMX in different culture media and kinetic of drug release

It is noteworthy that in the synthesis procedure the use of surfactants or stabilizing agents has been avoided and the stoichiometric molar ratios between the gold precursor and the selected thiol has been accurately controlled, thus obtaining AuNPs with almost monodispersed size.⁵² In our case, the choice of 3MPS, a small length alkyl chains thiol with charged terminal sulphonate group, allows to obtain high monodispersed AuNPs with high stability in terms of low aggregation phenomena in water suspension, up to 1 month. Moreover, we choose a molar ratio Au/S = 1/4 to be confident to balance the possible effect of high drug loading on the stability of the final conjugated system.⁵³

In fact, after careful purification of the AuNP-3MPS, DLS data from water suspensions confirmed the nanodimension of gold nanoparticles, 〈2R_H〉 = 14 ± 2 nm, and the negatively charged surface, ζ potential = −38 ± 2 mV. The negative zeta potential value, due to the presence of negative charges on the AuNPs surface, prevents nanoparticles' interactions, limits their aggregation and allows the persistence of the gold colloids up to 1–3 months, as reported for analogous systems.⁵⁴ After the synthesis of AuNP-3MPS and AuNP-3MPS/DXM⁴⁷ the stability of both nanostructures in different biological media (HEPES, RPMI-1640, complete RPMI, FBS and HS) was assayed up to 15 days.

When AuNP-3MPS and AuNP-3MPS/DXM were incubated in the different media, the stability of drug-conjugated nanoparticles was far superior to that of empty AuNP-3MPS in terms of dispersion, as shown in Fig. 1a and b.

Fig. 1 〈2R_H〉 for gold nanoparticles in different media (H₂O, HEPES, RPMI-1640, complete RPMI, FBS, HS) and at different temperatures (25 °C ; 37 °C ): (a) AuNP-3MPS; and (b) AuNP-3MPS/DXM. Error bars quote the standard deviation of at least 3 independent measurements.

In fact, after few minutes in the biological media (t = 0), the hydrodynamic diameter 〈2R_H〉 of AuNP-3MPS at 25 °C became bigger than in water, up to 210 nm in RPMI 1640. On the other hand, the AuNP-3MPS/DXM, showed good stability in all tested media and especially in complete RPMI and full FBS, maintaining 〈2R_H〉 = 10 ± 3 nm at 25 °C, (Fig. 1b). At the same time, temperature can have an effect on aggregation phenomena, but in our system only in some media this behavior was confirmed, such as in RPMI and in complete RPMI, due to the nanoparticles' interaction with the media components.⁵⁵ The stability of Au-3MPS/DXM in all culture media was investigated during two weeks at two different temperatures, 25 °C and 37 °C, as reported in Fig. 2a–c.

Fig. 2 Size distributions by DLS data for AuNP-3MPS/DXM at different temperature (25 °C fill and hollow 37 °C) in different media: (a) H₂O and HEPES ; (b) RMPI-1640 and complete RPMI ; (c) FBS and HS (error bar quote the standard deviation of at least 3 independent measurements).

We can conclude therefore that the AuNP-3MPS quickly aggregate into the culture media tested while the AuNP-3MPS/DXM are stable over time. These data are in perfect agreement with our expectations. In fact, RPMI-1640, the media currently used for culturing mammalian primary as well neoplastic cell lines, is based on the RPMI-1630 series of media utilizing a bicarbonate buffering system and alterations in the amounts of amino acids and vitamins,⁵⁶ a highly polar mix that definitely favors the aggregation of nanoparticles.⁵⁷ Moreover, an effect of the cooperative action of thiol and amine on aggregation of AuNPs have been recently investigated, due to their coexistence in biological samples.⁵⁸ The conjugation of AuNPs to a polar molecule, such as a peptide or a drug, promotes the spatial separation between particles, in addition, stability of AuNPs over time also depends on their initial size and on the composition of the suspension medium.^56,59 Therefore AuNPs aggregation can be avoided by adding FBS or by over-coating AuNPs with other biomolecules such as lipids, or zwitterionic NPs of variable hydrophobicity.^60,61

In our case the long-term (two weeks) stability of AuNP-3MPS/DXM may be due to the presence of a carboxylic group in DXM molecules that may create a polar coating on AuNPs surface thus avoiding particles' interactions and aggregation phenomena in culture media. These data are extremely encouraging since stability is a strong primary requirement for further applications due to the fact that the aggregation state of the AuNPs strongly influences their cellular uptake, cellular toxicity and sub-lethal toxicity.⁶⁰

Moreover, the release in complete RPMI was investigated to compare these results with analogous studies performed in PBS (Fig. 3).

Fig. 3 Drug release profile for AuNP-3MPS/DXM sample in complete RPMI medium () and in PBS () during 14 days. Error bar quote the standard deviation of at least 3 independent measurements.

The drug release rate from AuNP-3MPS/DXM in complete RPMI medium at 37 °C was analysed over time and compared with previously observed drug release rates in PBS buffer (PBS, pH 7.4 and 37 °C).⁴⁷ As shown in Fig. 3, DXM is continuously released from AuNP-3MPS/DXM in complete RPMI, although at lower rate than in PBS, and reaches a plateau (40–45%) at 200 hours from culture initiation. The different DXM release in complete RPMI medium is probably to be ascribed to the weak interactions between the medium proteins (e.g., FBS proteins) and the nanoparticles.⁶² In fact, the hindrance that occur induce a slower drug release, as already shown in similar situations.⁶³

Taken together, these results indicate that the conjugation of an hydrophobic drug to AuNP-3MPS may have three favourable effects: first, it enhances the stability of AuNP-3MPS by preventing their aggregation in all the different culture media tested; second, due to the fact that AuNP-3MPS are water-soluble, conjugation enhances the solubility of DXM in the biological media tested; third, the slow release of DXM molecules over time may favour the continuous drug uptake by the cells and a prolonged bio-activity, as compared to unconjugated DXM.

3.2. AuNP-3MPS and AuNP-3MPS/DXM cytotoxicity assessment

The rapid development of nanotechnology for biomedical applications has raised concerns about the potential toxicity of nanomaterials. Although AuNPs are characterized by a higher grade of biocompatibility respect to other available metal nanoparticles, their chemical and physical properties including size, shape and surface charge may result in cytotoxic effects.^9,12 For this reason, the cyto-compatibility of AuNP-3MPS/DXM and of AuNP-3MPS was tested in vitro on two distinct cell lines: the human carcinoma HeLa and the murine lymphoma EG.7-OVA. Cells were treated with scalar doses of empty or DXM-conjugated AuNP-3MPS (dose range 50–0.08 μg mL⁻¹) and their viability was determined after 48 hours of culture by the high-throughput screening assay for cell viability MTT. Both AuNP types proved to be non-toxic, in fact cell viability in the treated wells was comparable to that of untreated wells for both cell lines (Fig. 4 and ESI Fig. 1†).

Fig. 4 Cyto-compatibility assessment of empty and DXM-loaded AuNP-3MPS. HeLa (a) and EG.7-OVA (b) cell lines were cultured in the presence of decreasing concentrations of either empty or DXM-conjugated AuNP-3MPS. After two days culture, cell viability was evaluated by MTT assay. Data are expressed as mean OD ± SEM (n = 6).

3.3. Evaluation of AuNP-3MPS/DXM pharmacological activity

Having demonstrated the absence of cytotoxicity of the AuNP-3MPS system, we decided to assess the pharmacological activity of AuNP-3MPS/DMX in vitro on DXM-sensitive cells.

DXM is known to induce up-regulation of the membrane immune-inhibitory receptor programmed death-1 (PD-1) in activated murine lymphocytes.⁴⁶ After performing a dose-finding experiment to identify the appropriate DXM concentrations (ESI Fig. 2†), primary murine lymphocytes isolated from spleen were treated with the polyclonal activating stimuli Concanavalin A (ConA) and concomitantly with comparable doses of either AuNP-3MPS/DXM (loading efficiency = 80%) or DXM. After a 3 days culture, PD-1 expression on the cell membrane of live T lymphocytes (NIRnegCD3+) was evaluated (Fig. 5a). As expected, ConA-stimulated T lymphocytes up-regulated PD-1 respect to their unstimulated counterparts (resting T cells) and a further upregulation was induced by AuNP-3MPS/DXM with a similar efficiency to free DXM, as shown by comparable median fluorescence intensity (MFI) values (Fig. 5b). As expected, treatment of activated T lymphocytes with empty AuNP-3MPS did not affect PD-1 expression (Fig. 5b).

Fig. 5 AuNP-3MPS/DMX-induced PD-1 upregulation on primary murine lymphocytes. Primary murine lymphocytes were activated by treatment with ConA and concomitantly incubated with either AuNP-3MPS/DMX or with unconjugated DXM and empty AuNP-3MPS as controls. Median fluorescence intensity (MFI) of PD-1 expression on live lymphocytes (NIR-CD3+) is indicated within each histogram. One representative experiment out of two independent is shown.

To confirm the biological activity of AuNP-3MPS/DMX and to investigate whether the slow release rate of DXM molecules from AuNP-3MPS could prolong the pharmacological activity of DXM, we compared the pro-apoptotic activity of unconjugated DXM or AuNP-3MPS-conjugated DXM on the human lymphoma cell line Karpas-422, in which DXM is known to induce growth inhibition and apoptosis.^45,46

We first cultured Karpas-422 cells with decreasing concentrations of DXM (70–0.7 ng mL⁻¹) to identify the minimum effective dose of DXM to be used in the subsequent experiments (ESI Fig. 3†). Cells were counted daily for three days and the dose of 7 ng mL⁻¹ was identified as the minimum effective dose inducing a decrease in the cells' proliferative rate. Karpas-422 cells were then cultured for 10 days in the presence of 7 ng mL⁻¹ of DXM or an equivalent dose of AuNP-3MPS/DXM. Untreated or AuNP-3MPS-treated cells were considered as controls. The proliferation rate (Fig. 6a) and the levels of apoptosis (Fig. 6b) in Karpas-422 cells were evaluated at the indicated time-points after culture initiation.

Fig. 6 AuNP-3MPS/DMX pharmacological activity in Karpas 422 cell line. (a) Viable cell count by trypan blue exclusion and (b) apoptosis detection by Annexin V/propidium iodide (PI) staining at different time points after treatment with 7 ng mL⁻¹ of DXM or an equal concentration of AuNP-3MPS/DMX or empty AuNP-3MPS (c) pictures of cell cultures in the different experimental conditions (day 3). One of two representative experiments is shown. *p = 0.05, **p < 0.01, ***p < 0.005.

As expected, AuNP-3MPS did not affect cell proliferation nor cell viability, as evidenced by increasing cell numbers (Fig. 6a) and by the low apoptotic rates detected at all time points which are mainly due to cell contact-dependent apoptosis at confluence (Fig. 6b). These data corroborate the previous observations on AuNP-3MPS cyto-compatibility (Fig. 4).

On the contrary, treatment with 8.4 ng mL⁻¹ AuNP-3MPS/DXM (corresponding to 7 ng mL⁻¹ of DXM), significantly impaired tumor cell proliferation in a way comparable to soluble DXM, as evidenced by cell counts (Fig. 6a) and, indirectly, by the lower apoptotic rates at day 3, as compared to non-DXM treated cultures, reflecting a delay in cell culture confluence establishment. Viable cell count data were corroborated by the absence of cell proliferation clusters in the cultures treated with DXM or AuNP-3MPS/DXM, as compared to control cultures in which many clusters of proliferation were instead already visible on day 3 (Fig. 6c). Interestingly, at early time points, DXM and AuNP-3MPS/DXM induced comparable levels of apoptosis, while at later time points from culture initiation (day 6–8), the pro-apoptotic effect of AuNP-3MPS/DXM was higher with respect to unconjugated DXM.

These data support the hypothesis that small bioactive amounts of DXM are constantly released from AuNP-3MPS/DXM over time and that this event may prolong the pharmacologic activity of the drug over time, thus translating into stronger antitumor effects. In fact, it is tempting to speculate that due to the 3D structure of AuNP-3MPS/DMX conjugates, DXM molecules are inactive as far as the they are bound to AuNP-3MPS because of steric hindrance. Bledsoe et al.⁶⁴ have shown that the high affinity binding of DXM to the glucocorticoid receptor (GR) is due to the extensive hydrophobic and hydrophilic interactions between the ligand and its receptor. One or more hydrophobic residues within the GR protein contact nearly every atom of the steroid core of DXM and the hydrophilic groups of DXM form hydrogen bonds with different protein residues. In addition, the fluorine of DXM also makes direct interactions with a residue (F623). As a consequence, we may assume that when the drug is conjugated to the AuNP-3MPS, the thiols are arranged around it and cause considerable steric hindrance, thus preventing DXM binding to its receptor. Upon drug release, the receptor binding site of DXM is made accessible and the drug acquires full bioactivity.

According to these data, we may conclude that this drug formulation is a biocompatible and efficient system to enhance the pharmacological properties of a suboptimal dose of DXM due to the prolonged release of small amounts of bioactive DXM in vitro over time. These results may be particularly relevant for cancer patients undergoing radiation therapy as AuNP-3MPS/DMX could act as radiosensitizing agents while delivering DXM to the irradiated lesion to control radiation-induced edema. Due to the so called EPR effect⁶⁵ i.e., the capability of AuNP to permeate leaky angiogenic endothelium thus passively accumulating within tumor lesions, AuNP-3MPS/DMX could prove particularly effective as targeting agents in tumor-bearing subjects.

4. Conclusions

AuNP are extremely promising tools for biomedicine due to their high stability and ease of conjugation. The hydrophobic drug DXM can be efficiently loaded and slowly released in biological media, showing that the conjugation of an hydrophobic drug to AuNP-3MPS has favourable effects (enhancement of the stability of AuNP-3MPS, enhancement the bioavailability, prolonged bio-activity as compared to unconjugated DXM). Most importantly, we showed that AuNP-3MPS/DXM continuously release bioactive DXM molecules in culture media that efficiently induce cell proliferation arrest and apoptotic cell death on a DXM-sensitive lymphoma cell line and upregulation of the DXM-inducible programmed cell death molecule on mouse T cells.

These data confirm that the conjugate AuNP-3MPS/DXM is a promising tool for drug delivery and slow release and open interesting perspectives for further in vivo evaluation on multiple disease models.

Acknowledgements

We gratefully acknowledge the University “La Sapienza” of Rome, (Ateneo Sapienza 2015/C26A15H5J9 and 2015/C26A15LRMA projects) and AIRC (MFAG13058 to LB) for financial support. We are grateful to Mr Antonio Di Virgilio and Mr Teodoro Squatriti for technical assistance and to Mrs Rosina Bellizzi for secretarial assistance. Ilaria Fratoddi acknowledges the Dept of Chemistry, Sapienza University of Rome, for Support Research Initiative.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra19561j

‡ These authors contributed equally.

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