Fluorescent (rhodamine), folate decorated and doxorubicin charged ...

J Mater Sci: Mater Med (2012) 23:1697–1704 DOI 10.1007/s10856-012-4634-2

Fluorescent (rhodamine), folate decorated and doxorubicin charged, PEGylated nanoparticles synthesis B. Silvestri • D. Guarnieri • G. Luciani A. Costantini • P. A. Netti • F. Branda

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Received: 5 December 2011 / Accepted: 26 March 2012 / Published online: 7 April 2012 Ó Springer Science+Business Media, LLC 2012

Abstract PEGylated silica nanoparticles, giving very stable aqueous sols, were successfully functionalised with rhodamine, one of the more stable fluorophore; they were also decorated with the targeting agent folic acid (FA) and charged with the well known drug doxorubicin. Rhodamine functionalization required a modification of the synthesis route of the nanoparticles (NP). Functionalization with FA required activation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Folate decorated NP were easily charged with doxorubicin. The experimental results proved the successfulness of the functionalization. The bond to the NP does not reduce the therapeutic efficacy of the drug. The calculated encapsulation efficiency (32 %) was only a little lower than the value (47 %) reported for the very popular PEGylated PLGA NP.

1 Introduction Silica nanoparticles (SiO2 NP) were recently recognised as very promising nanocarriers matching all the demands for their use in the field of nanomedicine [1]. In particular the possibility to functionalize their surface with stimuli B. Silvestri (&) G. Luciani A. Costantini P. A. Netti F. Branda Department of Materials and Production Engineering, University of Naples Federico II, 80125 Naples, Italy e-mail: [email protected] B. Silvestri G. Luciani A. Costantini P. A. Netti F. Branda Interdisciplinary Research Centre on Biomaterials (CRIB), University of Naples Federico II, Piazzale Tecchio, 80, 80125 Naples, Italy D. Guarnieri P. A. Netti Center for Advanced Biomaterials for Health Care (CABHC), Istituto Italiano di Tecnologia, Largo Barsanti, Naples, Italy

responsive groups, nanoparticles (NP), polymers, and proteins makes silica nanoparticle a promising platform for various biotechnological and biomedical applications [1]. It is worth reminding that, when dealing with therapeutical scopes, delivering the drug precisely and safely to its target site to achieve the maximum therapeutic effect remains a yardstick in the design and development of novel drug delivery systems [2, 3]. This is particularly important, in the oncology field when dealing with metastatic states [4–6]. Moreover, trackable fluorescent nanoparticles have the potential to radically improve cancer diagnostics for early tumour detection [7]. The other expected outstanding application of NP is in the field of sensing, identifying and tracking intracellular structures [8–11]. Core–shell fluorescent silica NP may provide a safe, bright and more photostable alternative [12] to the proposed systems, i.e. quantum dots, whose use is nowadays highly questioned [13–15]. Monodisperse systems of silica spheres of different size (from a few nm to 1 lm) can be obtained through the Sto¨ber method [16]. Silica NP produced through this sol– gel method [17] have received very great attention in recent years for biomedical applications [18–24]. Recently, amino functionalized silica NP were obtained by the authors [25] using the Sto¨ber method [16, 26], starting from mixtures of Tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTS). Amino groups functionalised NP can be very interesting for biomedical applications, since they can be easily coupled to a wide range of biomolecules [27, 28] as well as synthetic hydrogels [29]. Subsequently the authors were able [30] to produce PEGylated amino functionalized silica NP through a similar synthesis route. This functionalization is important to avoid the undesired interactions with the immune system producing allergic or rejective reactions [31–35]. The authors showed [30] that by properly selecting the PEG

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content of the reaction batch, PEGylated silica NP smaller than 150 nm and stable towards aggregation in water media up to 6 months were obtained. The experimental results suggested also that the obtained NP consisted of nanosilica (a few nanometers in size) dispersed into a PEG matrix, produced by entanglement of PEG chains [30]. It is worth reminding that, stable dispersions of monodisperse fluorescent colloidal silica spheres containing fluorescein isothiocyanate (FITC) were easily produced [36] by coupling the dye FITC to a silane coupling agent, APTS. Moreover the chemical synthesis was also described of polyethylene glycol (PEG)-functionalized magnetite (Fe3O4) NP, which were activated with a stable ligand, folic acid (FA), and conjugated with an anticancer drug, doxorubicin [37]. It is worth reminding that folate receptors are overexpressed on the surface of some cancer cells and receptor mediated endocytosis is the route to exploit for delivering the drug precisely and safely to its target site and to achieve the maximum therapeutic effect [37–41]. In this paper we show that synthetic routes, inspired by previous works reminded above [36, 37], allow to modify PEGylated silica NP [25, 30] in order to have 1.

2.

Fluorescent PEGylated silica NP containing the fluorophore, rhodamine B isothiocyanate (RBITC) (instead of fluorescein). RBITC is reported to be one of the most stable fluorophore [42]. Folate decorated PEGylated silica NP charged with the anticancer drug doxorubicin (instead of folate decorated PEGylated doxorubicin charged magnetite NP).

2 Materials and methods 2.1 Materials Tetraethoxysilane (TEOS), APTS, Ammonia solution in ethyl alcohol, Polyethylene glycol 5000 monomethyl ether, RBITC, FA and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich (Milan, Italy). Ham’s F12 medium and Dulbecco’s Modified Eagle Medium (DMEM) were received from Gibco. Faetal Bovine Serum (FBS), penicillin and streptomycin were purchased by HyClone, UK. Doxorubicin hydrochloride (DOX; purity [99 %) was obtained from Discovery Fine Chemicals (Wimborne, UK). 2.2 Synthesis 2.2.1 Synthesis of nanoparticles (SiO2 NP and PEG–SiO2 NP) Silica nanoparticles (SiO2 NP) and PEGylated silica nanoparticles (PEG–SiO2 NP) were obtained through the

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previously reported [25, 30] synthesis route reminded below. In particular the reaction batch composition that gave sols stable more than 6 months was used for the (PEG–SiO2 NP) [27]. All the NP had also an amino functionalization [25, 30] because of the use, in the reaction batch composition, of a mixture of alcoxides (TEOS ? APTS). In detail: amino functionalised SiO2 NP were synthesised through a modified Sto¨ber method from a mixture of TEOS and APTS (2 % mol) [25]. This mixture was added drop-wise to an alcoholic (ethanol) solution of ammonia and water in order to have final ammonia, water and total alcoxides (APTS ? TEOS) concentrations equal to 0.1, 8.8 and 0.15 M, respectively. The system was kept at room temperature under magnetic stirring for 24 h. The particles were centrifuged several times in water at 11,500 rpm for 10 min to remove unreacted monomers ethanol and ammonia; finally they were dried at room temperature. PEG–SiO2 NP were simply obtained [30] by previously dissolving PEG in the solution of ammonia and water in ethanol, before addition of alcoxides and keeping it stirred at room temperature for 1 h, to have the complete solubilisation of the polymer. The PEG polymer amount was such to have a final concentration of 0.15 9 10-1 M that gave sols stable more than 6 months [30]. All other concentrations and recovery procedures were the same as for the synthesis of SiO2 NP. 2.2.2 Synthesis of fluorescent nanoparticles (RB–PEG–SiO2 NP) In order to have fluorescent PEGylated silica nanoparticles (RB–PEG–SiO2 NP) an hybrid rhodamine/APTS molecule was used obtained by allowing APTS to react, in a first step, with rhodamine as reported in literature, for the fluorescein functionalised SiO2 NP [36]. A mixture of 1.06 M in APTS and 0.40 mM in RBITC in ethanol was prepared (Solution 1). The reaction was allowed to proceed for 18 h in the dark with magnetic stirring. Finally solution 1 was added altogether with TEOS to the ethanol–PEG–ammonia and water one in the right amount to have the same final concentration as for the PEG–SiO2 NP synthesis. The subsequent steps were the same as for the PEG–SiO2 NP. 2.2.3 Synthesis of fluorescent and non-fluorescent folate decorated PEGylated silica nanoparticles (RB–FA– PEG–SiO2 NP and FA–PEG–SiO2 NP) The fluorescent (RB–PEG–SiO2 NP) and non-fluorescent (PEG–SiO2 NP) PEGylated silica nanoparticles were folate decorated following a procedure similar to the one that allowed PEGylated magnetite nanoparticles to be synthesised successfully [37].


Carboxyl group of FA was first activated with EDC: 4.5 mg of FA was dissolved in 5 ml of dimethyl sulfoxide (DMSO). EDC was added to the solution corresponding to a FA:EDC molar ratio of 1:1 and stirred for 2 h. 20 mg of PEG–SiO2 or RB–PEG–SiO2 NP were added and continuously stirred for 2 h and the obtained FA modified particles (FA–PEG–SiO2 NP and RB–FA–PEG–SiO2 NP) were then washed according to the same procedure reported above. 2.2.4 Preparation of DOX loaded nanoparticles (DOX–FA–PEG–SiO2 NP) The anticancer drug doxorubicin (2 mg) was dissolved in 2 ml of tris(hydroxymethyl) aminomethane buffer solution at pH 7 and 18 mg of FA–PEG–SiO2 particles were added to the solution and stirred over night at room temperature. The obtained Nanoparticles (DOX–FA–PEG–SiO2 NP) were then washed twice with water and dispersed in DMEM. The DOX–FA–PEG–SiO2 NP were, in particular, prepared by suspending FA–PEG–SiO2 NP in a solution containing DOX in weight ratio to NP about 1/10 as elsewhere reported [43]. 2.3 Physicochemical characterisation Fourier Transform Infrared (FTIR) transmittance spectra were recorded in the 400–4,000 cm-1 using a Nexus FT-IR spectrometer. Scanning Electron Microscopy (SEM) images of samples were obtained on Leica Stereoscan 440 microscope. The RB–PEG–SiO2 sample, after centrifugation and drying, was re-dispersed in water and sprayed on a gold coated glass and then they were gold-coated before observation. 2.4 Cell culture Human ovarian adenocarcinoma cell line, IGROV-1 were used to test biological effects of NPs. IGROV-1 cells were cultured with a complete medium, based in RPMI 1640 medium (Lonza), containing 10 % (v/v) FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin (HyClone, UK). Cells were maintained in 100-mm diameter cell culture dish in a humidified atmosphere at 37 °C and 5 % CO2. 2.5 Quantification of nanoparticle intracellular uptake To evaluate cell uptake of NPs as a function of NP surface functionalization with FA, about 5 9 104 cells were seeded in a 96-well. RB–FA–PEG–SiO2 NP and RB–PEG–SiO2 NP were dispersed in cell culture medium with and without free FA. Cells were incubated with NP suspensions for 24 h. After incubation, cells were rinsed with PBS and

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lysed with 1 % Triton X100 in PBS. Cell lysates were TM analysed by a spectrofluorometer (Wallac 1420 Victor2 , Perkin–Elmer, USA) to measure the amount of internalised NPs. The wavelength was 543 nm that is the excitation length of rhodamine. 2.6 Encapsulation efficiency (EE) DOX–FA–PEG–SiO2 NPs (2.47 mg) were dissolved in 1 ml of HF under stirring. DOX entrapped was quantified TM by spectrofluorometric analysis (Wallac 1420 Victor2 , Perkin–Elmer, USA) at 488 nm (the excitation wavelength of DOX). The fluorescence assay was performed in 96-well black flat-bottom plates (Corning, USA) after calibration by standardised solutions of DOX in HF. The encapsulation efficiency was calculated as EE ¼

DOXentrapped 100 DOXtotal

where DOXtotal is the total amount used when NP were DOX charged. 2.7 Cytotoxicity assay The cytotoxic activity of DOX-loaded SiO2 NPs against IGROV-1 cells was compared to non-treated cells, free drug and nanoparticles functionalised or not with FA (RB–PEG– SiO2 NP and RB–FA–PEG–SiO2 NP), as controls, using Alamar Blue Assay to quantify cell survival. 1 9 105 cells were added in 200 ll of cell culture medium to each well of a 96-well plate and allowed to recover for 24 h. After recovery, free drug, blank nanoparticles, or drug-loaded nanoparticles were added to the wells in 100 ll medium, and non-treated cells received 100 ll medium. Cells were treated for 24 h, after which Alamar Blue Assay was performed. Absorbance of the Alamar Blue reagent solution was read at 570 and 600 nm wavelengths by a plate reader (Wallac Victor 1420, Perkin Elmer). Data were reported as fraction of cell viability normalised to non-treated cells. 2.8 Statistical analysis The statistical significance of the results was assessed by one-way analysis of variance. ANOVA test at the significance level P \ 0.05 was used to identify statistically different groups by using Excel 2007 software package.

3 Results Figure 1 shows SEM micrographs of SiO2 NP (Fig. 1a), RB–PEG–SiO2 NP (Fig. 1b) and RB–FA–PEG–SiO2 NP (Fig. 1c). As can be seen the SiO2 NPs are about 150 nm in

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Fig. 1 SEM micrographs of SiO2 NP (a), RB–PEG–SiO2 NP (b) and RB–FA–PEG–SiO2 NP (c)

diameter and sometimes they aggregate forming micrometric clusters; whereas RB–PEG–SiO2 NP (Fig. 1b) and RB–FA–PEG–SiO2 (Fig. 1c) are about 150 nm in average diameter. Rhodamine does not influence the morphology of nanoparticles therefore micrographs of non-fluorescent samples are not reported. The DLS results of RB–PEG– SiO2 NP reported in Fig. 2 confirm the average nanoparticles size of about 150 nm in diameter and it is representative of all PEGylated samples (PEG–SiO2 NP, FA–PEG–SiO2 NP and RB–FA–PEG–SiO2).

Fig. 2 DLS analysis of RB–PEG–SiO2 nanoparticles

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The FTIR of SiO2 NP (Fig. 3a), PEG–SiO2 NP (Fig. 3b), FA–PEG–SiO2 NP (Fig. 3c) are reported in Fig. 3. The NP spectra are dominated by the bands of Silica. The organic are hardly visible. However, the bands at 2,900, 1,467 and 1,360 cm-1 are characteristic of PEG

Fig. 3 FTIR of SiO2 NP (a), PEG–SiO2 NP (b), FA–PEG–SiO2 NP (c)


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Fig. 4 Nanoparticles uptake in IGROV1 cells as a function of NP surface functionalization with folic acid (FA). Cells incubated for 24 h with 0.4 mg/ml RB–PEG–SiO2 NP and RB–FA–PEG–SiO2 NP dispersed in cell culture medium without free FA and with free FA. P \ 0.05

Fig. 5 Cytotoxity assay of 0.4 mg/ml DOX–FA–PEG– SiO2 NP loaded with 2 lg of drug in IGROV1 cells compared to 2 lg/ml free drug (Doxo free), 0.4 mg/ml RB–PEG–SiO2 NP and 0.4 mg/ml RB–FA– PEG–SiO2 NP after 24 h of incubation. P \ 0.05

[44]. A difference is observed in the intensity of the bands at 1,630 and 3,500 cm-1 due to H2O and OH absorption. Their intensity in the FA–PEG–SiO2 NP spectrum is greater than in the SiO2 NP but lower than in PEG–SiO2 NP one. The best proof of rhodamine functionalization is the pink colour of the NP and the possibility to see them in confocal microscopy using fluorescence excitation at k = 543 nm characteristic of rhodamine. This was exploited to study NP uptake into cancer cells IGROV1, as described in the experimental section. The results are reported in Fig. 4. The experimental results obtained in the presence of free FA are also reported in the Fig. 4. As can be seen FA–PEG–SiO2 were better internalised than PEG– SiO2. However, the weight ratio of internalised FA–PEG– SiO2/PEG–SiO2 was reduced when free FA was present (from 1.6 to 1.25). The DOX–FA–PEG–SiO2 NP were red coloured and emitted at the typical excitation wavenumber of Doxorubicin (k = 488 nm). This allowed to evaluate the encapsulation efficiency and to perform the cytotoxicity tests described in the experimental section. The encapsulation

efficiency resulted to be 32 % and the doxorubicin/NP weight ratio resulted to be 5.71 lg of doxorubicin per mg of NP. Figure 5 shows the cell viability after incubation in the presence of PEG–SiO2, FA–PEG–SiO2 and DOX–FA– PEG–SiO2 NP. The cell viability after incubation in the presence of free DOX (in concentration equal to the one obtained if the total amount entrapped in the DOX–FA– PEG–SiO2 NP would be instantaneously released) is also reported. As can be seen, in the case of free DOX, the same cell viability was appreciated as in the presence of DOX– FA–PEG–SiO2 NP: the bond to the NP does not reduce the therapeutic efficacy of the drug. There is no difference in cell viability of cells exposed to RB–PEG–SiO2 NP and RB–FA–PEG–SiO2.

4 Discussion As it is known the Sto¨ber method is based on the basic hydrolysis and polycondensation reactions of Sol–Gel chemistry:

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Si ðORÞ4 þ n H2 O ! Si ðORÞ4n ðOHÞn þn ROH Si OH þ HOSi ! Si O Si þ H2 O Si OH þ ROSi ! Si O Si þ ROH Sto¨ber proved [16, 24] that, in some conditions of TEOS, ammonia and water concentrations in ethanol, particles may be obtained with size ranging from a few nanometers to micron. As Si–C bonds are not easily hydrolysable, the use of APTS allows to have aminofunctionalised nanoparticles [17, 25]. PEG grafting can be easily obtained when PEG is present in the reaction batch through the occurrence of the following trans-esterification reaction [45, 46]. Si OR þ OHðCH2 CH2 OÞn CH3 $ Si OðCH2 CH2 OÞn CH3 þ ROH As a matter of fact very stable (more than 6 months) PEG–SiO2 NP were easily obtained [27], by this way, by the authors in the conditions described in the experimental section. PEGylation is well supported by the FTIR spectra. The bands at 2,900, 1,467 and 1,360 cm-1 are in fact characteristic of PEG [44]. The pink colour of the RB– PEG–SiO2 NP and possibility to see them in confocal microscopy using fluorescence emission at k = 543 nm characteristic of rhodamine well proves that the synthesis route described in the experimental section was successful. The synthesis route is similar to the one reported in the literature for fluorescein doped NP [36]. We can admit that the dye RBITC was covalently attached to the coupling agent APTS by an addition reaction of the amine group with the thioisocyanate group so as reported for fluorescein [36]. The use of this hybrid molecule does not appear to affect the final NP size: SEM analysis (Fig. 1) and DLS analysis (Fig. 2) show nanoparticles having a diameter less than 150 nm comparable to the PEG–SiO2 NP previously reported [30]. Folic acid is difficult to conjugate to the surface of PEG. It was recently reported [37], for PEGylated magnetite NP, that stable conjugation may be performed when the carboxyl group of FA was activated with dicyclohexylcarbodiimmide (DCC). The same authors reported that NP could be successively easily charged with doxorubicin, with a direct bond to folate [37]. The experimental results reported in this paper confirm that this route was successful also for the PEGylated silica NP. The FTIR proves this indirectly. In fact PEG possesses a strong hydrophilicity. This justifies the increase of the bands at 1,630 and 3,500 cm-1 (due to H2O and OH absorption) for the PEG– SiO2 NP with respect to SiO2 NP. The decrease of the same bands observed in the FA–PEG–SiO2 NP spectrum with respect to the PEG–SiO2 NP one is just consistent with the hypothesis reported elsewhere [37] that folate decoration

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does occur just through interactions with PEG molecules. Otherwise the increased NP uptake shown by Fig. 4 in the case of RB–FA–PEG–SiO2 is a clear indication that the NP folate decoration was successful. It is known that intracellular uptake can occur through liquid phase endocytosis, receptor mediated endocytosis and phagocytosis. In particular the folate receptor-mediated endocytosis is a well assessed mechanism and the folate receptor targets are considered appropriate for tumour selective drug delivery [38, 47]. This receptor is a highly selective tumour marker as it is expressed in various types of human cancers but largely absent in most normal tissues [38, 47]; because of the small size and presumed lack of immunotoxicity of FA, folate conjugation has received considerable attention in the targeted delivery of drugs, bioactive molecules and radiopharmaceuticals to cancer cells [38, 47]. The intracellular uptake involves [48] entry of a folate receptor complex into the cell as a group, where it is fused with a lysosome in which the complex is processed. Next the folate is released from the lysosome and the receptor recycled back to the cell surface to capture more folate [48]. The results in Fig. 4 about the intracellular uptake in the presence of free FA in the cell culture admit a simple tentative explanation: in this case there is less folate receptor devoted to creating the group with the decorated NP (RB–FA–PEG–SiO2 NP) and the differences between the decorated and the undecorated ones is depressed. These data need, of course, considering in more detail. DOX–FA–PEG–SiO2 NP had a red colouration and absorbed light at k = 488 nm characteristic excitation wavelength of Doxorubicin. This is a clear proof that doxorubicin was effectively charged. It is worth reminding that, as reported in Sect. 2.2.4, the NP were washed twice after DOX loading. Therefore we admit that a good conjugation does occur so as reported elsewhere [37]. The result of Fig. 5 may be explained. A cell viability reduction was instead expected and observed after exposure to Doxorubicin charged NP. It is worth underlining the similarity of the cell viability results obtained in the case of exposure to DOX–FA–PEG–SiO2 NP and free Doxorubicin: the therapeutic efficacy of the drug does not appear to change after bond to the NP. Of course, in the case of IGROV1 cells, a low cytotoxic effect of free Doxo and Doxo-loaded NP was observed (a decrement of about 20 % of cell viability). It’s worth reminding that, as reported elsewhere [49] IGROV1 cells show a very low doubling time and are quite resistant to drug treatments. Our results, therefore, confirm the well known resistance to treatment of IGROV1 cells. Drug encapsulation efficiency is an important index for drug delivery systems. This is especially true for expensive drugs. The DOX–FA–PEG–SiO2 NP were prepared by suspending FA–PEG–SiO2 NP in a solution containing


DOX in weight ratio to NP 1/10 as reported for PLGA NP [43]. The calculated encapsulation efficiency (32 %) was only a little lower than the value (47 %) for the very popular PEGylated PLGA nanoparticles [43]. The doxorubicin/NP weight ratio was quite low: 5.71 lg of doxorubicin per mg of NP. This appears low if one takes into account the used FA/PEG molar ratio (FA/PEG = 10) and the Doxorubicin molar weight (544), about ten times lower than PEG 5000: if all the FA molecules would have bonded DOX molecules, the loaded doxorubicin should almost equal the PEG content which is [30] 86 lg per mg of NP. The low doxorubicin charge can be explained taking into account the structure of the PEG–SiO2 NP. They would consist [30] of nanosilica (a few nanometers in size) dispersed into a PEG matrix, produced by entanglement of PEG chains. We can admit that FA molecules would easily diffuse in the nanoparticle and bound to the PEG molecules in the interior of the NP as supported by the strong decrease of FTIR bands at 1,630 and 3,500 cm-1 (due to H2O and OH absorption) for the FA–PEG–SiO2 NP with respect to PEG–SiO2 NP. On the contrary the bigger doxorubicin molecules are expected to exploit only the surface FA groups.

5 Conclusions The experimental results prove that the proposed synthesis route allows to modify PEG–SiO2 NP in order to have: (1) fluorescent PEG–SiO2 NP containing the fluorophore, RBITC which is reported to be one of the most stable fluorophore; (2) folate decorated PEG–SiO2 NP charged with the anticancer drug doxorubicin. The bond to the NP does not reduce the therapeutic efficacy of the drug. The calculated encapsulation efficiency (32 %) was only a little lower than the value (47 %) reported for the very popular PEGylated PLGA nanoparticles.

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