Applied Radiation and Isotopes 69 (2011) 1692–1697

Contents lists available at ScienceDirect

Applied Radiation and Isotopes

0969-80

doi:10.1

n Corr

E-m

Geraldja

journal homepage: www.elsevier.com/locate/apradiso

Design, development and characterization of multi-functionalized goldnanoparticles for biodetection and targeted boron delivery in BNCTapplications

Subhra Mandal a, Gerald J. Bakeine b,n, Silke Krol c, Cinzia Ferrari d, Anna M. Clerici d, Cecilia Zonta d,Laura Cansolino d, Francesca Ballarini e, Silva Bortolussi e,f, Subrina Stella e, Nicoletta Protti e,Piero Bruschi f, Saverio Altieri e,f

a Department of Tumor Immunology, Radboud University Nijmegen Medical Centre, The Netherlandsb Department of Internal Medicine and Therapeutics—Section of Clinical Toxicology, University of Pavia, Piazza Botta 10, 27100 Pavia, Italyc Institute of Neurology, Fondazione IRCCS Carlo Besta, Milan, Italyd Department of Surgery, Laboratory of Experimental Surgery, University of Pavia, Italye Department of Nuclear and Theoretical Physics, University of Pavia, Italyf National Institute of Nuclear Physics (INFN), Section of Pavia, Italy

a r t i c l e i n f o

Available online 18 May 2011

Keywords:

BNCT

Functionalized gold nanoparticles

Targeted boron delivery

Folate receptor

Optical molecular imaging

Biocompatibility

43/$ - see front matter & 2011 Elsevier Ltd. A

016/j.apradiso.2011.05.002

esponding author.

ail addresses: Jamesbakeine1@yahoo.com,

mes.bakeine@unipv.it (G.J. Bakeine).

a b s t r a c t

The aim of this study is to optimize targeted boron delivery to cancer cells and its tracking down to the

cellular level. To this end, we describe the design and synthesis of novel nanovectors that double as

targeted boron delivery agents and fluorescent imaging probes. Gold nanoparticles were coated with

multilayers of polyelectrolytes functionalized with the fluorescent dye (FITC), boronophenylalanine and

folic acid. In vitro confocal fluorescence microscopy demonstrated significant uptake of the nano-

particles in cancer cells that are known to overexpress folate receptors.

& 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Boron neutron capture therapy (BNCT) is a binary anti-cancertherapy that is designed to exploit the cytotoxic effect of the ionizingalpha and lithium particles. These heavily charged particles has arange of about one cell diameter so the radiation damage will havean effect mainly in the cell from which they arise.

In order for BNCT to have therapeutic value, it is critical todeliver 10B atoms within target tumor cells in therapeuticallyrelevant concentrations while maintaining a very high ratio tohealthy tissue. Consequently, the ability to monitor the tempo-spatial distribution and concentration of 10B in tumor and normaltissue down to the cellular level is critical for accurate treatmentdecision making, treatment planning and follow up.

To date only two boron compounds, boronophenylalanine(BPA) and sodium borocaptate (BSH), are used for 10B deliveryin BNCT clinical trials. However, their use has been associatedwith mixed results. The limiting factors of these agents include:(1) failure to universally attain high tumor to healthy tissue

ll rights reserved.

ratios; and (2) current tools like neutron autoradiography andmass spectroscopy used for the ex-vivo determination 10B con-centration and distribution are challenging and lack spatialresolution at the cellular level (Altieri et al., 2008; Wittig et al.,2008). There is, thus, a need for improvement of boron deliveryvehicles to maximize the tumor/healthy tissue ratio and efficientmodalities to facilitate the tracking of boron concentration anddistribution down to the sub cellular level.

A rational approach to overcome these limitations is the develop-ment of boronated carriers that are simultaneously conjugated withligands, that selectively bind to antigens or receptors that are usuallyabundantly or uniquely expressed on the tumor cell surface, andimaging probes that allow their tracking with the same specificity.

Colloidal gold nanoparticles (GNPs) have recently received con-siderable scrutiny as possible vehicles for drug delivery largelybecause of their desirable qualities that include biocompatibility,low toxicity, small size and tunable surface functionalities (Youet al., 2006, 2005). In order to target the GNPs to the tissue of choice,folic acid presents advantages as a selective tumor targeting moietybecause high affinity folic acid receptors are frequently overex-pressed on the surface of human cancer cells (Franklin et al., 1994;Weit et al., 1992). Furthermore, the folate receptor is efficiently cellinternalized after binding with its ligand (folic acid) (Antony, 1992).In this regard, we propose the use of gold nanoparticles (GNPs)

S. Mandal et al. / Applied Radiation and Isotopes 69 (2011) 1692–1697 1693

multi-functionalized with BPA, folic acid and the organic fluorescentdye fluorescein isothiocynate (FITC) to enable their tracking.

In this report we describe the design and results of synthesis ofbiocompatible GNPs coated with multilayers of boronated poly-

Fig. 1. Schematic representation of the model design

Fig. 2. (a) Characterization of GNPs by hydrodynamic size, surface charge and PDI-val

spectrophotometry. On the right is the typical 10BPA absorbance spectra. (c) and (d)

solution and 10% BSA solutions by DLS and PDI (c) and UV–visible spectrophotometry

electrolytes with the purpose of improved targeted boron deliveryand simultaneous optical imaging. Initial in vitro studies byconfocal microscopy confirmed the substantive and selectiveuptake of these nanovectors in cancer cells.

and synthesis of the multi-functionalized GNPs.

ue analysis after every layer deposition. (b) the verification of BPA upload by UV-

Comparison of particle (Fo-0.5 M 71bPNG) stability in MQ-water, saline, Ringer’s

(d).

Fig. 3. (a) TEM micrograph depicting the structure of the resulting GNPs with a

nanogold core and a polyelectrolyte shell. Bar line 200 nm. (b) Neutron auto-

radiography validated the electrostatic binding of BPA to the GNPs.

S. Mandal et al. / Applied Radiation and Isotopes 69 (2011) 1692–16971694

2. Materials and methods

Multilayer coated GNPs were synthesized by the depositionof oppositely charged boronated polyelectrolytes according to amodified protocol described by Mandal et al. (2011). Briefly, boro-nated poly-fluorescein allylamine hydrochloride (10B-FITC-PAH) wasprepared by dissolving 3 mg polyfluorescein allylamine hydrochloride(FITC-PAH; MW 15 kDa [Sigma]), along with 0.5 mg of BPA permilliliter of MQ-water. While boronated polysodium 4-styrenesulfo-nate (10B-PSS) was prepared by dissolving 10 mg polysodium4-styrenesulfonate (PSS; MW 4.3 kDa [Sigma]) along with 2 mg ofBPA per milliliter of MQ-water. These boronated polyelectrolytesolutions were prepared one day in advance to allow maximumboron conjugation.

10B-FITC-PAH and 10B-PSS were adsorbed consecutively in alayer-by-layer fashion on GNPs (15 nm diameter) in the followingsequence [Au(10B-FITC-PAH/10B-PSS)2/10B-FITC-PAH] to obtain5 layers. In a typical procedure, GNPs stock solution was addeddrop-wise to the boronated polyelectrolyte solution under contin-uous vortexing and kept overnight at room temperature in the dark,to allow for sufficient and homogenous coating of the gold particle.The GNPs were then rinsed twice in MQ-water by centrifugationfollowed by removal of the supernatant and resuspended inMQ-water. After each layer deposition, the hydrodynamic size,surface charge and particle monodispersity were determined bydynamic light scattering (DLS), zeta-potential measurement andpolydispersity index (PDI), respectively. Binding of BPA was deter-mined by UV–visible spectrophotometry. GNPs coated with poly-mers not functionalized with BPA acted as controls. Eachmeasurement was repeated at least 5 times.

In order to avoid the aggregation of coated GNPs in biologicalfluids as well as to conceal the exposure of BPA on the surface ofthe growing GNPs (since BPA equally enhances uptake in normalproliferating cells), The 6th and 7th layers were deposited using10 mg/ml poly-styrene-4-sulfonate sodium (PSS) and 3 mg/mlpoly-allylamine hydrochloride (PAH) dissolved in 0.5 M NaClsolution, respectively. To further functionalize the resultingnanoparticles for cancer specific uptake, the outermost layer ofthe particles were conjugated electrostatically with folic acid (FA)by adding drop-wise folic acid solution (0.007 mg/ml) tothe suspension of these nanoparticles under constant vortexingfollowed by 6 h incubation in the dark at room temperature. Theunbound folic acid was removed by rinsing as described above.The whole synthesis procedure was carried out in triplicates.

2.1. Biological characterization

The stability of the coated nanoparticles was tested after 4 hincubation in the following representative physiological solutions;normal saline (0.9% NaCl), bovine serum albumin (10% BSA) andRinger’s solution. Particle monodispersity, hydrodynamic size andUV–visible absorbance was determined as above.

In vitro receptor-mediated endocytosis of the nanoparticles wasevaluated in three different cancer lines: hepatocellular carcinoma(JHH6); leukemic (HL60); and breast cancer (MDA MB 231). Non-cancer cell lines: immortalized human hepatocyte (IHH) and macro-phages (J774.2) acted as controls. Each cell line was treated with thenanoparticles at a concentration of 0.29 M for 24 h at a density of20�104 cells per plate using standardized cell culture protocols.Uptake efficiency (percentage of cells that internalized the functio-nalized GNPs) was determined by confocal fluorescence microscopicimaging. Each experiment was repeated at least three times (foreach experiment 100 cells were evaluated).

To evaluate normal cellular viability after particle internalization,mitochondrial function was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

2.2. Optical imaging by fluorescence microscopy

Image acquisition was performed with a Nikon C1si laserscanning confocal unit attached to an inverse fluorescence micro-scope (Nikon D-eclipse C1, Japan). Images were acquired andprocessed using the NIKON software EZ-C1.

Data are expressed as means7standard deviation (SD) whereindicated. Statistical analysis was performed by Graphpad Prism5 and Origin 8 software.

3. Results and discussion

3.1. Synthesis of GNPs coated with multilayers of boronated

polyelectrolytes and their physico-chemical characterization

Fig. 1 schematically represents the model of the layer-by-layerelectrostatic coating of the GNPs with oppositely charged functiona-lized polyelectrolytes. The 7th and outermost layer is denominated asFo-0.5 M 7ABPNG. After every coating with a polyelectrolyte layer,the hydrodynamic size, particle monodispersity and the surfacecharge were determined. The results are summarized graphically inFig. 2. Particle diameter gradually increases with the increment in thenumber of layers deposited. As anticipated, the 6th and 7th layerswere coated with polyelectrolytes dissolved in 0.5 M NaCl, thesecounter ions resulted in the collapsing of the polyelectrolyte linearmolecule into a random coil causing further increase in the hydro-dynamic diameter but with a reduced surface charge. The finalparticle diameter and surface charge after coating with the 7th layerwere 14075 nm and þ2573 mV, respectively. The PDI valuessuggest that the GPNs did not aggregate during the synthesis process.The overall architecture of a single nanogold core with the polymercoating and its monodispersity was visualized by TEM imagingFig. 3(a). This further confirms the PDI analysis that the particlesdid not aggregate during and after synthesis. The upload of BPA andhence by inference 10B was evaluated by UV–visible spectrophoto-metry and the results are graphically summarized in Fig. 2(b).UV-absorbance signal at 278 nm due to phenylalanine of BPAindicates its successful binding. BPA has phenylalanine group inwhich the p electrons of the phenyl ring can stack with otheraromatic systems and often do within folded polyelectrolytes, adding

Fig. 4. Merged gray transmission images with fluorescence microscopy images reveals negligible green FITC GNPs uptake in noncancerous cell lines indicated by arrows

(a)IHH and (b) J774.2, while significant uptake is observed in tumor cell lines JHH (d), HL60 (e) and MDA MB 231 (f). Bar line 10 mm. The percentage of the cells that

internalized the nanoparticles and hence BPA are further graphically presented in (c). (For interpretation of the references to color in this figure legend, the reader is

referred to the web version of this article.)

S. Mandal et al. / Applied Radiation and Isotopes 69 (2011) 1692–1697 1695

Fig. 5. Cell toxicity profile of the synthesized nanoparticles using the test cell

lines. There was no significant difference between the controls.

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to the stability of the structure. 10B uploading was further validatedby neutron autoradiography. Densely packed a particles tracksimplied a remarkable uploading efficiency (Fig. 3(b)).

3.2. Biological characterization

3.2.1. Stability in physiological solutions

One of the major obstacles in the development nanoparticlebased systems for boron delivery in BNCT is poor stability inbiological fluids where slow aggregation leads to precipitation ofthe particles (Kennedy et al., 2009). We therefore tested mono-dispersion stability of the coated GNPs using representativephysiological solutions namely normal saline, 10% bovine serumalbumin and Ringer’s solution and the findings are graphicallysummarized in Fig. 2(c). No significant change was observed inthe presence of these ionic solutions. Nevertheless some changescould be seen in Ringer’s solution, where the hydrodynamicdiameter increased from about 128 to 180 nm. This could bedue to the swelling of poly-allylamine hydrochloride in thepresence of various types of ions. These findings imply that theGNPs are stable in basic solutions. This property suggests theirsuitability for use in in vitro and in vivo systems in which the pHis tightly controlled at 7.4.

3.2.2. Optimization of selective cancer cell targeting of GNPs by

folic acid functionalization

A number of malignant cells are known to overexpress the folatereceptor of their surface membrane. We sought to exploit this uniquebiological address by electrostatically binding folic acid (the naturalligand of the folate receptors) to the outmost layer of our GNPs toenable selective targeting of folate-receptor-positive cancer cells.Given the fact that the receptor recognizes the pteroic acid moietyin the folic acid molecule, the molecule was electrostatically boundvia a- and g-COOH (glutamic acid moiety) to the positively chargedPAH outermost layer in order to have the right orientation of the folicacid molecules for receptor recognition. An indication of successfulfolic acid electrostatic binding on the GNPS surface was given by DLSand zeta-potential studies (Fig. 2(a)). The particle hydrodynamicdiameter increased from 12070.6 to 13472 nm and surface chargedecreased from þ35.971 to þ27.370.7 mV, upon folic acid bind-ing. The presence of folic acid was further validated by UV–visibleabsorbance.

3.2.3. Optical imaging guided evaluation of the interaction of GNPs

with cancer cells

The uptake of the GNPs was followed in three different cancercell lines i.e. JHH6, HL60 and MDA MB 231, which are known tooverexpress the folate receptor, and compared to non-canceroushepatic and macrophages cells. As shown in Fig. 4(a), (b) and (d)–(f), fluorescence microscopy imaging enabled the tracking of theirinternalization by visualizing the number of cells fluorescentlymarked by GNPs, their intracellular spatial distribution and theintensity of fluorescent signal. Graph 4(c) shows that nearly 90%of folic acid positive cancer cells internalized the GNPs (po0.01).Critically, a significant tumor cell to normal cell uptake ratio of5:1 was observed (po0.01). This is a remarkable result whencompared to our experience with BPA, in which tumor to normaltissue ratio is inconsistent and varies from 1–4. Furthermore wefound that after 24 h treatment most of the cancer cells had theinternalized particles accumulating in the perinuclear region,whereas in the non-cancerous cell lines a non-significant numberof cells had taken up the particles. The observed high uptake bycancer cells of folic acid functionalized GNPs is in good agreementwith other studies in the literature for folic acid-poly-ethylene-glycol conjugates (Cho et al., 2005; Gabinzon et al., 1999).

Macrophage recognition is the main reason for fast bloodclearance of charged nanoparticle based drug delivery systems.As shown in Fig. 4(c) we observed that only about 20% of themacrophages had internalized the GNPs. We hypothesize that thequasi neutral surface charge of the polyelectrolyte shell couldhave prevented recognition by the macrophages. Furthermore,cell viability analysis by MTT after 3 days of incubation suggeststhat the GNPs have minimal toxic effects compared to thecontrols (Fig. 5) This is in an improvement because carboranelabeled nanoparticles for targeted boron delivery have beenreported as cytotoxic (Kennedy et al., 2009).

4. Conclusion

Taken together, the results of this study provide a proof-of-concept for the synthesis of a novel boron delivery system by thelayer-by-layer coating of GNPs with oppositely charged polyelec-trolytes previously functionalized with BPA. This protocol isattractive because multiple stratification permits greater controlof particle size and the boron payload per particle, while thepolymer shell confers improved particle stability in physiologicalsolutions. Furthermore the GNPs particles allow for an easy, fastand stable electrostatic functionalization for targeted delivery asshown by the binding of the model targeting molecule, folic acid.The integration of boron delivery, targeting and optical imaginghas the added value of providing a direct link of information ontumor identification, anatomy and nanoparticle distribution.Importantly, electrostatic binding of multiple functional mole-cules opens up the possibility to bind a broader spectrum ofspecific target molecules corresponding to the specific tumormarkers unique to each tumor type. This may be a promisingroad to personalized medicine.

Given the favorable biocompatibility the GNPs and the opti-mized tumor cell to normal cell uptake ratio of GNPs, furtherstudies are warranted to correlate the intensity of the fluorescentsignal to the concentration of 10B in order to provide a histologicalmap of 10B boron uptake concentration and determine the in vivotumor localizing potential of these boron carriers in folic acidpositive tumor cells.

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