ASP
AMERICAN SCIENTIFIC PUBLISHERS
Anti-hMC2RL1 Functionalized Gold Nanoparticles for Adrenocortical Tumor Cells Targeting and Imaging
Thiago Demetrius Woiski1, Lisiane de Castro Poncio1, Juliana de Moura2, Alexandre Orsato3, Arandi Ginane Bezerra-Jr4, João Carlos Minozzo5, and Bonald Cavalcante de Figueiredo1,*
1 Instituto de Pesquisa Pelé Pequeno Príncipe. Avenida Silva Jardim, 1632, Água Verde, Curitiba, PR. CEP: 80.250-200, Brazil
2 Departamento de Patologia Básica, Universidade Federal do Paraná. Avenida Francisco H. dos Santos, 100, Jardim das Américas, Curitiba, PR. CEP: 80.250-200, Brazil
3 Departamento de Química, Universidade Estadual de Londrina. AC Jardim Bandeirante, Jardim Portal de Versalhes 1, Londrina, PR. CEP 81.531-990, Brazil
4 Departamento de Física, Universidade Tecnológica Federal do Paraná. Avenida Sete de Setembro,
3165, Centro, Curitiba, PR. CEP: 86.057-970, Brazil
5 Centro de Pesquisa e Produção de Imunobiológicos (CPPI). Rua Piquiri, 170, Rebouças, Piraquara, PR. CEP: 80 230-140, Brazil
The low rate of cure of adrenocortical carcinomas (ACC) in children and adults is related to germ line TP53 mutation, late diagnosis, incomplete surgical resection, and lack of an efficient adjunctive therapy. To provide a new approach for the improvement of ACC diagnosis and therapy, the present study aimed to explicitly target ACC cells using gold nanoparticle (AuNP) probes bound to specific antibodies. Immunohistochemistry of ACC and positive and negative control tissue micro-sections under light microscopy was used to test a purified polyclonal antibody raised against the 80-93, outer loop 1 position of the human melanocortin receptor 2 (hMC2R). Both this and a control commercial antibody were found to specifically target cells known to express hMC2R. These were bound to FITC-labeled AuNPs and tested via direct immunofluorescence using the H295R ACC cell line. Both probes recognized only cells expressing hMC2R and exhibited very low background. Further studies are required to ascertain the potential of AuNPs bound to ACC cells for tumor diagnostics via imaging analysis or as a delivery device for targeted therapy.
KEYWORDS: Gold, Nanoparticle, Supra-Renal, Carcinoma, Antibody, Staining, Imaging, Probe.
INTRODUCTION
Adrenocortical tumors among children, although rare, are frequently malignant, with an annual incidence ranging from 0.20 to 0.30 new cases per million children in the United States.1 In contrast, benign adrenocortical tumor among adults is one of the most common tumors, but adrenocortical carcinoma (ACC) incidence is only 1.7 to 2 cases per million, occurring more frequently between the third and the seventh decades.2 Most cases of ACC in young children are associated with Li-Fraumeni syn- drome, commonly originating from germ line mutations in the gene for the tumor suppressor p53.3 The highest ACC incidence among children occurs in Southern Brazil
*Author to whom correspondence should be addressed.
Email: bonaldf@yahoo.com.br
Revised/Accepted: 30 August 2016
because of the germ line TP53 R337H mutation,4,5 which demonstrates a founder effect in this region.6
Recently, a neonatal screening study involving 171.649 newborns in the state of Paraná, Brazil, confirmed that the incidence of benign and malignant adrenocortical tumors among children is 15 to 18 times higher than that in other countries.7 Adrenocortical tumor diagnosis is mainly based on clinical manifestations, levels of adrenal hormones, and imaging analyses8, 9 that provide additional information about the size of the tumor and the involve- ment of adjacent organs, which are important for proper treatment. The most commonly used methods are com- puted tomography (CT) and magnetic resonance imaging (MRI), which are also used to identify metastases and for distinguishing benign and malignant tumors.10, 11 Although these techniques are sensitive they cannot identify adrenal lesions owing to the lack of specific tumor markers.12 In cases where there are lesions smaller than 1 cm, the CT
technique, for example, cannot distinguish a metastasis from an adrenal tumor and nodular hyperplasia.13
Recent advances in nanotechnology have allowed new approaches to be tested for the diagnosis and treatment of diseases such as cancer. For example, gold nanopar- ticles (AuNPs) with sizes below 100 nm exhibit optical, electrical, and thermal characteristics of great interest in biological and medical fields. Because AuNPs are easily distinguished from biological tissue by CT14, 15 when cou- pled to molecules that specifically bind to tumor cells, they can be used in cancer diagnostics.16-18 This potential has recently been analyzed using clinical CT abdominal and skull imaging protocols, yielding satisfactory results. Thus, this finding indicates that gold nanoparticles produced by laser ablation over approximately 1.5 nm can be easily distinguished from the bone and soft tissues even at low concentrations.19
One of the protein classes most commonly used in the functionalization of nanoparticles comprises the immunoglobulins (antibodies), which are a group of glyco- proteins responsible for the highly specific defense mech- anisms of vertebrate animals.18-20 Antibodies are usually attached directly to the surface of AuNPs through a chemi- cal interaction between gold and sulfhydryl (SH) or amino (NH2) groups.21 For example, the antibody can be split in half by reduction with 2-mercaptoethylamine to sepa- rate the two heavy chains, then covalently attaching the SH group to gold.18 It is also possible to activate the car- boxyl group terminal Fc portion and use another molecule, such as cysteamine, to connect the gold nanoparticles to the intact antibody.22
In this context, the melanocortin 2 receptor (MC2R) has been shown to represent a potential specific target for adrenal cortical tumors. MC2R, a member of a family consisting of five transmembrane proteins (MC1R-MC5R), is primarily expressed in the adrenal gland and despite sharing 38, 42, 46, and 44% identity with the amino acid sequences of MC1R, MC3R, MC4R, and MC5R, respectively, it is restricted to signaling by the adrenocor- ticotropic hormone and thus is also known as the adreno- corticotropic hormone receptor (ACTH-R).23-26
Surface proteins such as MC2R have the advantage of being located in an area of easy access,27 which facilitates their use as targets for the development of new approaches to assist in the diagnosis and treatment of diseases. A well- studied example is the case of the human epidermal growth factor receptor type 2 (HER2), which is overexpressed in breast cancer cells. Notably, specific antibodies against this target are capable of blocking breast cancer develop- ment or of quantifying the HER2 protein for prognosis assessment.28-30
To determine the utility of MC2R as a target for ACC, in this study we raised polyclonal antibodies in rabbits immunized with a peptide corresponding to the extracel- lular loop 1 portion of human MC2R. These antibodies were purified in an affinity-column and their specificity
when used as anti-MC2R functionalized gold nanoparti- cles was determined. These particles offer the potential to target ACT cells in vitro and in vivo as a candidate tool for both the diagnosis and treatment of ACT.
MATERIAL AND METHODS Materials
The peptide was synthesized by Peptron Inc. (Daejeon, South Korea). Bovine serum albumin (Imject™ Maleimide- ACTivated BSA), succinimidyl-4-N-maleimidomethyl- cyclohexane-1-carboxylate (SMCC), PD10 desalting column, sepharose column (Resin Coupling SulfoLink®), protein sample concentrator (20 KD MWCO; 7.0 mL); SlowFade® Gold Antifade Mountant with DAPI, penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scien- tific Inc., (Waltham, MA, USA). Freund’s complete and incomplete adjuvant, fluorescein isothiocyanate (FITC), N-(3-dimethylaminopropyl)-n-thylcarbodiimide hydrochloride (N-hydroxysuccinimide; EDC), N-(3- dimethylaminopropyl)-n-ethylcarbodiimide hydrochlo- ric (NHS), cysteamine, albumin, Dulbecco’s modified Eagle medium (DMEM) F12, and anti-hMC2RN-ter (SAB4501839) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Antigenic recovery solution, Immuno Retriever, and Enzyme Advance HRP Link reagents were purchased from Dako® North America, Inc. (Carpinteria, CA, USA).
Anti-hMC2R Production
Antigen Preparation
A synthetic peptide from amino acid (aa) residue 80-93 of the human melanocortin receptor 2 (hMC2R) was synthe- sized with a cysteine addition (CENILIILRNMGYLK) and designated here as hMC2RL1. The cysteine (C) sulfhydryl group was used to couple the hMC2RL1 to activated BSA obtained through the cross- linker SMCC. Accord- ing to the manufacturer, activated BSA was obtained by adding 0.1 mL of an SMCC dimethylformamide solution (10 mg/mL) to 1 mL of a BSA ultrapure water solution (10 mg/mL) that was maintained under mild magnetic stir- ring for 1 hour at room temperature and with protection against light. A PD10 desalting column was used to sep- arate activated and non-activated BSA. Samples were col- lected, analyzed according to absorbance (280 nm), and the first peak obtained referent to BSA-SMCC was used for subsequent experiments. For coupling the activated BSA to the peptide, 0.1 mL dimethyl sulfoxide (DMSO) was used to solubilize the hMC2RL1 in 754 pL ultrapure water (5 mg/mL). Then, 155 µL BSA-SMCC was added to the solution, which remained under mild magnetic stir- ring for 2 hours at room temperature under light protec- tion. Finally, the free amino groups were blocked with a cysteine solution (0.001M).
Animal Immunization
Animal maintenance and the immunization process were carried out in the Centro de Pesquisa e Produção de Imunobiológicos (CPPI-Piraquara, Paraná, Brazil) accord- ing to institution protocols under the approval of the Committee of Ethics in Research in Animals. Adult female rabbits were immunized with the antigen (hMC2RL1- BSA-SMCC) through seven subcutaneous injections at 14 day intervals. The first emulsion injection was prepared by using 100 µL antigen solution (5 mg/mL), 500 ML Fre- und’s complete adjuvant, and 400 µL phosphate buffered saline (PBS; pH 7.4; 0.05M) solution. The remaining injections were prepared using Freund’s incomplete adju- vant. The immunological response of each animal was monitored according to the serum concentration of anti- hMC2RL1 antibodies via an indirect ELISA. The animal sera were collected and the immunoglobulins were precip- itated with a saturated ammonium sulfate solution under mild stirring in an ice bath.
Anti-hMC2RL1 Affinity Column Purification
The purification of anti-hMC2RL1 antibodies was per- formed using a sepharose-based affinity chromatography column with 2.0 mg synthetic peptide (hMC2RL1) immo- bilized via thiol groups (SH) in a final volume of 2 mL. Approximately 50 mg precipitated immunoglobulins were diluted in 4 mL PBS, pH 7.4 and incubated in the col- umn under mild stirring at room temperature for 1 hour. After incubation, the column was exhaustively washed by gravity flow, allowing antibodies that did not recog- nize the hMC2RL1 sequence to be easily eluted. Spe- cific antibodies were then eluted and collected by adding small volumes (150 µL) of glycine/hydrochloric acid solu- tion (0.2 M, pH 2.5) until an immunoglobulin absorbance (280 nm) peak was obtained.
The total amount of specific antibodies was filtered with a syringe filter (0.22-um, Kasvi, Brazil) and con- centrated using a protein sample concentrator. The final concentration of the anti-hMC2R solution was determined using a NanoVue Plus spectrophotometer at A280 nm (GE-Healthcare). Samples were stored in 25 and 50 AL PBS/sodium azide (0.02%) solution aliquots at -20 ℃.
Anti-hMC2RL1 Specificity
The specificity of anti-hMC2RL1 antibodies were evalu- ated through immunohistochemistry (IHC) and immuno- cytochemistry (ICC). Human tissue specimens had been discarded during surgical procedures and their use in the present study was approved by the Ethic’s Committee. We tested four types of tissues using IHC; a childhood adreno- cortical carcinoma (cACC), adult skin, liver, and smooth muscle tissue embedded in paraffin were used in triplicate to compare the anti-hMC2RL1 (1:200) and the commer- cial antibody anti-hMC2RN-ter (1:200) (Sigma-Aldrich,
St. Louis, Mo, USA). After deparaffinization and rehydra- tion of the sectioned tissues (4-um thick), the endogenous peroxidase was inactivated with 5% hydrogen peroxide solution. Antigenic recovery was carried out by leaving the samples dipped in Immuno Retriever in the bath at 99 ℃ for 40 min. After cooling and washing, samples were incu- bated with antibodies (anti-hMC2RL1 or anti-hMC2RN- ter) overnight at 4 ℃ into the moist chamber. The slides were then incubated with Advance HRP Link for 30 min at room temperature. The Enzyme Advance HRP Link was incubated for 30 min at room temperature, washed, and stained with 3, 3-diamino-benzidine and hematoxylin.
The ICC tests were performed using the human adreno- cortical tumor cell line H295R.31 Additional details are summarized below (In vitro hMC2RL1-F-c-AuNP tests).
Synthesis and Characterization of AuNPs
In the present study, AuNPs were prepared by laser abla- tion synthesis in liquid solution.21 A gold metal plate (>99.99% pure) was placed on the bottom of a glass vial filled with 10 mL bi-distilled water and irradiated with a Nd: YAG laser (Model 117, Quantronix Corp., Santa Clara, CA, USA) emitting 200 ns pulses at 1064 nm and energy of 3 mJ/pulse at 1.5 kHz. The laser was focused onto the target at 1 mm below the liquid surface with a spot size of 40 um. The target was randomly moved for 10 min in the focusing plane to obtain identical surface conditions during laser ablation. 14
The absorption spectra of the samples were measured using an Ocean Optics USB2000+ spectrometer (Dunedin, FL, USA), 200-1050 nm, and 1 nm resolution.
Dynamic light scattering was measured with a Micro- trac model Nanotrac Ultra NPA-252 (Montgomeryville, PA, USA). A transmission electron microscope, JEOL JEM2010-200KV (Tokyo, Japan), was employed for the micrographs of the nanoparticles. For this, drops of the colloidal solution were deposited on copper grids and left to dry in air.
Synthesis and Characterization of Gold Anti-hMC2RL1 Probes
Cysteamine/Fluorescein Functionalized-Gold Nanoparticles (F-c-AuNPs)
For this, first, the volumes of the Au atom and single AuNP were estimated by the following equation:
3 . 7 . 13 V = 4
where V is the volume (nm3) and r is the atomic radius (nm) of the Au atom or the radius of the AuNP (nm). The number of gold atoms comprising nanoparticles was estimated by the following equation:
At Au
=
V AuNP V Au
where At Au is the number of atoms, VAuNp is the volume of a single AuNP, and VAu is the volume of an Au atom. The mass of 1 mol of AuNP was estimated by the following equation:
AuNPmoi = AU mol . At Au
where AuNPmol is the mass corresponding to 1 mol of Au nanoparticles (g), Aumol is the mass corresponding to 1 mol of Au atoms (g), and AtAu is the amount of Au atom compounds in a single nanoparticle.
Each milliliter of the nanoparticle suspension used con- tained 0.2 mg of Au. The mol/mL AuNP value was then determined. From this value, equivalent molar ratios of AuNP suspension, cysteamine, FITC, and antibody could be calculated.
Briefly, 2 mL AuNP suspension (1.992 nmol) was mixed with 306 uL cysteamine ultrapure water solution (1992 nmol) and maintained under mild magnetic stir- ring overnight at room temperature with light protec- tion (Fig. 1(A)). The c-AuNPs were then labeled with FITC (Fig. 1(B)) by adding 388 ₪L FITC/DMSO solu- tion (996 nmol) at a concentration of 1 mg/mL, with stir- ring overnight at room temperature and light protection. To remove the unbound cysteine and FITC, the sample was then divided and centrifuged at 21460 g for 45 min at 10 ℃. The supernatants were separated carefully from
the precipitate and centrifuged again using a new tube under the same conditions described above.
The supernatant from the second centrifugation was discarded and 3 ML distilled water was added to the remaining precipitate. These samples were submitted to two alternating cycles of homogenization on a vortex mixer and sonication using an ultrasonic bath (Embra- sol, LS-0,8D-2/X. Itanhaém, SP, Brazil). The F-c-AuNP precipitated samples were collected and resuspended by previously described process. The suspension was washed and centrifuged three times following the procedure of homogenization and sonication after each spin. Finally, after washing, the F-c-AuNPs were resuspended in 200 µL PBS, pH 7.4, and stored at 4 ℃.
Immobilization of Anti-hMC2RL1 Onto F-c-AuNPs (hMC2RL1-F-c-AuNP)
Prior to binding to c-F-AuNPs, the carboxyl group of the antibodies was activated using NHS and EDC22 (Fig. 1(C)). Initially, 1.2 mL anti-MC2RL1 solution (1.5 mg, 9.96 nmol) was dialyzed in 200 mL PBS (0.05 M, pH 7.4) overnight at 4 ℃ under mild magnetic stir- ring. After dialyzing, EDC (2 mg, 9.96 umol) and NHS (1.2 mg, 9.96 umol) were added and the solution remained under moderate stirring on hematologic shaker at room
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temperature, protected from light, for 1 hour. Prior to the end of antibody activation, the F-c-AuNP suspension was heated to room temperature and sonicated briefly to remove aggregates. The activated antibodies were mixed with the F-c-AuNPs and subsequently stirred for an addi- tional 5 hours under the same stirring, light protection, and temperature conditions, then dialyzed overnight in 250 mL PBS at 4 ℃ to remove the residual reagents. Finally, a BSA/PBS solution (5%, 500 µL) was added to the probe suspension (hMC2RL1-F-c-AuNP) and kept at 4 ℃ (Fig. 1(D)).
In Vitro hMC2RL1-F-c-AuNP Testing
Direct immunofluorescence using deparaffinized tissues and one cell strain was performed. To test the tissues, 4-um thick tissue sections from childhood ACC and adult liver were mounted on positively charged slides (Starfrost®, UK. Biogen, SP, Brazil). The staining protocol was adapted from Robertson et al.,32 Briefly, after deparaf- finization and rehydration, antigenic recovery was carried out as described previously. After cooling and washing, the samples were incubated with a BSA/PBS solution (5%) for 20 min at room temperature and incubated with hMC2RL1-F-c-AuNP probes (1:500) at the same tem- perature in a moist chamber for 1 hour with protection from light. After washing, the slides were mounted using SlowFade® Gold Antifade Mountant with DAPI.
H295R cells were initially seeded at 2 × 103 cells/well on previously cleaned and sterilized glass circular cov- erslips (13 mm) placed into 12-well plates. The cells were maintained in complete DMEM F12 medium sup- plemented with 10% FBS and the antibiotics penicillin (100 U/mL) and streptomycin (100 µg/mL). The cells were cultured for 24 hours at 37 ℃ in a fully humidified 5% CO2 atmosphere. The plate was then placed on ice for a few minutes and wells were carefully washed twice with culture medium (4 ℃, pH 7.4) without serum. The cells were incubated with the anti-hMC2RL1-F-c-AuNP or probed without anti-hMC2RL1 (F-c-AuNP, 1:500) in serum-free medium and cultured with 1% BSA for 40 min at room temperature. The cells were then washed twice with serum-free medium (4 ℃) and three times with PBS (4 ℃; pH 7.4). The cells (2 x 104) were loaded onto electrically charged slides using a cytological centrifuge (Cytospin, Thermo Scientific Inc.) at 134 g for 5 min. The mounted cells were fixed with paraformaldehyde (0.2 %) for 15 min, washed with PBS/glycine (0.1 M), and dried at room temperature. Appropriated negative controls with- out the addition of primary antibody were also stained. The remaining staining protocol was performed using the method as described above. The samples were washed five times with PBS and the coverslips were carefully removed from the wells, washed in ultrapure water, and mounted as described above. All Images and sample analyses were carried out using an Olympus BX51 microscope coupled to an Olympus DP72 digital camera.
RESULTS
Anti-hMC2RL1 Production and Validation
Approximately 4.5 mg anti-hMC2RL1 rabbit polyclonal antibodies was purified from rabbit serum. Both the anti- hMC2RL1 polyclonal antibody and a commercial anti- body (anti-hMC2RN-ter), used as the positive control for MC2R expression, presented similar immunoreactiv- ities for the epidermis and sebaceous glands and, as expected, both were negative when assayed on liver sam- ples. Both antibodies presented specific immunoreactivity to cACC cells and were negative for smooth muscle tis- sue, where MC2R is unexpressed (Fig. 2). No immunore- activity was observed in control samples stained without primary antibody.
Characterization and Validation of hMC2RL1-F-c-AuNP Probes
Figure 3(A) shows TEM image of AuNP and size dis- tribution. Figure 3(B) presents the UV-vis spectra of the utilized colloidal gold (AuNP) at 520 nm (Fig. 3(B-i)). After FITC labeling (Fig. 3(B-iii)) or the addition of anti-hMC2RL1 (Fig. 3(B-iv)) onto the surface of AuNP, the spectrum shifts from 523 nm to 521 and 540 nm, respectively, indicating AuNP functionalization. When the
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AuNPs are totally functionalized with antibodies and flu- orescein and blocked with BSA (hMC2RL1-F-c-AuNP) (Fig. 3(B-v)) the 520 nm spectrum is no longer observed, regressing to the wavelength of 496 nm. DLS measure- ments of the hydrodynamic diameters of nanoparticles have been made before and after conjugation; no sig- nificant changes were observed in the diameters. We therefore agree that nanoparticle aggregation could be disregarded.
The specificity of the hMC2RL1-F-c-AuNP probes as assessed by direct immunofluorescence is shown in Figure 4. The observed immunoreactive cells indicate that the probes recognize the tumor cells but not the liver cells, including at 4 ℃. Different incubation conditions (incubation duration, different temperatures, and different hMC2RL1-F-c-AuNP dilutions) were tested before select- ing the ideal experimental conditions. The observed stain- ing pattern in H295R suggests that hMC2RL1 binds to the cell surface; such staining was not observed in the neg- ative control (Fig. 5). Furthermore, no quenching effects
cACC (+)
Liver (-)
hMC2RL1-F-c-AuNPs
-
of the nanoparticles were observed used in the present study.
DISCUSSION
The search for new diagnostic and therapeutic approaches for ACC was motivated by the high adrenocortical tumor incidence among children in the States of Paraná5, 7,33 and São Paulo.34 Considering the large number of ACC cases among children and adults worldwide, the total population target is substantial. Furthermore, the available therapeu- tic options using pharmaceuticals have remain unchanged in the last 20 years, and their outcomes remain a major concern for advanced stage ACC.7,9-11
MC2R is a transmembrane protein that plays a key role in the synthesis of steroids and the development of the adrenal gland cortex. Its localization in the membrane pro- vides binding sites that can be easily accessed.27,26 Sev- eral antibodies are currently available against this protein; however, these usually recognize epitopes located in the intracellular N-terminal portion. In contrast, the antibody in this study was generated against an epitope in the first outer loop of the protein (Loop 1, L1).
The results obtained from the IHC tests showed that both antibodies recognized ACC cells (Fig. 2) as well as epidermal and sebaceous gland cells (Fig. 4), the primary sites of MC2R expression associated with the synthesis of steroids and with stress response, respectively, corroborat- ing other studies.35,26 MC2RL1 immunoreactivity was also observed in H295R cells (Fig. 5), which exhibit high levels of MC2R expression,36,37 whereas liver and smooth mus- cle tissue samples did not present any specific immunore- activity. The functionalization of AuNPs was analyzed based on surface plasmon resonance, a phenomenon that occurs when AuNPs carry free surface electrons excited by visible light, generating a collective oscillation at wave- lengths near 520 nm. This wavelength is observed in nanoparticles with sizes below 50 nm and changes upon plasmon resonance, e.g., wavelength shifts, can be used for the detection of surface modifications.38-41 In this study,
H295R
hMC2RL1
hMC2RL1-F-c-AuNPs
F-c-AuNPs
the characteristic AuNP resonance (523 nm) observed initially presented an emission band at 521 nm after flu- orescein (FITC) binding, whereas upon anti-hMC2RL1 attachment the band shifted to 540 nm.
Sharma et al.,22 employed the same functionalization method through the cysteamine linker and observed the same plasmon shifts. Raoof et al.,42 upon coupling 10 nm AuNPs to monoclonal antibodies, recorded these changes on the plasmon band at a lower intensity. Marangoni et al.,43 using AuNPs encapsulated in G4 PAMAM den- drimers, found the same offset with lower intensity after
protein (jacalin) and FITC coupling. The different inten- sities observed in plasmon shifts by the various stud- ies can be explained by the ratios between proteins and nanoparticles used in the conjugation processes, as well as the differences in the linkers and AuNPs used. In the present work, 1.5 nm nanoparticles were used to bind approximately 1000 molecules of cysteamine linker, 500 molecules of FITC, five anti- hMC2RL1 antibodies per each AuNP, and finally a blocking solution of 5% BSA. We observed that after the end of hMC2RL1-F-c- AuNP probe synthesis, the 523 nm plasmon AuNP band was not detected, decreasing to 496 nm. This phenomenon might have occurred because the experiment was designed to cover the entire nanoparticle surface. Thus, no free elec- trons might have been available to generate plasmon res- onance, a phenomenon that was not detected by the prior studies.
It was possible to detect strong immunostaining with- out background in cACC cells and skin micro-sections processed using the imunoperoxidase technique. The anti- body did not present any cross-binding causing non- specific staining in the liver, a tissue that is known not to express the MC2R protein (Fig. 4). The specificity was also confirmed in cultured cancer cells, which presented an immunofluorescence signal only in the presence of anti- bodies against MC2R (Fig. 5). Incubation of the probe without the antibody presented a weak fluorescence that 1: Mon, could be attributed to non- specific binding, autofluores- cence of cells, or artifacts associated with the technique. It is worth noting that FITC and antibody binding to AuNP was stable, with a minor degree of false positive bind- ing, suggesting that the observed strong fluorescence is related to the entire AuNP-FITC complex bound to the cells through a specific antibody. These results suggest that it might be possible to bring the AuNPs to the target cells and to utilize them for ACC identification in in vivo imag- ing tests in future studies. This ability would increase the probability of finding small metastases in areas of difficult access, e.g., metastasis in the chest. However, therapeutic radiotherapy, as well as imaging analysis using radiation, for patients carrying a germ line TP53 mutation should be used with caution due to increased risk for radiation- induced second primary tumors.
Several in vitro studies have demonstrated that the func- tionalization of AuNPs with molecules that specifically bind to particular epitopes generates a degree of specificity and consequently a higher accumulation in the cells of interest. Consistent with this hypothesis, Li et al. (2011)38 utilized glucose and folic acid, the receptors of which are overexpressed in certain tumor types. The authors observed that in normal human lung cells there was no accumulation of AuNPs, whereas cervical carcinoma cells showed a large accumulation. Similarly, Marangoni et al. (2013)43 observed that AuNPs functionalized with jacalin, a protein that recognizes a disaccharide present in 85% of
human tumors, recognized leukemic cells whereas normal mononuclear cells were not labeled.
The benefit of nanoparticle functionalization may also be observed during in vivo experiments. Hainfeld et al. (2011),44 for example, used two groups of mice; the first was inoculated with HER2 positive human breast tumor cells, and the second group was inoculated with HER2 negative breast tumor cells. After intravenous injection of AuNPs functionalized with an anti-HER2 antibody, the authors observed by microCT that in animals with HER2 positive tumors more AuNP accumulation was observed than those with HER2 negative tumors. Gao et al. (2004)35 using quantum dots combined with an anti-PSMA mono- clonal antibody and verified by fluorescence that tumors were clearly marked in mice inoculated with prostate can- cer cells, whereas the healthy animals had an insignifi- cant non-specific labeling. The authors concluded that the active labeling of tumors by means of specific molecules such as antibodies is much faster and more efficient than labeling via a passive tag, taking into account the perme- ability, retention capacity, and internalization of the tumor tissue.
CONCLUSION
The anti-MC2R antibodies produced against the segment of the external loop of this receptor (Loop1) showed sat- isfactory results by recognizing ACC cells and not tissue cells with low or no MC2R expression. It was possible to produce and characterize gold nanoparticles functionalized with antibodies and FITC, resulting in probes that had the same level of specificity as the free antibodies used for the recognition of cACC cells in ICC experiments. This combination allowed us to infer that AuNPs are impor- tant not only to facilitate the construction of the targeting probe but also to simultaneously confer great potential as a tracer in future studies of diagnostic imaging in vivo, such as in animal models with human ACC with detection most likely by using CT. Further studies are required to test this delivery device for targeted therapy in vivo.
Acknowledgments: This study was funded by a special grant from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for Nanotechnology (2009). We are grateful to Instituto de Pesquisa Pelé Pequeno Príncipe employees for their support.
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