Breast Cancer DOI 10.1007/s12282-014-0537-z

ORIGINAL ARTICLE

p53 gene therapy of human breast carcinoma: using a transferrin-modified silica nanoparticles Chandrababu Rejeeth • Soundrapandian Kannan

Received: 19 February 2014 / Accepted: 16 April 2014 Ó The Japanese Breast Cancer Society 2014

Abstract Background Nanoparticles have an enormous potential for development in biomedical applications, such as gene or drug delivery. In our study, we examined the efficacy of p53 gene therapy in human breast carcinoma (MCF-7) cells using silica nanoparticles (SiNPs) supplemented with transferrin. Methods MCF-7 cells were exposed to transferrin–SiNPs–p53 in vitro, and the growth inhibition rate, expression of p53 and bax, and induction of apoptosis were measured 48 h later. Results Treatment of MCF-7 cells with transferrin–SiNPs–p53 resulted in 60.7 % growth inhibition. Wild-type p53 expression and an increase in bax expression were observed following transfection with transferrin–SiNPs– p53, and 20.5 % of the treated MCF-7 cells were apoptotic. In vivo, the MCF-7 tumor transplanted into nude mice grew to 5–6 mm in diameter. Following growth of the tumor to this size, transferrin–SiNPs–p53 was locally applied to the peripheral tumor (day 0) and then applied once every 5 days for a total of six times. During the administration period, tumor growth did not occur, and the mean tumor volume on the last day of administration (day 25) was 10.0 % of that in the saline control group. Conclusion These results suggest that p53 gene therapy via transferrin-modified silica nanoparticles is an effective strategy for treatment of breast carcinoma.

C. Rejeeth (&)  S. Kannan Proteomics and Molecular Cell Physiology Lab, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India e-mail: [email protected]

Keywords Silica nanoparticles  Gene delivery  Breast carcinoma  p53  Transferrin (Tf)

Introduction Gene therapy opens a wide variety of new medical application if appropriate gene vectors are made available [1]. It is recognized as one of the most promising approaches for treatment of serious diseases, including monogenic diseases, infectious diseases and cancer [2]. Breast cancer is the second leading cause of cancer death in the world for all female cancers. The conventional therapeutic approaches for hormone refractory breast cancer include chemotherapy [3], radiation therapy [4] and surgery [5] chemotherapy and radiation therapy are supposed to kill many normal cells along with tumor cells. Despite surgical methods that can remove the primary and local sites of breast cancer, relapse at local or distant sites is still possible because of the presence of micro metastasis undetectable at the time of diagnosis [6]. Advances in understanding genetic alterations are associated with breast cancer progression. In this regard, the application of nanotechnology to biomedical research is expected to have a major impact leading to the development of new types of diagnostic and therapeutic tools. The viral gene delivery system shows a high transfection yield but it has many disadvantages, such as oncogenic effects and immunogenicity. Current research focuses on enhancing the targeting and specificity of nonviral vectors and control of transgene expression levels [7–13]. Many systems have been studied in the past, including peptides, liposomes, organic polymeric and dendrimers [14]. Transferrin receptor antibodies have also been used for site-specific drug delivery for various systems, including protein/toxin conjugates, polymer/drug conjugates,

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modified viral vectors, liposomes/polyplexes and nanoparticles (NPs), etc. [15, 16]. Transfection competence of cationic liposomes could be amplified when complexed with targeting ligands, such as transferrin (Tf), employing a receptor-mediated endocytosis apparatus [17]. Level of transferrin receptor (TfR) is established to be superior in various types of cancer cells [18], and it is concurrent with the aggressive or proliferative aptitude of tumor cells [19]. This is a transmembrane glycoprotein involved in iron uptake by internalization of transferrin. TfR exerts growth regulatory functions and is over-expressed in rapidly growing tumors. The expression of TfR is of prognostic significance for several tumor types. Ferrous iron can either be bound to transferrin or to other proteins before uptake [20]. The p53 tumor suppressor gene is the most commonly altered gene in human cancer [21]. It encodes for the transcription factor p53, which plays a central role in regulating cell cycle progression, senescence, differentiation, DNA repair, and apoptosis [22]. In response to DNA damage or other stress signals, p53 activity is up regulated to initiate a cascade of biological events that ultimately results in prevention of tumor development [23]. Mutations in p53 abrogate normal tumor suppressor functions, contributing to the survival and/or proliferation of abnormal cells with poorly differentiated phenotypes [24, 25]. Cancer cells containing mutant p53 are associated with more aggressive disease, increased resistance to chemotherapy and radiation therapy, and poor prognosis [26, 27]. As a result, there is a great interest in therapeutic strategies aimed at restoring the function of p53 for the treatment of cancer [28, 29]. In our previous study, MCF-7 cancer cells treated in vitro with p53 gene-loaded NPs (p53–NPs) demonstrated prolonged p53 gene expression, greater anti proliferative activity, and less toxicity p53 gene-loaded ORMOSIL nanoparticles [30]. The objective of this study was to evaluate NPs as vectors for p53 gene therapy in vivo for tumor inhibition. In this study, we did in vitro and in vivo introduction of a p53 gene into a transferrin receptor—expressing breast carcinoma cell line using a transferrin-modified silica nanoparticles and studied the subsequent growth inhibition and induction of apoptosis.

Materials and methods Silica precursors are surfactant (Aerosol-OT 98 %) and cosurfactant (n-butanol 99.8 %), vinyltriethoxysilane (VTES 97 %), 3-aminopropyltriethoxysilane (APTES 99 %), dimethyl sulfoxide (DMSO 99.5 %). Transferrin (Tf) (Sigma) was used without further purification. All the glasswares (glass bottle and small pieces of glass substrate)

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were cleaned and sonicated in ethanol for 5 min, rinsed with double-distilled water, soaked in a H2O/HNO3 (65 %)/H2O2 (1:1:1, v/v/v) solution, rinsed again with doubly distilled water, and finally dried in air. The pCMV plasmid DNA encodes the pCMV signaling probe, which is a fusion of enhanced p53 gene. The plasmid pCMV contains the lacZ gene under control of the SV40 promoter (Promega, Madison, WI). The p53 expression plasmid pCMVp53 containing wild-type human p53 cDNA under control of a cytomegalovirus promoter was purchased from (Clontech). Xenograft models used in this study, MAS98.06 and MAS98.12, represent luminal-like and basal-like subtypes of ductal carcinoma, respectively. Luminal-like subtype has been charcterised as estrogen receptor positive (ER?) and basal-like subtype as estrogen receptor negative (ER-). Basal-like subtype has demonstrated a worse prognosis than luminal-like subtype. Preparation of the transferrin–SiNPs–DNA complex The nanoparticles were synthesized in the non-polar core of Aerosol-OT/DMSO/water micelles as described in our previous studies [30]. In brief, the micelles were prepared by dissolving 0.44 g Aerosol-OT and 800 lL n-butanol in 20 mL of double-distilled water by vigorous magnetic stirring. One hundred microliters of DMSO was added. After that, 200 lL of neat VTES was added to the micelles system and the resulting solution was stirred for *30 min. Furthermore, the silica nanoparticles were precipitated by adding 10 ll of APTES and stirring for *20 h at room temperature using a magnetic stirrer. Formation of the nanoparticles is indicated by a white blue colour. Surfactant Aerosol-OT and co-surfactant n-butanol were removed by dialyzing the aqueous solution against distilled water in a 12to 14-kD cut-off cellulose membrane for 48 h. The dialyzed solution was filtered through a 0.2-lm cut-off membrane filter and used straight way for further experimentation. The silica nanoparticles were vortexed vigorously for 2 min. Transferrin (ion-saturated holotransferrin; Sigma, St. Louis) was dissolved in HBSS (Invitrogen). Transferrin (25 lg) in 100 lL HBSS and 100 lL silica nanoparticles (100 lmol/L) were mixed in a polypropylene tube and incubated for 15 min at room temperature. Then, OptiMEM (100 lL, Invitrogen) containing 2 lg plasmid DNA was added and gently mixed. After 15-min incubation, DMEM (700 lL) was added to the tube. The final DNA/ SiNPs/transferrin ratio was 1:10:12.5 (lg/nmol/lg). These formulations demonstrate in Fig. 1. Cell culture MCF-7 (Human breast cancer cell line) cells were obtained from National Centre for Cell Science (Pune, India). The

Breast Cancer Fig. 1 Elucidation of cancer targeting mechanisms of transferrin-modified silica nanoparticles. a Normal cell there is no transferrin receptor present in cell surface. b Cancer cell more number of transferrin receptors present on the surface of the cancer cell. Due to the presence transferring receptor conjugated with transferrin ligand and silica nanoparticles complex to deliver the p53 gene in cancer cells

cells were maintained at 37 °C fewer than 5 % CO2 in Dulbecco’s Modified Eagle’s medium (DMEM) high glucose, supplemented with 10 % (v/v) heat-inactivated fetal bovine serum, penicillin (100 U/mL), streptomycin (100 lg/mL) and L-glutamine (4 mM). Particle size and zeta potential measurements Nanoparticles zeta potential was determined by laser diffractometry using a Zetasizer Nano Series (Malvern Instruments) after resuspension of NP in 1 mM HEPES, 1 % (w/v) glucose (pH 7.4). All measurements were performed in triplicate for 60 s at 1,000 Hz and an electric current of 3 mA with zero field correction. In vitro transfection with the transferrin–SiNPs– pCMV–p53 Complex 5

Cells were plated on 24-well plates (1 9 10 per well) 24 h before transfection. After the cells were washed with PBS, transfection medium Dulbecco’s modified Eagle’s medium (DMEM) (1 mL) containing the transferrin–SiNPs– pCMV–p53 complex was added to each MCF-7 cell cultured well for a 5-h incubation period. The transfection medium was then replaced with fresh culture medium containing 10 % fetal bovine serum. After culturing for 48 h, b-galactosidase expression was measured by 5-bromo-4-chloro-3-indolyl-h-D-galactopyranoside staining and b-galactosidase activity. Transfected cells were washed with PBS and fixed with 2 % formaldehyde plus

0.2 % glutaraldehyde for 5 min at room temperature. The fixed cells were washed twice with PBS and exposed to 5-bromo-4-chloro-3-indolyl-h-D-galactopyranoside solution (1 mg/mL 5-bromo-4-chloro-3-indolyl-h-D-galactopyranoside) in PBS containing 5 mmol/L potassium ferricyanide, 5 mmol/L potassium ferrocyanide, and 2 mmol/L MgCl2, Cell and Molecular Technologies, at 37 °C overnight. After washing, cells stained blue were counted in five random fields under a microscope to determine the percentage of blue cells per 200 cells. H-Galactosidase activity was measured with an h-Galactosidase Enzyme Assay System (Promega) according to the manufacturer’s protocol.

Cytotoxic assay Cells were seeded on 96-well plates at a density of 1 9 104 cells per well. After culturing for 24 h, the cells were treated with five different agents (100 lL) as follows: (a) DMEM only (as the control), (b) transferrin–SiNPs, (c) pCMV–p53 alone, (d) SiNPs–pCMV–p53 complex and (e) transferrin–SiNPs–pCMV–p53 complex. After culturing for 5 h, the cells were washed with PBS and the medium was replaced by DMEM supplemented with 10 % fetal bovine serum. After culturing for another 48 h, the number of viable cells was measured by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. The results for each agent were compared with those for the control.

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Treatment of tumor xenografts in nude mice The tumor diameter reached 5–6 mm in all mice by 7–9 days after MCF-7 cell transfer. Tumor growth was monitored by measuring two perpendicular tumor diameters with an electronic digital caliper. The animals were divided into four groups with five mice per group, and each group was treated with one of the following: (a) saline, (b) pCMV–p53 alone, (c) SiNPs–pCMV–p53 complex, (d) transferrin–SiNPs–pCMV–p53 complex. The transferrin–SiNPs–pCMV–p53 complex contained 125 lg transferrin, 100 nmol SiNPs, and 10 lg pCMV–p53. Each agent (100 lL) was injected along the tumor margin using a 27-gauge needle. Different sites were used for each injection such that the injection points formed a circle that surrounded the tumor. The initial day of administration was defined as day 0. Administration was then repeated five times at 5-day intervals such that day 25 was the final day of administration. The tumor volume was measured during the administration period and for 25 further days after day 25.

Western blotting Cells were washed twice with cold PBS and lysed at 4 °C for 30 min in buffer (100 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 % Triton X-100, 0.1 % SDS, 0.5 % sodium deoxycholate, 20 lg/mL aprotinin, 20 lg/mL leupeptin). Insoluble material was removed by centrifugation at 4 °C for 30 min at 16,000g, and protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL). The protein contents of the cell lysates were adjusted to 4 mg/mL. The cell lysates were mixed by boiling in SDS sample buffer with reducing agents and applied to 10–15 % SDS–polyacrylamide gradient gels. The proteins were then electrophoretically transferred to polyvinylidene difluoride membranes using the Phast System. Blotted membranes were blocked with 10 % dried milk in TBS plus 0.1 % Tween 20 for 1 h at room temperature and incubated for 1 h with antibodies specific for p53, bax (mouse monoclonal anti-human antibodies, Oncogene Research Products, Boston, MA), or transferrin receptor (mouse monoclonal anti-human antibodies, Zymed Laboratories, Inc., South San Francisco, CA) diluted 1:1,000 in TBS plus 0.1 % Tween 20. After incubation, the membranes were extensively washed in TBS plus 0.1 % Tween 20 and incubated with anti-mouse IgG horseradish peroxidase—conjugated secondary antibody diluted 1:10,000 in TBS plus 0.1 % Tween 20. Band detection was carried out using an enhanced chemiluminescence system (Amersham Biosciences).

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Terminal deoxynucleotidyl transferase: mediated dUTP nick end labeling assay Apoptosis-related DNA fragmentation was detected by terminal deoxynucleotidyl transferase—mediated dUTP nick end labeling (TUNEL) using an Apop-Tag Plus Peroxidase In situ Apoptosis Detection Kit (Intergen, Oxford, United Kingdom). Cells were seeded in Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) at a density of 1 9 104 cells per well. After culturing for 24 h, the cells were treated with one of the above agents (100 lL) for 5 h. Another 48 h later, the cells were fixed by treatment with 1 % paraformaldehyde for 10 min. The fixed cells were washed twice with PBS and preserved in precooled ethanol plus acetic acid (2:1, v/v) for 5 min. After washing twice with PBS, an equilibration buffer was added to the cells. Subsequently, the cells were incubated at 37 °C for 1 h with working strength terminal deoxynucleotidyl transferase enzyme that contained digoxigeninlabeled dUTP. The reaction was stopped by addition of prewar med stop/wash buffer. After washing twice with PBS, an anti-digoxigenin antibody fragment carrying a conjugated peroxidase was added and the mixture was incubated in a humidified chamber for 30 min at room temperature. The peroxidase enzyme location in the cells was detected with 3, 30 -diaminobenzidine, which is a substrate for the enzyme. Histology and immunohistochemistry A portion of tissue samples extracted from nude mice tumors were fixed with a 10 % per solution of buffered formalin for 24 h (4 °C). After dehydration in graded alcohol solutions, the tissues were embedded in paraffin and sectioned (4 mm). The sections were stained with hematoxylin and eosin (HE) for histological observation with an optical microscope (magnification, 2009). Protein expression in section was examined by immunohistochemical (IHC) methods. Briefly, sections of formalin fixed, paraffin-embedded tissue were cut and subjected to de-paraffinization through a series of hydrations. Then sections were placed inside a plastic pressure cooker in a microwave oven for antigen retrieval procedure. The endogenous peroxidase activity of section was blocked by 3 % hydrogen peroxide followed by two washes in PBS. The sections were incubated with primary antibodies for 24 h at 4 °C. After 3 washes in PBS for 10 min, the sections were incubated in biotinylated IgG in PBS for 30 min at room temperature and washed 3 times in PBS. Then sections were added streptavidin biotin–peroxidase complex (SABC) for 20 min at room temperature and washed for 3 times. 3, 30 -diaminobenzidine (DAB) was used to detect the protein expression. The positive expression in

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section was expressed as percentage of the control (as 100 %). Statistical analysis The entire experiment was done in triplicate and the data were analyzed using Student’s t test. Ps \ 0.05 were considered statistically significant.

Results Physicochemical characterization of nanoparticles: particle size and zeta potential The synthesized nanoparticles were observed to be highly monodispersed, stable in aqueous suspension and also surface-functionalized with amino groups for binding of DNA. Such characterization was provided in the former articles presenting the preparation of the nanoparticles [30]. Since the same protocol has been rigorously followed, it did not appear as mandatory to the same data. These particles were characterized for physical properties, such as particle size and surface charge. We have also studied the surface charge of these nanoparticles by measuring their fpotential values. It can be seen from (Fig. 2) that the overall surface charge of the nanoparticles is dependent on the functional group present on their surface, being positive for NH2. To demonstrate ‘‘active targeting’’ of these nanoparticles to cells in culture, we have conjugated biotargeting molecules Transferrin (Tf). The conjugation was confirmed by agarose gel electrophoresis and measuring the f-potential values of the resulting nanoparticles (Fig. 3). The f-Potential measurements revealed that the net anionic charge on the nanoparticles is reduced as a result of bioconjugation, which

Fig. 3 Agarose gel electrophoresis showing (lane A) NPs alone (?5.53 mV), (lane B) p53 alone (-7.34 mV), (lane C) NPs/p53 (-8.36 mV) and (lane D) NPs/p53/Tf (-12.8 mV). Numerical values denote the corresponding f-potential

is attributed to the partial shielding of their surface carboxyl groups by the bioconjugated proteins. The bioconjugation is also evident from the reduced mobility of the bioconjugated nanoparticles toward the positive terminal through the agarose gel (lanes A–D), as opposed to the non-conjugated one (lane A). Transfection efficiency with the transferrin–SiNPs– pCMV–p53 complex Transfection efficiency using the transferrin–SiNPs was evaluated by measurement of the h-galactosidase activity and 5-bromo-4-chloro-3-indolyl-h-D-galactopyranoside staining. The h-galactosidase activity in MCF-7 cells transfected with the SiNPs–pCMV–p53 complex was 38.3 ± 12.0 mU/mg proteins, whereas that following transfection with the transferrin–SiNPs–pCMV–p53 complex was 171.0 ± 14.1 mU/mg proteins. H-Galactosidase activities of cells transfected with medium alone, transferrin–SiNPs alone, and pCMV–p53 alone were all below the detection limit. In the 5-bromo-4-chloro-3-indolyl-h-Dgalactopyranoside staining assay, 23.0 ± 4.1 % of cells treated with SiNPs–pCMV–p53 and 42.0 ± 6.0 % of cells treated with transferrin–SiNPs–pCMV–p53 were stained blue. Cytotoxic effects

Fig. 2 F-potential values of NPs alone (?5.53 mV), p53 alone (-7.34 mV), NPs/p53 (-8.36 mV) and NPs/p53/Tf (-12.8 mV)

The numbers of viable MCF-7 cells relative to the control in SiNPs–p53-treated cells and transferrin–SiNPa–p53treated cells were significantly lower than those found after other treatments (P \ 0.05). Growth inhibition rates of 45.6 and 61.5 % were observed for cells treated with SiNPs–p53 and transferrin–SiNPs–p53, respectively, and the growth of transferrin–SiNPs–p53-treated cells was significantly inhibited compared with that of SiNPs–p53treated cells (P \ 0.05; Fig. 4). In MCF-7 cells, each treatment had little effect on cell survival [31].

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Day Fig. 4 Viability of MCF-7 cells treated with transferrin–Silica NPs, p53 plasmid alone, Silica NPs–p53 and transferrin–Silica NPs–p53. The number of viable cells, relative to the control, was measured by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. P \ 0.05, growth of Silica NPs–p53–treated or transferrin–Silica NPs–p53-treated cells was significantly inhibited compared with the growth of cells that underwent other treatments. P \ 0.05, growth inhibition of transferrin–Silica NPs–p53-treated cells was significantly different to that of Silica NPs–p53–treated cells

Fig. 6 Tumor volume analysis of tumor-bearing mice after intratumoral injection of various agents. Each group included five mice. Administration was started on day 0 and repeated on days 5, 10, 15, 20, and 25, for a total of six times. Tumor volume was measured once every 5 d from the day of the last administration (day 25) until day 50. Tumor volume in transferrin–SiNPs–p53-treated animals on day 25 was significantly decreased compared with that on day 0 (P \ 0.05), but the tumor volumes in animals receiving SiNPs–p53 treatment (P \ 0.05) and in other treated groups (P \ 0.01) were significantly increased compared with day 0. On day 50, the tumor volume in transferrin–SiNPs–p53-treated animals was significantly suppressed compared with other treated groups (P \ 0.05)

Growth inhibitory effect of transferrin–SiNPs–p53 on tumors transplanted into nude mice

Fig. 5 Western blots of p53 and bax in MCF-7 cells. Expression of p53 and bax in each treatment group 48 h after completion of transfection. Lane 1, control; lane 2, p53 plasmid alone; lane 3, SiNPs–p53; lane 4, transferrin–SiNPs–p53. Arrow a, exogenous wildtype p53; arrow b, endogenous p53. Experiments were repeated in triplicate and gave similar results. b-actin was used as a loading control

Expression of p53 and bax As shown in Fig. 5, expression of exogenous wild-type p53 was detected in SiNPs–p53-treated cells (lane 3) and transferrin–SiNPs–p53-treated cells (lane 4) but not in cells that underwent other treatments. Furthermore, the expression of p53 following transferrin–SiNPs–p53 treatment was slightly higher than that following SiNPs–p53 treatment. On the other hand, elevated expression of endogenous mutated p53 was observed in transferrin–SiNPs–p53-treated cells compared with that in the control group. We also evaluated the expression of bax, which is a target gene in the p53-dependent apoptosis pathway. Bax expression increased distinctly in cells treated with transferrin–SiNPs– p53, but little change was observed in the SiNPs–p53treated cells.

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On day 0, each group of mice had an average tumor volume in the range 60–70 mm3. During the administration period, the tumors in the saline group continued to grow and had increased by 5.9 times to an average of 418.7 mm3 on day 25. Tumor growth was also seen in the SiNPs–p53 group during the administration period, with an increase in tumor volume of 1.6 times to an average of 118.0 mm3 on day 25; this, nonetheless, reflected relative growth inhibition compared with the saline group. In contrast, during administration of transferrin–SiNPs–p53, the tumor gradually reduced in size, with the tumor volume significantly decreased by 36.6 % from an average of 66.1 mm3 on day 0 to 41.9 mm3 on day 25. Continued observation from completion of administration (day 25) until day 50 revealed an even steeper tumor growth curve in the saline group and a growth rate in the SiNPs–p53 group comparable with that in the saline group, with the tumor volume on day 50 reaching 2,218.9 and 926.1 mm3 in the saline and SiNPs– p53 groups, respectively. In contrast, the tumors in the transferrin–SiNPs–p53 group only began to grow after day 35 and at a considerably slower rate compared with the other groups, with the volume on day 50 being no [186.4 mm3, 8.4 % of that in the saline group (Fig. 6).

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Fig. 7 Apoptotic cells were detected by the TUNEL assay (3, 3 Vdiaminobenzidine, brown) in MCF-7 cells: a control, b SiNPs–p53, and c transferrin–SiNPs–p53. TUNEL-positive cells were counted in five random fields under a microscope to determine the percentage of apoptotic cells per 200 cells. Percentages of apoptotic cells (apoptotic

index) were 3.6 ± 0.42 %, 12.2 ± 1.8 %, and 19.7 ± 1.2 % in the control, SiNPs–p53, and transferrin–SiNPs–p53 groups, respectively. Apoptotic index of transferrin–liposome–p53 group was significantly different from that of control (P \ 0.01) and SiNPs–p53 (P \ 0.05) groups. Original magnification, 9200

Fig. 8 TUNEL assay of tissue sections from tumors in the saline group (a), SiNPs–p53 group (b), and transferrin–SiNPs–p53 group (c). Tumors were extirpated 48 h after completion of administration (day 27). In saline-treated tumors, very few apoptotic cells were

found. In transferrin–SiNPs–p53-treated tumors, many apoptotic cells were detected at the periphery of the tumor. Original magnification, 9100

TUNEL assay

differences in TUNEL-positive cells were noted between these groups and the saline group (Fig. 8).

In vitro, TUNEL-positive cells that stained brown were found in each treatment group. The percentage of TUNELpositive cells in a field, which is called the apoptotic index, was 3.6 ± 0.42 % in the control but was markedly increased to 12.2 ± 1.8 % in SiNPs–p53-treated cells and 19.7 ± 1.2 % in transferrin–SiNPs–p53-treated cells (Fig. 7). In vivo, the tumor was extirpated 48 h after completion of the final administration (day 27) and observed by the TUNEL assay. TUNEL-positive cells were sparse in the physiologic saline group but were particularly prevalent in the periphery of the tumor in the SiNPs–p53 or transferrin–SiNPs–p53 groups in proximity to the local injection site. For more central regions of the tumor, no

Immunohistochemistry assay Immunohistochemical studies of p53 and Bax were performed. The results showed significant variant expressions between p53 and Bax in each experiment group (Fig. 9). Protein expression qualitatively, a greater number of positive cells were observed in tumors treated with transferrin–SiNPs–p53 complex compare to the other treated groups. These results indicate that transferrin–SiNPs–p53 greatly induces the apoptosis of the tumor tissue cells in vivo.

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Fig. 9 Immunohistochemical analysis of tissue sections from tumors in the saline group (a), SiNPs–p53 group (b), and transferrin–SiNPs–p53 group (c). Values represented were means SD of triplicates. *P \ 0.05 vs. control. **P \ 0.01 vs. control

Discussion Gene therapy holds great promise for the treatment of cancer through the delivery of genes that suppress and/or reverse tumor growth and disease progression. However, this strategy requires the development of safe, stable gene delivery systems that, ideally, can be delivered systemically to target tumor cells. We report on transferrin-modified silica nanoparticles gene vector for delivery of a prominent tumor suppressor gene, p53. We demonstrated that this vector is nontoxic and can be delivered locally or systemically to induce functional expression of p53 in tumor cells, resulting in growth inhibition of breast carcinoma with p53 mutations. Our previous studies have shown that NPs act as an intracellular depot, resulting in sustained DNA delivery and gene expression [30, 32]. Due to transferrin binding to the transferrin receptor, the efficacy of gene transfer with a transferrin-modified SiNPs increased by 3.7 times and the growth inhibition effect increased by 1.4 times compared with use of the unmodified SiNPs. The expression of exogenous wild-type p53 was higher following transfection with transferrin–SiNPs–p53 compared with transfection with unmodified SiNPs–p53. Therefore, the increase in the efficacy of p53 gene transfer and the subsequent increase in p53 expression seemed to have an effect on enhancement of

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MCF-7 growth inhibition. The results of TUNEL staining suggested that this growth inhibition effect is produced by induction of apoptosis through p53. Cells are directed to cell cycle termination or apoptosis by increased p53 expression, and the transcriptional activity of the bax gene is directly associated with the p53-dependent apoptosis pathway [33]. Many such studies have used viral vectors, and growth inhibition in vitro has been reported to be *60 % [34]. In the current work, we obtained a growth inhibition rate for MCF-7 cells of 60.7 % following transfection with transferrin–SiNPs–p53. Nonspecific growth inhibition by viral infection may be a consequence of gene therapy using viral vectors, but the nonspecific growth inhibition of MCF-7 cells caused by transfection with transferrin–SiNPs was only *10 % (Fig. 3). Furthermore, transferrin–SiNPs alone showed no cytotoxicity against normal gingivalderived fibroblasts. The in vivo antitumor effect of transferrin–SiNPs–p53 on the MCF-7 tumor was considerably higher than expected based on the in vitro results. Treatment with SiNPs–p53 inhibited tumor growth during the administration period but allowed tumor growth at a rate similar to that in the saline group after completion of administration, whereas growth after completion of administration of transferrin–SiNPs–p53 was relatively low. In the

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transferrin–SiNPs–p53 group, the tumor volumes on days 25 and 50 were 10.0 and 8.4 % of those in the saline group, respectively, showing a growth inhibitory effect higher than the *60 % growth inhibition observed in vitro. This difference is attributed to a single-dose administration in vitro compared with administration once every 5 days (a total of six doses) in vivo. In contrast, in vivo TUNEL images showed apoptotic cells only in limited regions of the tumor, such as those proximal to the local injection site, suggesting that the frequency of apoptosis induction is not necessarily correlated with growth inhibition. It has been reported that p53 gene therapy inhibits tumor vascularization, thereby exerting a far reaching effect even on nontransfected cells the so-called by stander effect [35]. Hence, these findings suggest that the in vivo antitumor effect of transferrin–SiNPs–p53 may involve not only apoptosis induction but also by stander effects, including inhibition of tumor vascularization. Further studies will be required to investigate this possibility. In addition to modification of SiNPs through addition of transferrin, the efficacy of gene transfer with SiNPs has been enhanced using ligands, such as epidermal growth factor, insulin, and lectin [36]. Efficacy of gene transfer is reportedly increased by 2–22 times by transferrin-modified silica nanoparticles, 10–23 times with epidermal growth factor, 3–18 times with insulin, and 5–28 times with lectin compared with SiNPs alone. These enhancement effects vary in different target cells, and one reason may be that expression of the receptor for each ligand varies depending on the cell type [37]. In the current study, enhancement of the efficacy of gene transfer occurred at 4.2 times the level of that with SiNPs alone, perhaps because transferrin receptor expression in MCF-7 cells is lower than in tumor cells used in other studies, although it is higher than in normal cells. The greatest benefit of the transferrin–SiNPs system is that it can be formulated by simply mixing each component with the plasmid DNA. First, the slightly negatively charged transferrin and the cationic SiNPs are electrostatically associated, and no chemical modification is necessary. The formulation can then be made by introduction of the negatively charged plasmid DNA, which will bind to the cationic liposome. In addition, it seems that the transferrin not only increases the uptake of DNA into cells but also plays an indirect role in enhancement of gene expression after uptake of the transferrin–SiNPs–DNA complex. The transferrin receptor is bound to the transferrin–SiNPs–DNA complex within cells, and transferrin may facilitate the escape of DNA from the endosome, because endosome escape is a normal physiologic process for transferrin and its receptor complex, thus making more DNA available for gene expression [17]. Binding of negatively charged transferrin to a cationic SiNPs reduces the

overall charge of the assembly compared with the cationic liposome alone, and most of the complexes formed between this assembly and plasmid DNA are of neutral charge [38]. Therefore, it is possible that a transferrinmodified cationic SiNPs might have a smaller amount of encapsulated plasmid DNA compared with a cationic SiNPs alone and that the transferrin–SiNPs–DNA complex might be less adhesive to the surface of cells. However, [39] have reported that the amount of plasmid DNA bound to a transferrin–SiNPs is similar to that bound to an unmodified SiNPs. Regarding cell adhesiveness, it is thought that the transferrin–SiNPs–DNA complex is compressed to less than a third of an unmodified SiNPs–DNA complex, making it more susceptible to endocytosis and overcoming the effects of the reduced charge [38]. The sensitivity of carcinomas to anticancer agents [34] and radio sensitivity [40] is increased by p53 gene therapy, so the effect of the therapy described in this study is likely to be further increased by combination with anticancer agents or radiotherapy. Regarding the transferrin–SiNPs– p53 administration schedule, administration once every 5 days inhibited tumor growth during the administration period (Fig. 6) and until 10 days after completion of administration, suggesting that long-term maintenance administration about once weekly may stop tumor growth; that is, it may induce ‘‘tumor dormancy.’’ Hence, a future challenge will be to investigate the antitumor effects of long-term administration of transferrin–SiNPs–p53 as well as to examine survival and safety in tumor-bearing mice.

Conclusions We have developed a non-viral, cationic NP-based gene vector that is effective in sustaining p53 expression in tumors and may be beneficial in the treatment of cancer. Transferrin–SiNPs–p53 complex methods and results of this study provide a foundation for further optimization of a promising gene therapy system. Acknowledgments The Authors are very much thankful to all faculty members of the Department of Zoology, Bharathiar University for their constant encouragement and timely help. This work was financially supported by RFSMS G2/6966/UGC NON-SAP New Delhi, Govt. of India. Conflict of interest

Authors no interest of conflict.

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p53 gene therapy of human breast carcinoma: using a transferrin-modified silica nanoparticles.

Nanoparticles have an enormous potential for development in biomedical applications, such as gene or drug delivery. In our study, we examined the effi...
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