Functionalized Silica Nanoparticles As an Alternative Platform for

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Functionalized silica nanoparticles as an alternative platform for targeted drug-delivery of water insoluble drugs Luciane Franca de Oliveira, Karim Bouchmella, Kaliandra de Almeida Gonçalves, Jefferson Bettini, Jörg Kobarg, and Mateus Borba Cardoso Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00214 • Publication Date (Web): 01 Mar 2016 Downloaded from http://pubs.acs.org on March 3, 2016

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Functionalized silica nanoparticles as an alternative platform for targeted drug-delivery of water insoluble drugs

Luciane França de Oliveira1, Karim Bouchmella1, Kaliandra de Almeida Gonçalves2, Jefferson Bettini,3 Jörg Kobarg,4 and Mateus Borba Cardoso1*

1

Laboratório Nacional de Luz Síncrotron (LNLS), CEP 13083-970, Caixa Postal 6192, Campinas, SP, Brazil. 2

Laboratório Nacional de Biociências (LNBio), CEP 13083-970, Caixa Postal 6192, Campinas, SP, Brazil. 3

Laboratório Nacional de Nanotecnologia (LNNano), CEP 13083-970, Caixa Postal 6192, Campinas, SP, Brazil. 4

Faculdade de Ciências Farmacêuticas e Departamento de Bioquímica e Biologia Tecidual - Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CEP 13083-970, Caixa Postal 6154, Campinas, SP, Brazil.

* Corresponding author (M.B.C.) E-mail: [email protected] Fax: +55 19 3512 1004 Tel: +55 19 3512 1045 1

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Abstract The selective action of drugs in tumor cells is a major problem in cancer therapy. Most chemotherapy drugs act non-specifically and damage both cancer and healthy cells causing various side effects. In this study, the preparation of a selective drug delivery system, which is able to act as carrier for hydrophobic and anticancer drugs is reported. Amino-functionalized silica nanoparticles loaded with curcumin were successfully synthesized via sol-gel approach and duly characterized. Thereafter, the targeting ligand, folate, was covalently attached to amino groups of nanoparticle surface through amide bond formation. The cytotoxic effect of nanoparticles on prostate cancer cells line was evaluated and compared to normal cells line (prostate epithelial cell). Cytotoxicity experiments demonstrated that folate-functionalized nanoparticles were significantly cytotoxic to tumor cells whereas normal cells were much less affected by the presence of these structures.

Keywords: silica nanoparticles; functionalization; insoluble drug load; tumor specific cytotoxicity

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1. Introduction Currently, chemotherapy is one of the key methods for the treatment of cancer. Most of the chemotherapeutic drugs are cytotoxic since they act harming the cellular mitosis. Thereby, they can cause damage to both tumor and normal cells, resulting in several side effects in the organism.1 Furthermore, chemotherapy drugs may not kill all cancer cells and the surviving cells may become resistant to the drug.2,3 It is therefore necessary to design new systems for the treatment of cancer which achieve specific drug accumulation at the tumor site and an increased efficacy with reduced side effects on healthy tissues and organs. In order to enhance the therapeutic effectiveness of the chemotherapeutics, the controlled delivery of anticancer drugs using nanoparticles (NPs) is under substantial research efforts.4–9 Nowadays, there are nanoparticles commercially available (Doxil and Abraxane)10–12 approved by the FDA which have shown to reduce the side effects if compared to the conventional chemotherapy without NPs. Decreasing the nanoparticle size may improve deep penetration and local accumulation of the drug in tumor tissue through the enhanced permeation and retention (EPR) effect.13–16 The optimal size of the nanoparticles to enhance the EPR effect is below 100 nm.17 However, a drug delivery system cannot be only based on the EPR effect since the presence of biological barriers hinders the effective penetration of NPs or drugs into tumors. One strategy used to increase tumor uptake is through nanoparticle surface functionalization with cancer-specific ligands (e.g., peptides,18–20 antibodies21,22 and folate23–26), which are selectively recognized by receptors over-expressed on the cancer cells surface increasing the drug delivery into tumor and their therapeutic success. Curcumin (diferuloylmethane - CCM), a yellow pigment obtained from the rhizomes of Curcuma longa, is a hydrophobic polyphenolic small molecule that has

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been proposed to have anticancer properties based on data from clinical evaluations.27–29 However, its clinical use is very restricted due to its low solubility, rapid degradation, low bioavailability, high metabolism rate as well as rapid systemic elimination.30 Thus, the CCM incorporation into drug delivery systems is necessary to allow its therapeutic application. Silica nanoparticles have been widely used for biological and medical applications.31–33 They can be used as nanocarriers for targeting drug delivery while avoiding drug resistance in cancer cells. One of the advantages of the use of silica is based on its easy surface tailorability which can be functionalized with different organic groups34–41 as cancer-specific ligands on the nanoparticles’ surface. Herein, we designed and synthesized a silica drug delivery system to act as a suitable carrier for hydrophobic drugs such as curcumin. The curcumin molecule was encapsulated inside of the amino-functionalized silica nanoparticles through a facile one-pot sol-gel synthesis method. The size of the nanoparticle was adjusted to around 70 nm to facilitate preferential accumulation in tumor sites by the EPR effect. Furthermore, to achieve tumor-selective targeting and reduce cytotoxicity to healthy cells, the nanoparticles were functionalized with folate groups through an amidation reaction. Folate receptors are over-expressed on the surface of human tumor cells42–47 and folate-conjugated formulations can be selectively recognized and trapped by these receptors.48–50 The cytotoxic effect of nanoparticles was evaluated on PC3 prostate cancer cells line and compared to the effect on PrEC normal cells line (prostate epithelial cell).

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2. Results and discussion 2.1. Synthesis and characterization of functionalized nanoparticles The monodisperse silica nanoparticles loaded with curcumin (CCM/SiO2-NH2) were generated by an ammonia-catalyzed hydrolysis of TEOS and then aminofunctionalized through a reaction with (3-aminopropyl)-triethoxysilane (APTES). Nanoparticles size was adjusted based on the amount of curcumin, TEOS and ammonia present in the reaction medium. This synthesis protocol is similar to the modified Stöber method recently reported by our group.51,52 Folate-functionalized nanoparticles were obtained through the surface modification of CCM/SiO2-NH2 using carbodiimide as coupling reagents. The use of water soluble carbodiimides (EDC) with Nhydroxysuccinimide (NHS) presents important advantages since the by-products of this reaction are water-soluble and can be easily removed from the system providing a stable and non-cytotoxic material. The carboxyl groups of folic acid were activated with EDC in the presence of NHS and reacted with the amino groups of CCM/SiO2-NH2 to form stable amide bonds. Then, folate-functionalized silica nanoparticles loaded with curcumin were obtained (CCM/SiO2-FO). The overall synthesis strategy steps are schematically presented in Figure 1.

Figure 1. Schematic representation of the synthesis steps for obtaining folate-functionalized silica nanoparticles loaded with curcumin. Curcumin is represented as orange spheres while folate as green spheres. This scheme is merely illustrative and is not in proportion to the actual size of the system.

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Nanoparticles were also synthesized in the absence of curcumin for comparison. These samples were named as SiO2-NH2 and SiO2-FO for the silica functionalized with amino and folate groups, respectively. It was possible to observe a color change of the amino-functionalized silica (SiO2-NH2, white solid) if compared to the sample in the presence of curcumin (CMC/SiO2-NH2, orange solid - Figure S1 in the Supporting Information). Silica nanoparticles were investigated by transmission electron microscopy (TEM). Figure 2 shows the typical morphology for CCM/SiO2-NH2 and CCM/SiO2-FO where almost spherical structures and particles size in the nanometer scale are seen. Comparison between these TEM images indicates that the functionalization protocol does not induce nanoparticles aggregation. All other particles presented similar structures and sizes and are omitted here for simplicity. TEM images of SiO2-NH2 and SiO2-FO are presented in Figure S2 (Supporting Information).

Figure 2. Transmission electron microscopy images of (A) CCM/SiO2-NH2 and (B) CCM/SiO2-FO.

Complementarily to TEM, silica nanoparticles size distribution was evaluated by small-angle X-ray scattering (SAXS). SAXS is a powerful technique to investigate nanoparticles since it allows their characterization in solution and avoids any possible

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sample aggregation due to the drying process. Figure 3A shows the SAXS curve for CCM/SiO2-FO sample and its corresponding fit.

Figure 3. (A) SAXS profile (open balls) of CCM/SiO2-FO as-synthesized nanoparticles. Spherical fit is shown as a solid red line. (B) Particle size distribution obtained from the fit shown in panel A.

It can be seen that the spherical model reasonably adjusts the experimental curve throughout its extension (Figure 3A). The subtle fit deviations in the low-q and oscillation regions are likely originated from a possible weak spatial interparticle correlation which is not able to affect our size distribution. Through the SAXS fit is possible to obtain the particle size distribution, which is shown in Figure 3B. For CCM/SiO2-FO, a distribution centered at 69 nm is seen. Due to the similarity, size distributions of SiO2-NH2, SiO2-FO, CCM/SiO2-NH2 are presented in Figure S3 (Supporting Information) while the average diameters together with the half-width at half-maximums (HWHM) of the distributions are presented in Table 1. Possible cytotoxic effects arising from the size variations were avoided since all synthesized systems exhibited similar sizes.

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Table 1. Average diameter and HWHM obtained by SAXS (aqueous suspensions) and zeta potential determined in a solution containing 40 mM KCl for the synthesized nanoparticles. Average diameter HWHM obtained by Zeta Potential Sample obtained by SAXS (nm) SAXS (nm) (mV) SiO2

52.6

9.3

-31.3 ± 0.9

SiO2-NH2

66.4

17.4

37.1 ± 1.1

CCM/SiO2-NH2

70.8

12.6

27.6 ± 1.2

SiO2-FO

61.1

16.4

-26.3 ± 1.0

CCM/SiO2-FO

69.0

13.1

14.5 ± 0.9

Textural properties of CCM/SiO2-FO were investigated by high-resolution transmission electron microscopy (HR-TEM) and nitrogen absorption/desorption techniques. Figure 4A shows an HR-TEM image of a cross-sectioned CCM/SiO2-FO sample where details of the specimen can be seen. Figure 4B is an amplification of the white demarked region in Figure 4A and the contrast intensity variation indicates that CCM/SiO2-FO sample is indubitably composed by silica and voids (pores). Due to the dark contrast on the sample edge, round shapes of about 1.7 nm are attributed to closely packed elementary silica particles (indicated by the white arrows) that stick together to form the overall nanoparticle structure. The size distribution of these elementary silica structures is presented in Figure S4 (Supporting Information). This result agrees well with the literature51,53 and with nitrogen adsorption-desorption results presented below. Consequently, CCM molecules are likely located in the pore space between neighbor elementary silica particles.

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Figure 4. HR-TEM of CCM/SiO2-FO at (A) low and (B) high magnifications. Inset white frame corresponds to enlarged region presented in B. The arrows indicate the presence of elementary silica particles. (C) Nitrogen adsorption-desorption isotherm of the CCM/SiO2-FO.

Nitrogen adsorption-desorption isotherm of the CCM/SiO2-FO material is shown in Figure 4C. The sample presents a type II isotherm with an H1 hysteresis loop that is often obtained for materials consisting of agglomerates or compacts of approximately uniform spheres.51 This result reinforces our HR-TEM interpretation where we suggest that our sample is formed by elementary silica spheres. The specific surface area calculated by the BET method was 30 m2 g-1 and is relatively small if compared to template assisted reactions.32,54 t-Plot analysis indicated that most of the specific surface area (90 %) was related to the sample mesopores (the specific micropore surface area was 3 m2 g−1). This result agrees well with the literature that describes Stöber silica particles containing microporous and mesoporous compartments in their structure.55 The quantification of CCM, amino and folate groups as well as the total weight loss for each obtained sample was determined by thermo-gravimetric analysis (TGA). Figure S5 (Supporting Information) shows the TGA curves of the synthesized samples while the total weight loss for all samples (after heating to 800 ºC in air at a rate of 5 ºC min-1) is reported as TGA weight loss in Table S1 (Supporting Information). All organic units were thermally stable up to 150 ºC and the quantification of the relative amounts of the loaded guest molecules was carried out between 150 and 800 ºC. When CCM is loaded in functionalized silica nanoparticles, the weight loss of the organic part 9


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in the hybrid framework occurs simultaneously with the drug molecules. Therefore, the CCM quantification is more complex than in conventional silica hosts and the quantification protocol used here is fully described in the Supporting Information. The SiO2 sample without any template or organic units was used as reference and showed a weight loss of 4.4 %. Weight loss of about 6 % was already reported for mesoporous silica preparation in the absence of template56 and can be attributed to unreacted ethoxy groups from TEOS. The total amount of amino (SiO2-NH2 and CCM/SiO2-NH2) and folate (SiO2-FO and CCM/SiO2-FO) were about 3 and 24 %, respectively. Thus, the CCM loading yields were 3.6 and 3.8 % for CCM/SiO2-NH2 and CCM/SiO2-FO, respectively. The curcumin-loaded nanoparticles were characterized by FTIR to evaluate the amino functionalization success as well as the folate chemical binding. The FTIR spectra of the SiO2-NH2, CMC/SiO2-NH2 and CCM/SiO2-FO are presented in Figure 5.

Figure 5. FTIR spectra of SiO2-NH2 (solid red line), CCM/SiO2-NH2 (solid blue line) and CCM/SiO2-FO (solid black line).

FTIR spectrum of SiO2-NH2 shows typical absorption bands of silica57 and the presence of amino groups was confirmed by the absorptions at 2918 cm-1, 2850 cm-1

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and 1473 cm-1 (an amplification of this region is presented in the Supporting Information – Figure S6A). These bands are usually assigned to C―H stretching and bending vibrations from the aminopropyl groups. In addition, the weak absorption at 1542 cm-1 was attributed to the typical bending vibration of NH2. For CMC/SiO2-NH2, the characteristic absorption peaks of curcumin were not observed (solid blue line). This result is expected since the amount of curcumin entrapped within the pores compared to silica is relatively small as already discussed. For CCM/SiO2-FO (solid black line), some characteristic absorption bands of folic acid can be observed (spectrum of pure folic acid is presented in the Supporting Information – Figure S6B). The bands at 2970 cm-1, 2944 cm-1 and 2866 cm-1 are attributed to –CH symmetric and asymmetric stretching vibrations. The absorption band at 1642 cm-1 corresponds to the C=O bond stretching vibration of –CONH2 group, while the bands at 1608 and 1539 cm-1 belong to the –NH bending vibration. Finally, the bands between 1513 and 1485 cm-1 are attributed to the phenyl ring. The spectrum of the CCM/SiO2-FO does not provide a direct evidence of the successful binding of folate to the amino-functionalized silica. However, the absorption band corresponding to the C=O of the carboxyl group of folic acid (1694 cm-1) is absent in the spectrum suggesting the formation of a covalent bond with the amino functionalized silica. Further, the zeta potential measurements were used to evaluate the surface charge of all synthesized materials (Table 1). Before zeta potential measurements, SAXS patterns in 40 mM KCl solutions were acquired and used to obtain nanoparticles size distribution and exclude any possible samples’ aggregation (Supporting Information – Figure S7). As expected, the zeta potential value for SiO2 is negative while the SiO2-NH2 has a positive zeta potential. It clearly indicates that the nanoparticles silanol groups were covalently bonded to APTES molecules. When the

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SiO2-NH2, positively charged, was functionalized with folic acid, the zeta potential became negative (-26.3 mV) suggesting that folate was grafted on the surface. Moreover, curcumin nanoparticles (CMC/SiO2-NH2 and CMC/SiO2-FO) also presented significant differences in their surface charges when compared to their respective materials without curcumin, indicating the presence of a curcumin fraction on the nanoparticles’ surface.

2.2. Drug release and cytotoxic effect in prostate cancer and epithelial cells Quantitative measurements of drug release from the folate-functionalized silica nanoparticles containing curcumin (CCM/SiO2-FO) were performed and Figure S8 (Supporting Information) shows the CCM release profile. For this purpose, CCM/SiO2FO sample was dispersed in a RPMI medium and the experiment performed at 37 ºC. The total CCM release from CCM/SiO2-FO was about 35 % after 48 h of incubation demonstrating the potential of this system as a drug delivery platform. The cytotoxic effect of nanoparticles was evaluated using PC3 prostate cancer and PrEC normal (prostate epithelial) cells. These cells were incubated for 24 and 48 h with nanoparticles containing equivalent curcumin doses and then examined by using high content imaging techniques. The resulting images of PC3 tumor cells in the absence (control) and in the presence of the CCM/SiO2-FO nanoparticles are shown in Figure 6.

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Figure 6. Fluorescent images of PC3 tumor cells acquired after 24 h of incubation. The images for control cells and the cells in the presence of CCM/SiO2-FO are presented. The nuclei and mitochondria were stained with DAPI and MitoTracker, respectively, while the overlay shows merged images of the nuclei and mitochondria. Scales bar = 20 µm.

In order to visualize the morphological changes of the cell nuclei, DAPI (4,6diamidino-2-phenylindole) was used as a dye that stains the nucleus (blue). The nuclei of both samples show similar features since their morphology was maintained. Moreover, no nuclear material was observed outside the nuclei. In parallel, the cytoplasm was represented by marking the mitochondria (MitoTracker – red). Cells treated with CCM/SiO2-FO showed very few normal cells and most of them exhibit deformations on the cell membrane that probably indicates the beginning of the apoptosis process (cell death). Finally, overlapped images (overlay) show simultaneously the nuclei and the cytoplasm where the overall cell morphology can be identified. These results suggest that treatment with CCM/SiO2-FO induces cellular structural changes likely associated with the apoptotic process. Quantitatively, the total number of nuclei was counted after 24 and 48 h of nanoparticles incubation and the results are shown in Figure 7. Comparative graphs of

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the percentage of viable tumor (Figure 7A) and normal (Figure 7B) cells when in contact with the synthesized materials are presented. The red bars represent measurements done after 24 h of nanoparticles-cells incubation while blue bars indicate incubation of 48 h.

Figure 7. Viability of (A) PC3 tumor cells and (B) PrEC normal cells after 24 (red bars) and 48 h (blue bars) of incubation. Viability results were obtained by comparison against those cells that were not treated (control) for each experimental time. Data are presented with mean ± standard deviation (SD) from six independent experiments. Statistical analysis was performed according to the t-test. The symbols indicate mean P

Functionalized Silica Nanoparticles As an Alternative Platform for

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