Home Antibody-functionalized nanoporous silicon particles as a selective doxorubicin vehicle to improve toxicity against HER2+ breast cancer cells
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Antibody-functionalized nanoporous silicon particles as a selective doxorubicin vehicle to improve toxicity against HER2+ breast cancer cells

  • Alma Rosa Oaxaca Camacho , Sara Elisa Herrera Rodríguez , Óscar René Ochoa Mojica , Vivechana Agarwal , Alexis Jazmín Preciado Pelayo , Gregorio Guadalupe Carbajal Arízaga , Ángel H. Álvarez , Zaira Yunuen García Carvajal , Moisés Martínez Velázquez EMAIL logo and Rodolfo Hernández Gutiérrez EMAIL logo
Published/Copyright: February 22, 2025
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Science and Engineering of Composite Materials
From the journal Science and Engineering of Composite Materials Volume 32 Issue 1

Abstract

Porous silicon (PS) has been widely used in biology, biomedicine, and nanotechnology. In recent years, its application in cancer therapy has received considerable attention. PS has shown low cytotoxicity, so it is reasonable to use it as an antibody conjugate for targeted anticancer drug delivery. In this work, we synthesized PS covalently attached to doxorubicin and covered by a nanometric polymer as a crosslinker between PS and the monoclonal antibody trastuzumab, using electrochemical etching for particle generation. The results revealed the formation of the antibody conjugate, obtaining particles with sizes around 250–350 nm, and a homogeneous size distribution determined by dynamic light scattering. Scanning electron microscopy (SEM) analyses showed the porous structure of the conjugate, while Fourier-transform infrared and transmission electron microscopy characterization demonstrated functionalization between PS and the antibody. The cytotoxic effect was evaluated against two breast cancer cell lines, SKBR3 (HER2+) and MCF-7 (HER2−), and one non-cancer cell line as control (HEK293). The cytotoxic effect on the SKBR3 cancer cell line was higher than on the MCF-7 and HEK293 cell lines. Notably, the targeted protein HER2 mediates the elevated cytotoxic activity exhibited by the antibody conjugate.

Graphical Abstract

Keywords: antibody conjugate; porous silicon; trastuzumab; particles; breast cancer; HER2

1 Introduction

Cancer is a leading cause of death worldwide [1,2]. In women, breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death [3]. Chemotherapy is one of the principal modes of cancer treatment, in particular for metastasis. For the latter, there is a limited therapeutic effect mainly due to two factors: the absence of specificity for the disease site and the need to use very high drug doses to achieve sufficient local concentrations, favoring non-specific toxicity and other adverse effects [4,5]. A new field in cancer therapy is emerging in nanomedicine [6]. This field utilizes nanomaterials to improve disease diagnosis, prevention, and treatment. Recent research has shown the development of functionalized particles that are covalently linked to biological molecules such as peptides, proteins, nucleic acids, or small-molecule ligands. These materials focus on increasing the treatment’s efficacy, reducing adverse effects, and overcoming drug resistance [7,8,9,10].

Porous silicon (PS)-based materials have been employed in biomedical applications because of their biodegradability [11,12,13], low cytotoxicity [14,15], biocompatibility [16,17], and cost-effective and easy fabrication procedure [18]. Porous silicon particles (pSiNp) have been used as nanocarriers due to their ability to load drugs and release them in a physiological environment [19]. These materials exhibit a large surface area with large pore volumes that allow the adsorption and deposition of active compounds, making them attractive for developing controlled active release and delivery systems [20]. The load of the active ingredient within the porous structures and its surface distribution will determine the speed of dissolution and, therefore, its release to a physiological environment of therapeutic interest [21].

The pSiNp have shown the ability to transport and release small molecules such as peptides, antibodies, oligonucleotides, proteins, and drugs [22]; therefore, they are considered promising nanocarriers against breast cancer [23,24,25]. However, there are still problems and limitations to be resolved for a targeted and synergistic therapy against breast cancer to exist and be successful [26]. Such limitations include untargeted delivery, low efficacy, increasing the tumor-homing capability while at the same time reducing the unwanted side effects and in vivo and in vitro cell biological interactions [27].

There are antibodies against the extracellular domain of overexpressed or mutant cell surface receptors (trastuzumab and biosimilars) and molecules capable of inhibiting the receptor’s kinase activity, which helps treat the disease [28]. The human epidermal growth factor receptor family member 2 (HER2) overexpression is correlated in some cases of breast cancer, with trastuzumab being a key drug in the treatment of HER2-positive breast cancer [29]. Furthermore, evidence indicates that the conjugation of trastuzumab with chemotherapy drugs, such as doxorubicin (Doxo), could be more effective for multiple targets [29].

In the present study, we focused on attempting to resolve these disadvantages by making an antibody conjugate composed of pSiNp charged with the drug Doxo. These particles were coated with a polymer that would not allow the drug to be released until the target was reached by a specific monoclonal antibody anti-HER2, anchored to the surface of the polymer (trastuzumab, a recombinant, humanized monoclonal antibody that is directed against the extracellular region of HER2 [30,31,32]). The advantage of the developed system is that it could be endocytosed by the cells, thereby solving the issue of cell absorption. Finally, breast cancer cells were exposed to the composite to determine the cytotoxicity level and to evaluate the ultrastructure of exposed cells by transmission electron microscopy (TEM).

2 Materials and methods

2.1 Materials

PS resistivity 0.001–0.002 (Montco Silicon Technologies, Inc.), HF 48% (Fermont SA de CV), trastuzumab (Herceptin, Roche), penicillin/streptomycin (Gibco), fetal bovine serum (FBS) (Byproducts), and Bio-Rad DC-Protein assay (Bio-Rad, Hercules, CA, USA). Cell lines SKBR3, MCF-7, and HEK293 were kindly provided by Dr. Luis Felipe Jave Suárez and Dr. Adriana del Carmen Aguilar Lemarroy (Centro de Investigación Biomédica de Occidente, Guadalajara, Jalisco, Mexico). Undecylenic acid 95%, N-ethyl-N′-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), Doxo hydrochloride, urea, citric acid, N-hydroxysuccinimide (NHS), bovine serum albumin (BSA), l-glutamine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ethylenediaminetetraacetate (EDTA), dimethyl sulfoxide (DMSO), sodium hydroxide, phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), all of these reagents, and chemicals were purchased from Sigma-Aldrich.

2.2 Nanoparticle synthesis

2.2.1 Preparation of pSiNp

Samples of pSiNp were prepared by the electrochemical etching of a single-crystal, oriented (100), p-type silicon wafer with a constant current density of 200 mA/cm2 for 180 s in a 3:1 (v/v) electrolyte solution of aqueous HF (48%)/ethanol.

A freestanding film of silicon-wafer nanostructure was removed from the crystalline silicon substrate by applying a current pulse of 4 mA/cm2 for 120 s in a solution of 3.3% (volume) 48% aqueous HF in ethanol. The freestanding hydrogen-terminated silicon-wafer film was placed in deionized water and fractured into multi-size particles by overnight sonication. Finally, to obtain pSiNp within a size range of less than 200 nm, the particles were centrifuged in deionized water at 14,000 rpm for 30 min. The supernatant containing smaller particles (<20 nm) was removed by SpeedVac, because the vacuum promotes solvent evaporation and prevents the oxidation of samples during the drying process.

The pSiNp were chemically modified using the thermal hydrosilylation of undecylenic acid employing microwave irradiation to form undecanoic acid terminated to enable covalent attachment of Doxo. Five milligrams of freshly prepared and dried pSiNp was placed in an Eppendorf tube with 200 μL of undecylenic acid 95% and then heated in a conventional 280-W microwave oven for 5 min. The resulting pSiNp was rinsed with hexane and ethanol (1:1) solution at room temperature to remove the excess of the unreacted reagent and then dried by SpeedVac.

2.2.2 Doxorubicin-loaded pSiNps (pSiNpDoxo)

The incorporation of Doxo into the pSiNp was through the binding of Doxo by the carbodiimide method. Five milligrams of pSiNp was placed in an Eppendorf tube and suspended in 600 μL of 10% v/v DMSO in a PBS solution containing 50 mM EDC and 5 mM N-hydroxylsulfosuccinimide. Afterward, 200 μL of Doxo solution (1 mg/mL) was added. The particles were mixed and stirred for 4 h at room temperature. After that, the mixed solution was washed twice with methanol, dried at room temperature for 24 h, and neutralized with 1 M hydrochloric acid (HCl).

2.2.3 Polymer coating by urea–citric acid reaction (pSiNpDoxoPol)

The polymer coating designed for the particles was intended to be a compatible network obtained by co-polymerizing citric acid and urea [33]. For this experiment, we followed a reaction in a liquid medium as follows [34]: 0.1 g of urea and 0.075 g of citric acid were dissolved in 18 mL of deionized water. Then, 10 mg of loaded silicon particles were added and stirred for 5 min. The solution was transferred to a hermetic Teflon vessel and sealed with an iron steel core. This reactor was heated at 120°C for 2 h. After that, the reactor was cooled down at room for 24 h. The final solution was centrifuged at 15,000 rpm for 30 min and stored at 4°C. One sample of the polymer was prepared without pSiNpDoxo as a control. The presence of Doxo in the pSiNpDoxoPol was determined by using a Nanodrop 2000 UV–VIS spectrophotometer at λ = 480 nm. pSiNpDoxoPol particle solutions were centrifugated, and the supernatant was discarded. Finally, the pellet was dissolved in 1 M sodium hydroxide (NaOH).

2.2.4 Trastuzumab antibody conjugation (mAbpSiNpDoxoPol)

About 5 mg of pSiNpDoxoPol particles were mixed in the antibody solution containing 800 μL of PBS solution with 50 mM EDC/5 mM NHS and 200 μL of trastuzumab solution (1 mg/mL) under constant stirring at room temperature for 24 h. The resulting solution was centrifuged at 13,000 rpm for 10 min, and the supernatant was discarded. The antibody conjugate (mAbpSiNpDoxoPol) was washed three times with PBS and suspended in 200 μL of PBS.

2.3 Dot blot assay

A dot blot assay was performed to determine the antibody binding to pSiNpDoxoPol by using a Bio-Dot SF microfiltration apparatus (Bio-Rad). 200 ng of either pSiNp or mAbpSiNpDoxoPol was suspended in 100 μL of PBS and placed in each well of the Bio-Dot SF apparatus and was subjected to vacuum pressure for 20 min, which allowed the Nps to be placed onto the nitrocellulose membrane. Next, the membrane was blocked with PBS-0.1% Tween 20–5% milk overnight. The membrane was washed three times with PBS–0.1% Tween 20 and then incubated with HRP-conjugated goat anti-human IgG as a secondary antibody diluted 1:2,000 in PBS–0.05% Tween 20–2.5% milk for 4 h at room temperature, followed by washing with PBS–0.1% Tween 20 solution. Finally, immunoreactivity was evidenced using the HRP Substrate and Detection kit (Opti-4CN; Bio-Rad).

2.4 Quantification of trastuzumab bound to pSiNpDoxoPol

Trastuzumab was quantified from the conjugate’s surface with the Bio-Rad DC-protein assay, a colorimetric assay for protein concentration determination, following detergent solubilization. This assay is based on the reaction of proteins with an alkaline copper-tartrate solution and the Folin reagent. Two steps lead to color development: the reaction between protein and copper in an alkaline medium and the subsequent reduction of the Folin reagent by the copper-treated protein. A standard curve was prepared, with five dilutions of a protein standard containing from 0.2 to 1.5 mg/mL BSA. Five microliters of standard and the samples were placed on a clean, dry microtiter plate and 25 μL of reagent A and 200 μL of reagent B were added into each well and then mixed for 5 s. After 15 min, and absorbance was read at 750 nm. The reaction was carried out in triplicate.

2.5 Physicochemical characterization

2.5.1 Dynamic light scattering (DLS)

Particle-size analysis was determined by the DLS method that utilized an electrophoretic light scattering spectrophotometer (Zetasizer Nano S90; Malvern Panalytical) at a fixed angle of 90°C at room temperature.

2.5.2 Fourier-transform infrared (FTIR) spectroscopy

Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy was performed using a NICOLET iS50 FTIR equipped in the wavelength region of 4,000–500/cm with a precision of 4/cm to confirm the composition of pSiNp, the chemical modification of the Si following thermal hydrosilylation, the presence of Doxo in the porous silicon particles (pSiNpDoxo), the copolymer formation using urea–citric acid reaction (pSiNpDoxoPol), and the antibody trastuzumab conjugation (mAbpSiNpDoxoPol).

2.6 Biological evaluation

2.6.1 Cell culture

The following three cell lines were used: human breast adenocarcinoma (SKBR3, HER2-positive), human breast adenocarcinoma (MCF-7, HER2 negative), and human embryonic kidney (HEK293) cells. The cells were maintained in a DMEM culture medium containing phenol red and 2 mM l-glutamine, 10% FBS, and 100 IU/mL penicillin/streptomycin. The cells were cultured in T-75 culture flasks, in a humidified incubator at 37°C, 5% CO2, and 100% humidity, until achieving a confluence of 90%. For passages, the cells were routinely detached using 0.25% trypsin with 0.2 g/L EDTA.

2.6.2 Cell exposure to particles

The cytotoxic assays were performed as follows: SKBR3, MCF-7, and HEK293 cells were seeded in Blackwall 96-well plates at cell densities of 5 × 103 cells per well and cultured for 24 h in DMEM supplemented with 10% FBS and 100 IU/mL penicillin/streptomycin before exposure to treatments. Cells were exposed to 0, 13, 26, and 52 μg/mL of mAbpSiNpDoxoPol from a stock solution. Furthermore, we evaluated the effect of pSiNp, pSiNpDoxo, pSiNpDoxoPol, Doxo, and cells without treatment to compare the results with the antibody nanoconjugate.

2.6.3 Measurement of cellular cytotoxicity (MTT assays)

Briefly, we used the antibody nanoconjugate as a Doxo carrier. The particles were loaded with Doxo, and then, the complex was coated with a polymer, which can bind to the monoclonal antibody trastuzumab by its Fc portion.

The MTT compound can be reduced by the dehydrogenase enzymes found within living cells, demonstrating cellular metabolic activity and cell viability [35]. Thus, it comprises one of the techniques utilized to measure the cytotoxic effect. We used it to evaluate the cytotoxic effect of the antibody nanoconjugate on breast cancer and control cells. Cells seeded in 96-well plates were separately treated with different concentrations of the antibody nanoconjugate, mAbpSiNpDoxoPol (0, 13, 26, and 52 μg/mL), or treated with pSiNp, pSiNpDoxo, pSiNpDoxoPol, or Doxo alone. The final volume of each well was 200 μL. The cells were then incubated for 24, 48, and 72 h at 37°C and 5% CO2. After incubation, the medium was removed, and 10 μL of MTT (5 mg/mL) and 190 μL of medium without FBS were added to each well; then, plates were incubated for another 4 h at 37°C. Purple formazan crystals were observed. Next, the medium was eliminated, and formazan crystals were dissolved in 100 μL of DMSO, with readings carried out at 570 nm utilizing a multi-well ELISA plate reader (XMark™; Bio-Rad). The absorbance of the control cells represented 100% cell viability. Cell viability was calculated as follows:

(1) Cell viability ( % ) = Sample abs Control abs × 100 .

3 Results and discussion

3.1 Characterization of pSiNp

The pSiNp were produced by the electrochemical etching of the Si wafer with HF and ethanol. FTIR confirmed the characteristic peaks of PS at 1,100/cm (Si–O–Si), 617/cm (Si–Si), and 897 and 780/cm (Si–H) (Figure 1a). The synthesized PS layer shows a high concentration of SixHy groups in the wave number of 2,112/cm (Si–H), which are highly reactive.

Figure 1 (a) FTIR spectra of pSiNp. (b) Size distribution analysis by DLS.
Figure 1

(a) FTIR spectra of pSiNp. (b) Size distribution analysis by DLS.

The characterization by DLS revealed the average size of pSiNp at 88 nm with a polydispersity index of 19.38 nm, measured by the percentage of the intensity of scattered light (Table 1; Figure 1b). Analysis by DLS confirmed that the particles possess a size suitable for targeted (anti-cancer) drug-delivery applications. This size is considered optimal for passive tumor targeting due to the enhanced permeability and retention (EPR) effect. Conversely, nanoparticles of a size below 10 nm can induce kidney damage [36].

Table 1

DLS determination

Samples Particle size (nm) hydrodynamic diameter Z-average (nm) Polydispersity index (PDI)
pSiNp 50–150 88 19.38
pSiNpDoxo 200–300 220.7 17.09
mAbpSiNpDoxoPol 200–700 323 115

3.2 Characterization of the drug-loaded sample

The loading capacity of Doxo on pSiNp was determined by the ultraviolet (UV) spectrum at 280 nm, which was calculated employing the difference in Doxo concentrations between the original Doxo solution and the supernatant solution after loading (equation (2)). According to the absorbance with a standard curve, the loading capacity of Doxo was calculated as 0.0653 ± 0.0075 mg/mL (Doxo reached 74% efficiency) in pSiNpDoxo, which was much higher than another PS system (3.24 μg/mg) [24]. The success of each reaction step was confirmed by FTIR spectroscopy (Figure 2). The Doxo spectrum reveals the characteristic peak of the vibrations of O–H at 3,322/cm, of C–H at 2,930/cm, of C═O at 1,722/cm, and of N–H at 1,436/cm [37,38,39]. On the other hand, the pSiNp spectrum revealed peaks at 1,100/cm (Si–O) and 600/cm (Si–H), respectively [40]. Finally, the pSiNpDoxo spectrum exhibited a shift of PS peaks at 1,000/cm due to the interaction with Doxo. A peak at 3,322/cm was detected and it corresponded to an O–H, while the peak at 2,930/cm corresponded to a C–H, and the peaks at 1,722 and 1,436/cm corresponded to C═O and N–H, respectively, confirming a load of Doxo onto the PS (Figure 2a). The FTIR shows that the peak at 2,112/cm disappeared, the Si–H bond, which reacted first with undecylenic acid and then with EDC and NHS, to then react with Doxo, as reported by Tabasi et al. [24]; finally, the appearance of the carboxyl link belonging to Doxo, as well as small amide peaks I and II.

Figure 2 (a) FTIR spectrum. Doxo; pSiNp; and pSiNpDoxo. (b) Size distribution analysis of pSiNpDoxo by DLS.
Figure 2

(a) FTIR spectrum. Doxo; pSiNp; and pSiNpDoxo. (b) Size distribution analysis of pSiNpDoxo by DLS.

Measurement by DLS revealed the average size of the pSiNpDoxo as 220.7 nm with a polydispersity of 17.09 nm, measured by the percentage of the intensity of scattered light (Table 1; Figure 2b). The size of particles continues to fall within the allowable range for the EPR effect to occur [41,42]. Vaccari et al. [23] developed Doxo-loaded PS bits, which exhibited cytotoxicity against LoVo and HT29 human colon adenocarcinoma cell lines. More recently, De Angelis et al. [43] developed water-soluble nanoporous silicon nanoparticles, loaded with a tumor-specific peptide, and evaluated their toxicity against murine B lymphoma A20 cell line, both in vitro and in vivo. They demonstrated an efficient tumor targeting and a sensible therapeutic effect.

(2) Load efficiency = ( concentration doxo ) i − ( concentration doxo ) f ( Concentration doxo ) i × 100 % .

3.3 Polymer synthesis

While synthesizing the polymer, it was observed that the methodology was easy, fast, and involved compounds compatible with the cells because the citric acid and urea can be degraded within the cell and can enter into the metabolic pathways. Therefore, it was decided to coat the pSiNp charged with Doxo with a polymer layer. The polymerization product between urea and citric acid was studied by FTIR spectroscopy. Figure 3 depicts the spectra of the citric acid, urea, and the polymer. For urea, it is possible to observe the bands corresponding to N–H at 3,195 and 1,575/cm [44,45] and the bands corresponding to C–N at 1,368/cm, while for citric acid, we can observe the O–H bands at 3,504/cm, C═O at 1,722/cm, and C–OH at 1,148/cm. When these compounds polymerize, forming peptide bonds, the infrared spectrum presents bands at 3,438/cm corresponding to O–H, while at 3,335 and 1,575/cm, these bands correspond to N–H. These bands present a shift and undergo a shortening due to the interaction between urea and citric acid. The band at 1,678/cm corresponds to C═O, which nearly disappears due to the formation of the new bonds between the citric acid-carboxyl groups and the urea-amine groups (Figure 3a). The presence of new, narrow, well-defined bands confirms the formation of a compound from the urea and citric acid reaction. The SEM micrograph of the polymer reveals a homogenous morphology with tiny holes through which Doxo could be released when the particle is endocytosed. This polymer is very useful because some experiments have demonstrated good biocompatibility, especially in assays with cell lines; however, its toxicity in complex organisms remains unclear. Once pSiNpDoxo was coated with the polymer, a drug-release study was performed. It was revealed that Doxo was maximally released at 48 h at a pH = 6.5 (Figure 4).

Figure 3 (a) FTIR spectra. Citric acid, urea, and polymer. (b) SEM micrograph of the polymer.
Figure 3

(a) FTIR spectra. Citric acid, urea, and polymer. (b) SEM micrograph of the polymer.

Figure 4 UV–Vis spectrum of the release of Doxo.
Figure 4

UV–Vis spectrum of the release of Doxo.

3.4 Antibody nanoconjugate

The next step was the conjugation of monoclonal antibodies on the surface of the pSiNpDoxoPol. Coupling between the pSiNpDoxoPol and the antibody involved a polymer functional linker, which was selected because it permits orienting the attachment of the antibody by means of its carbohydrate side-chain (the Fc fragment), thereby maintaining its active site accessible for potential biorecognition. The TEM micrograph reveals the morphology of the antibody nanoconjugate. At the center, the pSiNpDoxo coated with the polymer can be observed. The micrograph demonstrates small refringent white points attributed to Doxo, having a size of 7 nm (Figure 5a). The dot blot assay corroborated the trastuzumab binding to the conjugate’s surface (Figure 5b). The FTIR spectrum exhibited bands in the 1,700–1,600/cm region corresponding to the amide-I groups; here, trastuzumab is found. The band in 3,297/cm corresponds to ion N–H, while in 2,915/cm, we found a C–H and the characteristic porous silica (Si–O) bands in 1,116 and 625/cm (Figure 5c). It has been reported that the formation of a wide band near 3,000/cm is related to the bond between the antibody and the particle, corroborated with several peaks near 1,600/cm that are characteristic of amines I and II, in the same manner as indicated in the conjugation of antibodies with particles [46]. This allowed us to corroborate that the pSiNpDoxo was coated with the polymer (pSiNpDoxoPol) and, in turn, had interaction with the antibody trastuzumab. In addition, DLS revealed the average size of the mAbpSiNpDoxoPol at 323 nm with a polydispersity of 115 nm, measured by the percentage of intensity of scattered light (Table 1; Figure 5d). Analyzing data from Table 1, DLS measurements indicated the polymer coating produced an increase of around 100 nm in the radii of the particles, and this low-dimension thickness enables the release of the drug.

Figure 5 (a) A TEM micrograph revealing the appearance of mAbpSiNpDoxoPol. (b) Dot blot assay shows the presence of the mAb over the pSiNpDoxoPol. (c) FTIR spectrum of the mAbpSiNpDoxoPol. (d) Size distribution analysis of mAbpSiNpDoxoPol by DLS.
Figure 5

(a) A TEM micrograph revealing the appearance of mAbpSiNpDoxoPol. (b) Dot blot assay shows the presence of the mAb over the pSiNpDoxoPol. (c) FTIR spectrum of the mAbpSiNpDoxoPol. (d) Size distribution analysis of mAbpSiNpDoxoPol by DLS.

3.5 Effect of the antibody nanoconjugate on cell viability

After the physical and chemical characterization of the antibody nanoconjugate, we conducted experiments with the SKBR3 cell line, a HER2 overexpressing breast cancer cell line, MCF-7, a non-HER2-overexpressing breast cancer cell line, and HEK293, a non-cancer cell line. The in vitro cytotoxic effects of pSiNp, pSiNpDoxo, pSiNpDoxoPol, mAbpSiNpDoxoPol, and Doxo alone were evaluated at different concentrations and times. The experimental results showed more significant anticancer activity of mAbpSiNpDoxoPol against SKBR3 cells compared with MCF-7 and HEK293 cells, with time- and concentration-dependent cytotoxic activity. The cytotoxic effect of almost all examined treatments was observed from 24 h on all three cell lines (Figures S1 and S2). On the other hand, Doxo alone affected all the cell lines. The treatment with mAbpSiNpDoxoPol had slight cytotoxic effects on the SKBR3 cell line, at 24 and 48 h at 13 and 26 µg/mL concentrations, while at 52 µg/mL concentration, cellular viability decreased to around 75% (Figure S1). After 72 h, cellular viability decreased to 63, 57, and 48% at 13, 26, and 52 µg/mL concentrations, respectively (Figure 6). The cytotoxic effect of mAbpSiNpDoxoPol on the SKBR3 cell line was more pronounced than on the MCF7 cell line (Figure S2) and on the HEK293 non-cancer cells (Figure S3). The antibody conjugate exhibited higher in vitro cytotoxic activities than trastuzumab alone (Figure S4) and Doxo (Figures S1–S3, Figure 6).

Figure 6 Cellular viability of SKBR3 and MCF7 cells exposed to different treatments during 72 h. Data are presented as mean ± standard deviation.
Figure 6

Cellular viability of SKBR3 and MCF7 cells exposed to different treatments during 72 h. Data are presented as mean ± standard deviation.

In recent years, several alternative drug delivery systems have been developed to approach breast cancer treatment. One of them consisted of leukocyte-mimicking nanovesicles (leukosomes) loaded with Doxo. Leukosomes promoted a significant tumor growth inhibition compared with free Doxo in 4T1 breast cancer tumor-bearing Balb/c mice [47]. Following a similar approach, De Vita et al. [48] developed lipid-based nanovesicles chemically linked to an inhibitor of lysyl oxidase 1 enzyme and loaded with epirubicin. These conjugated vesicles exhibited superior inhibition of triple-negative breast cancer cell growth in MDA-MB-231 breast cancer tumor-bearing NU/NU nude mice, compared to free epirubicin and epirubicin-loaded nanoparticles. Moreover, Zou et al. [49] developed emodin-loaded polymer–lipid hybrid nanoparticles, which enhanced the sensitivity of MCF-7 breast cancer cells to Doxo.

Cellular uptake of the antibody conjugate complex and cytotoxicity appear to be dependent on HER2 expression: the higher expression, the greater uptake, and the specific cytotoxic effect were proved with the images of the TEM analysis. The cells endocytosed the mAbpSiNpDoxoPol as evidenced by vacuoles containing this material (Figure 7c), compared with cells exposed to antibody-free particles, which exhibited small amounts of endocytosed particles (Figure 7b).

Figure 7 TEM analysis. SKBR3: (a) control cells (not treated); (b) cells treated with pSiNp; and (c) cells incubated with the mAbpSiNpDoxoPol complex for 48 h.
Figure 7

TEM analysis. SKBR3: (a) control cells (not treated); (b) cells treated with pSiNp; and (c) cells incubated with the mAbpSiNpDoxoPol complex for 48 h.

We suggest that cytotoxicity is due to the trastuzumab binding to the HER2 receptor, which has two effects: (a) inhibition of the signaling cascade and (b) endocytosis of the complex bound to the receptor. In this case, when the antibody conjugate is endocytosed, trastuzumab is degraded, then Doxo is released after 48 h, and finally, the polymer is degraded as well. Some reports suggest that the microenvironment of the cells causes the pSiNp to pass to SiOH4, which is less toxic and biocompatible.

Although the findings made in this study are encouraging, we recognize that they are still preliminary results. It remains to discern the fine molecular mechanisms that underlie the processing and degradation of the components of the antibody nanoconjugate as well as delve into the molecular mechanisms that underlie the cytotoxicity caused by the antibody nanoconjugate. Furthermore, the efficiency of the antibody nanoconjugate in targeting breast cancer in vivo remains to be determined.

4 Conclusions

We have successfully demonstrated the potential of mAbpSiNpDoxoPol for the delivery of hydrophobic drugs to kill cancer cells. pSiNp were prepared by the electrochemical etching of Si. The average size of pSiNp was 88 nm. These particles were suitable for drug delivery.

Coating of pSiNpDoxo rendered stability to the structure and aided in the conjugation between the pSiNpDoxo and trastuzumab. pSiNpDoxoPol were conjugated with trastuzumab and were efficiently taken up by cancer cells overexpressing the HER2 receptor, whereas control cells with basal receptor expression interacted poorly with mAbpSiNpDoxoPol. Our results revealed that it is possible to obtain an antibody conjugate with a high affinity for breast cancer cells (HER2+). These preliminary results suggest that the complex can be employed as a drug immune-nanocarrier, with a specific affinity for tumor cells overexpressing a specific marker on the cellular surface. The antibody’s highest cytotoxic effect occurs only when it is used against SKBR3 breast cancer cells.

Acknowledgments

The authors are grateful for the reviewer’s valuable comments that improved the manuscript.

  1. Funding information: This research was partially funded by PEI-2016 (No. 233113) and Convocatoria para atender problemas nacionales (No. 215412) and partially by CONACYT-FORDECYT-PRONACES/568483/2020.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and consented to its submission to the journal, reviewed all the results, and approved the final version of the manuscript. Conceptualization, AROC and RHG; methodology, AROC, AJPP, and OROM; formal analysis, SEHR, ZYGC, and MMV; resources, RHG, VA, and GGCA; writing – original draft preparation, AROC and OROM; writing – review and editing, RHG, GGCA, ZYGC, MMV, and AHA; and funding acquisition, RHG.

  3. Conflict of interest: Authors state no conflict of interest.

  4. Institutional review board statement: Not applicable.

  5. Informed consent statement: Not applicable.

  6. Data availability statement: The datasets generated during the current study are available from the corresponding author on reasonable request.

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Received: 2024-08-21
Revised: 2024-10-31
Accepted: 2024-11-08
Published Online: 2025-02-22

© 2025 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/secm-2024-0049
Keywords for this article
antibody conjugate; porous silicon; trastuzumab; particles; breast cancer; HER2
Creative Commons
BY 4.0

Articles in the same Issue

  1. Research Articles
  2. Investigation on cutting of CFRP composite by nanosecond short-pulsed laser with rotary drilling method
  3. Antibody-functionalized nanoporous silicon particles as a selective doxorubicin vehicle to improve toxicity against HER2+ breast cancer cells
  4. Study on the effects of initial stress and imperfect interface on static and dynamic problems in thermoelastic laminated plates
  5. Analysis of the laser-assisted forming process of CF/PEEK composite corner structure: Effect of laser power and forming rate on the spring-back angle
  6. Phase transformation and property improvement of Al–0.6Mg–0.5Si alloys by addition of rare-earth Y
  7. A new-type intelligent monitoring anchor system for CFRP strand wires based on CFRP self-sensing
  8. Optical properties and artistic color characterization of nanocomposite polyurethane materials
  9. Effect of 200 days of cyclic weathering and salt spray on the performance of PU coating applied on a composite substrate
  10. Experimental analysis and numerical simulation of the effect of opening hole behavior after hygrothermal aging on the compression properties of laminates
  11. Engineering properties and thermal conductivity of lightweight concrete with polyester-coated pumice aggregates
  12. Optimization of rGO content in MAI:PbCl2 composites for enhanced conductivity
  13. Collagen fibers as biomass templates constructed multifunctional polyvinyl alcohol composite films for biocompatible wearable e-skins
  14. Early age temperature effect of cemented sand and gravel based on random aggregate model
  15. Properties and mechanism of ceramizable silicone rubber with enhanced flexural strength after high-temperature
  16. Review Article
  17. State-of-the-art review on the influence of crumb rubber on the strength, durability, and morphological properties of concrete
  18. Special Issue: Advanced modeling and design for composite materials and structures
  19. Validation of chromatographic method for impurity profiling of Baloxavir marboxil (Xofluza)
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