PTT-Mediated Inhibition of Cancer Proliferation and Tumor Progression by DARPin-Coated Gold Nanoparticles

Abstract

Targeting HER2-positive cancer cells with precision therapies is a critical challenge in oncology. Here, we present a study on gold nanoparticles (AuNPs) conjugated with DARPin_9-29, a designed ankyrin repeat protein with high specificity and affinity for HER2 receptors. In this study, we investigate the therapeutic potential of AuNP-DARPin_9-29 conjugates, which was synthesized and characterized by us earlier, for photothermal therapy (PTT). By combining AuNP-DARPin treatment with visible light illumination, we show selective inhibition of HER2-positive cancer cell proliferation and tumor progression in a murine model. The results highlight the effectiveness of AuNP-DARPin in disrupting cancer cell viability and reducing tumor growth, providing a cost-effective and targeted approach for combating HER2-positive cancers.

1. Introduction

Cancer continues to be a leading cause of mortality [1,2], prompting significant global research efforts aimed at precisely targeting and eradicating cancer cells. Targeted therapy [3,4] is a major focus in cancer research due to its ability to preferably attack cancer cells, leaving healthy tissues largely unaffected. Current treatment options, including radiation, chemotherapy, chemodynamic therapy, and synergistic therapies integrating chemotherapy with chemodynamic approaches [5], are widely used for preventing various types of cancer. However, despite significant progress, there is still a need for innovative therapies that can selectively eradicate cancer cells. Gold nanoparticles (AuNPs) possess unique optical, chemical, and physical properties, making them highly valuable for a wide range of biomedical applications, including imaging, drug delivery, and photothermal therapy (PTT) [6,7,8,9,10].

Designed ankyrin repeat proteins (DARPins) are a relatively novel class of non-IgG scaffolds derived from naturally occurring ankyrin repeats [11]. DARPins are small (13–20 kDa), highly water-soluble proteins that are stable under a range of experimental conditions and exhibit exceptionally high affinity for their target proteins [11,12,13,14], making them ideal ligands for directing AuNPs and other nanoscale materials to tumor-specific receptors [15]. Compared to conventional antibodies, DARPins offer several practical advantages, including reduced immunogenicity, higher tissue penetration due to their smaller size, and simpler production processes. These properties make DARPins a versatile tool in precision medicine [16].

The combination of AuNPs and gold nanorods (GNRs) with DARPins has emerged as a promising strategy for developing effective cancer treatments. It has been shown that gold nanostructures functionalized with DARPins can be directed to tumor-specific markers with high selectivity, leading to enhanced cellular uptake and selective accumulation in tumor tissues [15,17]. This is particularly true for conjugates of gold nanostructures, with DARPins targeting HER2 (human epidermal growth factor receptor 2), a receptor frequently overexpressed in breast and ovarian cancers [12,18].

We have previously reported the synthesis of a conjugate between AuNPand DARPin_9-29, which targets the HER2-positive breast adenocarcinoma cell line (SKBR-3), and demonstrated specific interaction of the conjugate with SKBR-3 cells and its internalization into the cells via endocytosis [19]. Despite extensive characterization of the conjugate’s structure and demonstration of its high-affinity binding to HER2 receptors, its impact on cell viability and tumor progression remains unexplored. In this work, we fill this gap by showing that the conjugate specifically eradicates cancer cells and leads to a marked reduction in tumor growth in animals.

2. Materials and Methods

2.1. Cell Lines

For in vitro and in vivo experiments, SKOV-3 ovarian adenocarcinoma stably expressing the NanoLuc luciferase gene [20] and BJ-5TA (immortalized hTERT fibroblasts derived from human foreskin cells) were used. SKOV-3 cell lines were characterized by HER2-overexpression (106 receptor per cell), while BJ-5TA cells were used as the HER2-negative control.

SKOV-3/NanoLuc and BJ-5TA cells were cultured in RPMI 1640 (PanEco, Moscow, Russia) in a humidified atmosphere with 5% CO₂ at 37 °C. All mediums used were supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 2 mM L-glutamine (PanEco), 10 U/mL penicillin (PanEco), and 10 μg/mL streptomycin (PanEco).

2.2. Protein Production

DARPin_9-29 specific to subdomain I of HER2 receptor was produced in bacterial BL21(DE3) cells by the autoinduction methodology [21] and purified using Ni²⁺-NTA affinity chromatography as described in [19]. The concentration of the purified protein was determined by spectrophotometry (UV Spec 900, GE, Buckinghamshire, UK) using an extinction coefficient of 4600 M⁻¹cm⁻¹ at 280 nm.

2.3. Synthesis of AuNPs and DARPin-AuNP Conjugates

The citrate-stabilized 5 nm AuNPs were synthesized as described in [19] and concentrated using a K10 Ultra Centrifugal Filter unit (Amicon, Birlington, MA, USA) to a final concentration of 18 µM (concentration was determined spectrophotometrically using an extinction coefficient of 10⁷ M⁻¹cm⁻¹ at 520 nm). The particles were incubated with DARPin_9-29 (final concentration of 180 µM) in 2 mM NaPi (pH 7.5) for 2 h under ambient temperature. Then, NaPi concentration was increased to 100 mM, followed by 16 h incubation under ambient conditions. The unbound protein was separated from the conjugate using size-exclusion chromatography on a Sepharose 4B column (1 × 16 cm) equilibrated in 10 mM NaPi (pH 7.5). The conjugate was eluted in the column’s void volume, before the DARPin.

2.4. Flow Cytometry

DARPin-AuNP or BSA-AuNP conjugates were incubated with 10 molar excess of FITC or Cyanine 3.5 NHS ether (Lumiprobe, Moscow, Russia) for 1 h in 20 mM NaPi (pH 8.0). The unbound dye was separated from the dye/protein conjugates using gel filtration on a NAP 5 column (Cytiva, Little Chalfont, UK) equilibrated with 20 mM NaPi (pH 7.5), 150 mM NaCl.

HER2-positive SKOV-3/NanoLuc cells or HER2-negative BJ-5TA cells (200,000 cells per probe) were incubated with 150 nM of AuNP-DARPin-FITC or AuNP-BSA-FITC for 7 min at ambient temperature. The cells were then washed 3 times with PBS and analyzed on a NovoCyte 3000 (ACEA Biosciences, San Diego, CA, USA) cytometer using a 488 nm laser for excitation and a 530 ± 30 nm filter for emission.

2.5. Confocal Microscopy

SKOV-3/NanoLuc cells were seeded at a density of 30,000 cells/mL (100 µL per well) into 96-well glass-bottom plates (Cell imaging plates, 96 wells, Eppendorf, Hamburg, Germany) and incubated overnight. Then, 150 nM of AuNP-DARPin-Cy3.5 or AuNP-BSA-Cy3.5 was added to each well and incubated for 10 min, 30 min, or 1 h at room temperature. The wells were subsequently washed with PBS. The nuclei were stained with Hoechst 33342 (10 nM) and lysosomes were labeled with LysoTracker Green (20 nM, Thermo Fisher Scientific, Waltham, MA, USA). The cells were then imaged using a LSM 980 (Carl Zeiss LSM-980, Jena, Germany) confocal microscope equipped with a 63× oil Plan-Apochromat immersion objective. Hoechst33342 was excited at 405 nm and detected between 410 and 520 nm, Cy3.5 was excited at 561 nm and detected at 580–683 nm, and LysoTracker Green was excited at 488 nm and detected between 497 and 562 nm.

2.6. Dynamic Light Scattering

The hydrodynamic sizes and ζ-potentials of AuNPs and AuNP-DARPin conjugates were analyzed using a Zetasizer Nano ZS analyzer (Malvern Panalytical, Malvern, UK). Measurements were carried out in 0.1% PBS at 25 °C. Zeta potential values were calculated using Smoluchowski approximation.

2.7. In Vitro Cell Cytotoxicity Measurements

SKOV-3/NanoLuc and BJ-5TA cells were seeded in 96-well plates at a density of 35,000 cells/mL and 25,000 cells/mL, respectively, and incubated overnight. AuNP-DARPin, previously sterilized using a 0.22 µm filter, were added to the cells at different concentrations (from 5 µM to 76 nM) and incubated for 3 h before illumination. Illumination was conducted for 10 min using a 532 nm laser (100 mW, KLM-532-200 Optronic, Moscow, Russia). After illumination, the medium was replaced with a fresh one, and the cells were incubated at 37 °C in 5% CO₂ atmosphere for 72 h. An MTT assay was performed as described in [22].

2.8. Animals

Female NU–A/A Tyrc/Tyrc Foxn1nu/Foxn1nu mice (of 25–28 g weight, 6–8 weeks old) were purchased from the SPF (specific-pathogen-free) licensed nursery of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences and housed in a specific-pathogen-free facility in individually ventilated cages with ad libitum access to standard sterilized food and water. All experimental procedures were approved by the Animal Care and Use Committee of the Shemyakin-Ovchinnikov Institute (protocol no. 368/2022, 19 December 2022).

To generate tumors, 2 × 106 SKOV-3/NanoLuc cells were resuspended in serum-free medium containing 30% Matrigel (Corning, Glendale, AZ, USA) and inoculated subcutaneously in the right flank of the mouse.

When the tumor sizes measured with a caliper reached ~120 mm³, 6 mice with SKOV-3/NanoLuc s.c. tumors were randomly divided into two groups (3 mice per group) and treated as follows: (1) the control group (initial tumor volume 123.37 ± 6.13 mm³) was injected with 100 μL of PBS; (2) PTT group (initial tumor volume 124.76 ± 4.24 mm³) was treated with 2 nmole of DARPin-AuNPs in 100 µL PBS (every other day, with a total of 8 injections) via the retro-orbital sinus and 4 h later were illuminated with a 532 nm laser (100 mW, KLM-532-200 Optronic, Moscow, Russia) for 10 min; irradiation was carried out daily. For injection, insulin syringes with a 29G needle were used.

Tumor growth was measured based on luminescence signal using an IVIS Spectrum CT system (PerkinElmer, Hopkinton, MA, USA) with open filter binning = 8, field of view = 13.4 × 13.4 cm, f/stop = 1, and auto exposure time. For bioluminescence imaging, furimazine was injected into the retroorbital sinus at a dose of 250 μg/kg in 100 µL PBS. During bioimaging, mice were kept under isoflurane anesthesia (1.5%). For quantification of light output, regions of interest (ROI) were manually selected in squares, and the flux in photons per second (p/s) was calculated using Living Image software 4.5 (PerkinElmer).

3. Results

3.1. Binding of DARPin-AuNPs to Cells

A conjugate of a 5 nm (in diameter) AuNP with DARPin_9-29 (DARPin-AuNPs) was synthesized as described in Section 2. The particles were incubated with HER2-specific DARPin_9-29, which interacts with high affinity with subdomain I of the HER2 receptor [18]. The binding of DARPin_9-29 to the nanoparticle’s surface induced an increase in the nanoparticle’s diameter from 4.1  ±  1.2 to 12.7 ± 3.9 nm (Figure 1A) and a reduction in the absolute value of the ζ-potential from −13.8 ± 0.1 to −5.5 ± 1.7 mV (Figure 1B). These results are consistent with the structure of the conjugate reported by us earlier [19]. Based on these data, the conjugate is estimated to consist of approximately 35 DARPin molecules attached to the nanoparticle’s surface. This surface modification results in a noticeable increase in particle diameter and alters the conjugate’s overall charge. At neutral pH, DARPin_9-29 carries a very low positive charge (less than +1, according to ExPASy calculations), leading to a strong reduction in the net negative charge of the AuNPs initially stabilized by citrate molecules (each carrying a charge of −3).

The ability of the DARPin in the conjugate to specifically interact with HER2 receptors on the cell surface was confirmed by flow cytometry and confocal microscopy (Figure 2 and Figure 3, respectively). To enable detection using these fluorescence-based methods, the non-fluorescent conjugate was labeled with the fluorescent dye—FITC, as detailed in Section 2. Flow cytometry data (Figure 2A) demonstrate that SKOV-3/NanoLuc cells, which overexpress the HER2 receptor, exhibit a median fluorescence intensity (MFI) eight times higher when treated with DARPin-AuNPs (green curve in Figure 2A) compared to the autofluorescence baseline (red curve in Figure 2A). In contrast, treatment with bovine serum albumin (BSA)-coated nanoparticles did not result in an MFI increase above the autofluorescence level (blue curve in Figure 2A). Furthermore, no difference in MFI was observed between DARPin-AuNP- and BSA-AuNP-treated BJ-5TA cells, which do not express the HER2 receptor (green and blue curves in Figure 2B, respectively). These results indicate the specific binding of DARPin-AuNPs to cells overexpressing the HER2 receptor, with minimal non-specific binding to the cell membrane or other membranal proteins.

Confocal microscopy further supports these conclusions. After 10 min incubation with Cy3.5-labeled DARPin-AuNPs (DARPin-AuNPs-Cy3.5), bright red staining of the cell membrane was seen in HER2-positive SKOV-3/NanoLuc cells (Figure 3A). In contrast, incubation with Cy3.5-labeled BSA-AuNP conjugate did not result in membrane staining (Figure 3B), confirming the lack of this conjugate binding to the cells. Longer (30–60 min) incubation resulted in redistribution of DARPin-AuNPs-Cy3.5 inside the SKOV-3/NanoLuc cells. The Cy3.5 signal, initially localized to the cell membrane (Figure 3A), was no longer visible on the surface but appeared in the cytoplasm instead (Figure 3C). This suggests that the dye associated with the conjugate was internalized. The colocalization of the red Cy3.5 signal (Figure 3C) with the LysoTracker green signal (Figure 3D), resulting in yellow dots in the merged image (Figure 3E), indicates that the conjugate is localized within lysosomes, confirming that internalization proceeds through endocytosis.

3.2. Effect of DARPin-AuNPs on Viability of HER-2-Positive Cells

To examine the light-induced phototoxicity of DARPin-AuNPs, HER2-positive and HER2-negative cells were treated with varying concentrations of the conjugate for 3 h, followed by exposure to green light illumination. The results (Figure 4) demonstrate that the phototoxicity of DARPin-AuNPs is significantly higher in HER2-positive SKOV-3/NanoLuc cells compared to HER2-negative BJ-5TA fibroblast cells. It can be suggested that the specific binding and subsequent internalization of the conjugate into HER2-positive cells enhance their sensitivity to green light, which is efficiently absorbed by the particles. The absorbed photon energy is converted into heat, resulting in cell death through PTT.

3.3. Effect of DARPin-AuNPs on Tumor Progression and Growth in a Mouse Model

To investigate the effects of DARPin-AuNPs on tumor progression and growth, we used a mouse model with xenograft subcutaneous tumors formed from HER2-positive ovarian adenocarcinoma SKOV-3/NanoLuc cells. 12 days after tumor inoculation the mice were randomly divided into two groups (n = 3); conjugate injection and photoillumination were performed as described in Section 2. As the route of DARPin-AuNP administration, we selected the retroorbital sinus as an alternative to tail vein injection for several reasons: (1) tail vein administration is time-consuming; (2) it typically involves prolonged handling of each mouse, including warming the tail, restraining the mouse, and performing the injection without anesthesia; and (3) this procedure, performed without anesthesia, induces measurable stress in the mice, particularly if repeated. In contrast, the retroorbital sinus is a safe and effective site for consecutive injections over a period of up to two weeks. Tumor progression was monitored using IVIS Spectrum CT (Perkin Elmer) on days 1, 8, and 16 of treatment (Figure 5A).

On day 16, bioluminescence intensity from the tumors and surrounding regions (Figure 5A) was 6.7-fold lower in the group treated with DARPin-AuNPs compared to the control group (Figure 5B). These results demonstrate the positive effect of DARPin-AuNP-mediated PTT on tumor growth in this animal model. The observed effect paves the way for the exploration of nanoparticle-DARPin conjugates as promising therapeutic strategies.

4. Discussion

The results of this study demonstrate the potential of DARPin_9-29-AuNP for PTT of HER2-positive cancers. We have confirmed by flow cytometry and confocal microscopy that DARPin_9-29, a HER2-specific ligand, facilitates precise targeting and internalization of AuNPs into HER2-positive cancer cells. This selective targeting minimizes off-target effects and underscores the advantage of DARPin-based therapeutics over traditional antibody-based therapies. The conjugate’s high stability under physiological conditions, compact size compared to conventional antibodies, and relatively low production cost position DARPins as highly promising candidates for anticancer therapies in general and PTT in particular. The significant photothermal cytotoxicity observed in vitro highlights the potential of DARPin-AuNP conjugates in eradicating HER2-positive cancer cells without affecting HER2-negative ones. By converting absorbed light into localized heat, the internalized AuNPs in the conjugate selectively destroy HER-positive cells via PTT. The selective effect of the conjugate on HER-positive cancer cells was also confirmed in vivo. The treatment with DARPin_9-29-AuNPs led to marked tumor growth inhibition, as evidenced by bioluminescence imaging. These findings align with previous studies demonstrating the therapeutic potential of gold nanostructures functionalized with DARPin molecules for targeted cancer therapy [15,17] and pave the way for future research focused on translating this promising approach into clinical applications.