Carbonic anhydrase-IX targeted copper-doped carbon dots as a theranostic probe for treating triple negative breast cancer

Abstract

Herein, we demonstrate selective diagnosis and chemodynamic therapy in triple negative breast cancer (TNBC) cells by carbonic anhydrase-IX targeted acetazolamide-functionalized copper-doped carbon dots (CuCD-Az). CuCD-Az showed ∼3-fold higher cytotoxicity to TNBC compared to normal cells by ROS-mediated apoptosis via Fenton-like reaction.

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Breast cancer is a major global health challenge, mostly diagnosed in females and accounting for approximately 25% of cancer cases. Triple-negative breast cancer (TNBC), which represents ∼15–20% of cases, designates a distinct subtype of breast cancer characterized by the absence of oestrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2) expression.¹ Despite significant progress in breast cancer survival rates, conventional chemotherapy and radiotherapy suffer from severe side effects and lack of target specificity.

In this context, target specific diagnostic probes with therapeutic potential offer a promising strategy to enhance the efficacy of cancer treatment. Several drug delivery vehicles such as liposomes, hydrogels, dendrimers, carbon nanotubes, nanoparticle-based theranostics etc. have been explored for targeted cancer therapy, each with benefits and limitations.^2–4 In the recent past, carbon dots with intrinsic fluorescence, photostability and biocompatibility have made remarkable contributions in therapeutics, sensing, bioimaging, drug delivery, and others.^5–8 Carbon dots hold strong potential to serve as theranostic tools due to their easy synthesis and tailor-made surface functionalization.⁹ Alongside this, the drawbacks of conventional therapies, such as drug resistance and tumour recurrence, have escalated the need for new strategies for treating cancer. To this end, chemodynamic therapy (CDT), a term proposed by Bu et al. in 2016, has gained significant attention as an alternative therapeutic strategy.¹⁰ CDT utilizes Fenton or Fenton-like reactions to convert intracellular H₂O₂ into reactive oxygen species (ROS), thereby inducing apoptosis in cancer cells.¹¹ The efficiency of ROS-based therapies is restricted by a vital intracellular antioxidant, glutathione (GSH), which maintains the redox homeostasis in cells and protects them from the effect of ROS.^12,13 Cancer cells contain a higher concentration of GSH that neutralizes a substantial amount of ROS, resulting in lower efficacy of CDT. Therefore, a significant amount of ROS generation along with the depletion of GSH is required for improvement in CDT.¹³ In this context, the Cu²⁺ ion plays a key role in converting GSH to GSSG and gets reduced to Cu⁺ ion, which reacts with H₂O₂ to produce ROS via Fenton-like reaction and the plummeted level of GSH leads to the accumulation of ROS.^14,15 Therefore, Cu²⁺-ion based CDT agents are gaining notable attention. To date, several CDT agents have been studied, such as dual cation-containing Zn nitroprusside, CsMnP metal organic frameworks (MOFs), Cu²⁺–pyropheophorbide a–cystine conjugate, cancer cell-coated copper sulfide nanoparticles etc. (Table S1). Glucose oxidase-loaded nanocarriers alone or in combination with metal ions (GOD encapsulated Cu doped ZIFs) have also played a significant role in CDT.¹⁶ However, the complex synthetic procedures and stability issues restrict the clinical applicability of these CDT agents. In this regard, copper-doped carbon dots can be a potential CDT agent due to their intrinsic characteristics. Despite these advantages, reports on Cu–CDs in CDT remain scarce, highlighting a promising route to be explored in cancer therapy.

Targeted therapies focusing on tumor biomarkers offer promise for early detection, prognosis, and selective treatment of TNBC. Carbonic anhydrase-IX (CA-IX), a Zn containing metalloenzyme, is one such biomarker.¹⁷ CA-IX, one of the 15 human isoenzymes of the α-carbonic anhydrase family, is a tumour-associated transmembrane protein that is involved in the regulation of intracellular CO₂ levels. CA-IX facilitates reversible hydration of CO₂ producing proton (H⁺) and bicarbonate ions (HCO₃⁻), which balances intracellular as well as extracellular pH levels to promote tumour cell survival and enhance migratory potential.¹⁸ CA-IX can be a potential biomarker for targeted therapy because of its overexpression in aggressive breast cancer in comparison to normal tissues.¹⁸ Therefore, copper-doped carbon dots with a CA-IX targeting motif could be a novel theranostic agent for improved cancer treatment through CDT. To date, there are very few reports on Cu²⁺-based nanocatalysts, and Cu(II)–BODIPY photosensitizers for combination and photodynamic therapy with the assistance of a CA-IX targeted inhibitor. Some additional attempts were made using CA-IX targeting nanovesicles, silica/iron nanoparticles and small molecule drug conjugates for cancer treatment.^19–25 However, currently there is almost no report on CA-IX targeted Cu²⁺ ion-doped carbon dots as theranostic probes. Keeping this in mind, we aimed to develop CA-IX targeted copper-doped carbon dots as a target specific theranostic probe for treating TNBC via CDT.

In order to synthesize CA-IX targeted carbon dots, carboxyl rich surface-functionalized carbon dots (CD) were prepared from citric acid and diethylenetriamine (1 : 0.3 mol ratio) via hydrothermal method.²⁶ Copper-doped carbon dots (CuCD) were prepared from citric acid, diethylenetriamine and copper nitrate (1 : 0.3 : 0.5 mol ratio) through optimization following the similar protocol. Both carbon dots were surface-modified with the hydrolysed product of acetazolamide (CD-Az and CuCD-Az, Fig. 1a, c and Fig. S1, S2) via EDC-NHS coupling (Scheme S1 and S2) to make it CA-IX target specific. Acetazolamide is a sulfonamide-derived CA-IX inhibitor capable of binding with the active zinc centre of CA-IX.¹⁷ The size of CD-Az and CuCD-Az was around ∼3–5 nm as determined from the respective TEM and AFM images (Fig. 1b, d and Fig. S3). The ζ-potential of the carbon dots was found to be −0.65 mV for CD-Az and +0.61 mV for CuCD-Az. According to the UV-vis spectra, the absorbance maxima of CD and CD-Az were at 350 nm, while the same for copper-doped carbon dots (CuCD and CuCD-Az) got slightly shifted to 345 nm (Fig. 2a) due to copper doping. Additionally, a shoulder peak was observed at around 275–280 nm for both CD-Az and CuCD-Az, which accounted for the carbon dot surface tethered acetazolamide moiety having absorbance in a similar range (Fig. 2a). Therefore, the UV-vis spectra confirmed the successful functionalization of the carbon dots with acetazolamide. Both carbon dots showed blue fluorescence in the visible region under UV torch irradiation (excitation at 365 nm, Fig. 2b and Fig. S4). The emission maxima for CD-Az and CuCD-Az were observed at λ_em = ∼455 nm and ∼460 nm, respectively upon excitation at λ_ex = 350 nm (Fig. 2b and Fig. S4). According to the excitation dependent emission, both carbon dots showed emission maxima at λ_em = ∼460 nm and ∼465 nm for CD-Az and CuCD-Az, respectively, upon excitation at λ_ex = 370 nm (Fig. 2b and Fig. S4). The quantum yields of CD-Az and CuCD-Az were found to be 6% and 5% with respect to the quinine sulfate.

Fig. 1 (a) Structure and (b) TEM image of carbon dot CD-Az and (c) structure and (d) TEM image of carbon dot CuCD-Az.

Fig. 2 (a) UV-vis spectra of the carbon dots and acetazolamide and (b) excitation dependent emission spectra of CD-Az. Inset: image of blue fluorescence of carbon dots under UV torch irradiation.

To ascertain the successful copper doping as well as surface functionalization with acetazolamide, XPS analysis was performed for both CDs. The XPS spectra of CuCD and CuCD-Az showed peaks at 284 eV, 400 eV and 530 eV that denoted C(1s), N(1s) and O(1s) orbitals (Fig. S5a and c). Moreover, two peaks were observed at 931 eV and 951 eV corresponding to Cu 2p_3/2 and Cu 2p_1/2 in the high resolution XPS spectra of both the carbon dots (Fig. S5b and d), which confirmed the successful doping of Cu²⁺. Another peak in the XPS spectra of CuCD-Az was observed at 163 eV, denoting S 2p (Fig. S5a), which was absent in the XPS spectra of CuCD, and ensured the successful functionalization of the carbon dots with acetazolamide. Furthermore, the XPS spectrum analysis of CuCD-Az confirmed that CuCD-Az contained C (48%), N (13%), O (35%), Cu (2%) and S (2%) whereas CuCD contained C (47%), N (8%), O (41%) and Cu (4%). The presence of sulphur in CD-Az was confirmed from EDX data ensuring the existence of an acetazolamide moiety (Fig. S6). FTIR study of CD-Az and CuCD-Az showed two sharp peaks at ∼1180 cm⁻¹ and ∼1350 cm⁻¹ due to the symmetric stretching and asymmetric stretching of S=O bonds in sulfonamides, which is also present in the FTIR spectra of acetazolamide (Fig. S7). Similarly, two sharp peaks at ∼585 cm⁻¹ and 650 cm⁻¹, were observed in the fingerprint region of both CD-Az and CuCD-Az. Thus, the IR spectra supported the presence of acetazolamide on the surface of both the carbon dots. Also, a peak ranging from 20° to 30° in the XRD spectra (Fig. S8) confirmed the amorphous nature of CD-Az and CuCD-Az.

Next, the chemodynamic function of CuCD-Az was tested upon reaction with GSH. The efficacy of CDT is hindered by a natural antioxidant, GSH, which is present in a high concentration in cancer cells and degrades the accumulated ROS to maintain optimal oxidative stress. Cu²⁺ ion is capable of oxidizing GSH into GSSG, that results in the depletion of GSH concentration. Here, the GSH depletion ability of CuCD-Az was studied using DTNB (5,5′-dithiobis-2-nitrobenzoic acid) as a probe. DTNB has an absorbance maxima at ∼325 nm. GSH reacts with DTNB producing 5-mercapto 2-nitrobenzoic acid (TNB), which is yellow in colour and absorbs at ∼410 nm (Scheme S3). The Cu²⁺ ion converts GSH into GSSG, which is unable to react with DTNB and as a result, the absorbance peak for TNB at 410 nm decreases (Fig. 3). Here, with increasing concentration of CuCD-Az (20–200 μg mL⁻¹), the intensity of the absorbance peak for TNB at ∼410 nm steadily decreased, while the absorbance peak intensity for DTNB at ∼325 nm gradually increased. Cu²⁺ in CuCD-Az converted GSH into GSSG leading to the inefficient conversion of DTNB to TNB due to the depleted level of GSH. Notably, at 200 μg mL⁻¹ of CuCD-Az, almost all GSH was converted to GSSG by Cu²⁺ ions as no peak of TNB was observed and only the significant presence of unreacted DTNB was noted (Fig. 3). This ensured the potential candidature of CuCD-Az in CDT.

Fig. 3 DTNB UV-vis study for GSH depletion using CuCD-Az.

Next, we investigated the ability of the acetazolamide-functionalized carbon dots (CD-Az) to selectively target triple negative breast cancer cells over normal cells. Prior to that, the endogenous expression of CA-IX in TNBC, MDA-MB-231 and 4T1 cell lines was confirmed from western blot analysis (Fig. 4a, b, raw data, Fig. S9). The selective uptake of CD-Az in comparison to the non-functionalized CD by cancer cells was also assessed from CA-IX expression (Fig. 4a and b). In the case of CD-Az treated MDA-MB-231 and 4T1 cells, the expression of CA-IX decreased, whereas for non-functionalized CD treated cells, the expression of CA-IX remained unaltered in comparison to that in untreated cells (Fig. 4a and b). The reduced CA-IX expressions in TNBC cells depict the target-specific successful binding of acetazolamide-functionalized carbon dots on the surface of the respective cancer cells. Next, the intrinsic fluorescence of the carbon dots was utilized for selective bioimaging of cancer cells. Fluorescence microscopy revealed the bright blue fluorescence for both MDA-MB-231 and 4T1 cells treated with CD-Az (Fig. 4c and f). On the other hand, faint blue fluorescence was observed in HEK-293 and NIH3T3 cells when treated with CD-Az (Fig. 4i and l). In contrast, when the cells were treated with non-functionalized CD, comparable blue fluorescence was observed in all four cell lines (Fig. S10a–h). It ensured the lack of target specificity of the carbon dots without an acetazolamide moiety. The CA-IX specificity of CD-Az was further confirmed by blocking experiments in 4T1 and MDA-MB-231 cells. Very faint blue fluorescence was observed in both acetazolamide pre-treated 4T1 and MDA-MB-231 cells (Fig. S11) in comparison to untreated cells (Fig. 4c and f). Binding of an acetazolamide moiety to CA-IX prevented it from conjugating with CD-Az resulting in the failure of CD-Az internalization into cancer cells. This further confirmed that the selective internalization of CD-Az in TNBC took place through the interaction between CA-IX and the acetazolamide moiety. To further support the target specificity of CD-Az towards cancer cells, flow cytometric analysis was performed for all four cell lines treated with CD-Az. For MDA-MB-231 and 4T1 cells, significantly high mean fluorescence intensity values of ∼33 600 and ∼48 100 were observed, respectively, whereas for HEK-293 and NIH3T3 cells, they were considerably low at ∼11 500 and ∼12 000 (Fig. 4e, h, k and n, Fig. S12). According to flow cytometry data, ∼3–4-fold selectivity of CD-Az towards TNBC cells was observed in comparison to HEK-293 and NIH3T3 cells (Fig. S12). This is due to the binding of the acetazolamide moiety of CD-Az with overexpressed CA-IX enzyme in TNBC cells leading to bright blue fluorescence inside the cells, while feeble blue fluorescence was observed in non-cancerous cells due to the unavailability of the CA-IX enzyme. All these experiments ascertained that the target specificity of CD-Az toward selective diagnosis of TNBC cells was achieved due to the presence of the acetazolamide moiety.

Fig. 4 Immunoblot analysis of carbonic anhydrase-IX levels in (a) MDA-MB-231 and (b) 4T1 treated with CD and CD-Az for 24 h. β-actin levels are shown as a loading control. Fluorescence microscopic images, brightfield images and corresponding flow cytometric analysis plots of CD-Az treated (c)–(e) MDA-MB-231, (f)–(h) 4T1, (i)–(k) HEK-293 and (l)–(n) NIH3T3 cells. Scale bars correspond to 20 μm. Mean fluorescence intensity values are mentioned in the insets.

After ensuring the target specificity of CD-Az, we have explored the selective cytotoxicity of CuCD-Az via CDT. As mentioned, in the presence of GSH, Cu²⁺ gets reduced to Cu⁺, which upon reaction with intracellular H₂O₂ produces ROS that induces apoptosis in cancer cells. Alongside this, the efficacy of CDT may also increase due to the reduced level of GSH that neutralizes ROS. MTT assay showed that both CD and CD-Az exhibited ∼80% cell viability for up to 3 mg mL⁻¹ (Fig. S13) after 24 h of incubation in all four cell lines ensuring their notable biocompatibility. Furthermore, the hemocompatibility of CuCD-Az was evaluated, which showed dose-dependent hemolysis of RBCs maximum up to ∼35% at the highest concentration of CuCD-Az (3 mg mL⁻¹, Fig. S14) confirming minimal toxicity and potential biocompatibility of CuCD-Az. In case of both CuCD and CuCD-Az, the cytotoxicity got steadily enhanced from ∼10% at 50 μg mL⁻¹ to ∼90% at 3 mg mL⁻¹ for 4T1 cells (Fig. 5a and b). A similar trend was observed for MDA-MB-231 cells, where both CuCD and CuCD-Az induced ∼5% killing at 50 μg mL⁻¹, which increased to ∼85% at the highest concentration of carbon dots (3 mg mL⁻¹). Higher cytotoxicity of CuCD and CuCD-Az in cancer cells arises due to the enhanced CDT by means of efficient conversion of Cu²⁺ to Cu⁺ by overexpressed GSH and subsequent Fenton-like reaction with abundant H₂O₂ that led to the generation of a greater amount of ROS. Notably, CuCD exhibited ∼55% and ∼65% killing in normal HEK-293 and NIH3T3 cells, respectively, while CuCD-Az exhibited only ∼30–35% killing in both of these normal cells (Fig. 5a and b). Acetazolamide functionalization of carbon dots made CuCD-Az more target specific towards TNBC, which resulted in ∼2.8–3 fold higher cytotoxicity of CuCD-Az towards TNBC cells in comparison to HEK-293 and NIH3T3 cells. The scatterplot based on Annexin V-FITC/PI staining showed that in case of CuCD-Az, the majority of treated cells resided in the Q4 quadrant for MDA-MB-231 and 4T1, while for CD-Az they were mostly in the Q3 quadrant (Fig. S15). Thus, the cells underwent apoptosis through the early apoptotic pathway upon treatment with CuCD-Az. Furthermore, an intracellular GSH assay revealed that in the presence of CD-Az (3 mg mL⁻¹), almost no change in GSH concentration was observed in 4T1 and MDA-MB-231 when compared with untreated cells (Fig. S16). However, when the cells were treated with CuCD-Az (3 mg mL⁻¹), ∼70% depletion of GSH concentration was observed in both cell lines (Fig. S16). Hence, there is depletion in intracellular GSH content in the presence of CuCD-Az. According to DCFH-DA staining, bright green fluorescence of 2′,7′-dichlorofluorescein (DCF) was observed in TNBC cells when treated with CuCD-Az (Fig. S17). In contrast, almost no green fluorescence was observed in CD-Az-treated cells (Fig. S17). Thus, DCFH-DA staining confirmed the significant amount of ROS generation by Cu⁺ ions inside cancer cells leading to apoptosis. Therefore, intracellular GSH assay and ROS generation study together confirmed the proposed CDT mechanism that intracellular GSH converted Cu²⁺ to Cu⁺ ions, which produced ROS upon reaction with intracellular H₂O₂.

Fig. 5 Cytotoxicity of (a) CuCD and (b) CuCD-Az in 4T1, MDA-MB-231, HEK-293 and NIH3T3 cells determined by MTT assay. Values are expressed as mean ± SEM from three independent experiments. ***p < 0.001 vs. CuCD and ***p < 0.001 vs CuCD-Az treated 4T1, MDA-MB-231 HEK-293, and NIH3T3 cells.

In summary, we have developed CA-IX targeting acetazolamide-functionalized copper-doped (CuCD-Az) intrinsically fluorescent carbon dots, which showed considerable selectivity in the diagnosis of TNBC cells. CuCD-Az was capable of depleting GSH followed by ROS generation through Fenton-like reaction resulting in ∼3 fold higher killing of cancer cells in comparison to normal cells. Thus, CA-IX targeting CuCD-Az has emerged as a potential theranostic probe for cancer cells via CDT.

A. H. K. and S. P. acknowledge CSIR, India and UGC, India for the respective research fellowships.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the SI. Supplementary information: Experimental methods, synthetic scheme, table on literature survey, ¹³C spectra, HRMS, AFM, fluorescence and XPS spectra of the carbon dots, EDX spectra of CD-Az, IR spectra, XRD spectra, raw data of western blots, bioimaging using CD, bioimaging after pre-treatment with acetazolamide, cytocompatibility, hemolytic assay, flow cytometric plots, intracellular GSH assay, and ROS generation using DCFH-DA staining. See DOI: https://doi.org/10.1039/d5cc04653j.

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