Tumor‐Targeted Radioiodinated Glyconanoparticles for Doxorubicin Delivery and Auger‐Chemotherapy in Triple‐Negative Breast Cancer

Abstract

This study focuses on developing a tumor‐responsive core‐shell glyconanoparticle formulation targeting galectin‐3 (Gal‐3) in triple‐negative breast cancer (TNBC) for Auger‐chemotherapy. The formulation utilizes citrus pectin dialdehyde (CPDA) to create nanoparticles capable of tumor‐responsive release of the drug Doxorubicin (Dox) for chemotherapy and incorporating the radionuclide, Iodine‐125 for Auger therapy. CPDA‐based core‐shell nanoparticles (CPDANP) are engineered for drug Dox release in the tumor microenvironment using boronate ester links between polymeric chains of CPDANP and imine linkages for conjugating Dox to CPDANP (CPDANP‐Dox). The nanoparticles are characterized for their size, effective charge, and core‐shell like structure by DLS and TEM and radiolabeled with ¹²⁵I. Gal‐3 specific targeting of CPDANP‐Dox and ¹²⁵I to the nucleus of tumor cells is confirmed by fluorescence microscopy and estimating radioactivity in the isolated nucleus of cells. In vitro and in vivo studies demonstrated that the combination therapy with [¹²⁵I] I‐CPDANP‐Dox exhibited enhanced cytotoxicity in TNBC cells. SPECT imaging and biodistribution studies showed rapid clearance of the formulations from the bloodstream, and tumor regression studies in 4T1 tumor‐bearing mice confirmed the therapeutic superiority of the combined treatment over individual therapies. This study highlights the potential of CPDANP as a dual‐targeting platform for efficient Auger‐chemo therapy in the treatment of TNBC.

Combining multiple anti‐tumor therapeutic modalities using nanoparticles allows harnessing their therapeutic potential in combination with minimal side effects at lower doses. This study focuses on the development of tumor‐responsive core‐shell glyconanoparticle formulation targeting galectin‐3 in triple‐negative breast cancer (TNBC) for Auger‐chemotherapy. The formulation utilizes citrus pectin dialdehyde (CPDA) to create nanoparticles capable of tumor‐responsive release of the drug Doxorubicin (Dox) for chemotherapy and incorporating the radionuclide, Iodine‐125 for Auger therapy.

1. Introduction

Auger electron therapy is envisioned as the future of precision medicine due to its ability to deposit energy within 2 to 500 nm of tissue near the deposition site with high linear energy transfer (LET) ranging from 4 to 26 keV µm. This selective energy deposition minimizes cytotoxicity to healthy cells.^{[ 1 ]} The integration of targeting molecules, such as peptides, small molecules, or nanoparticles, which localize in the nucleus, enhance the effectiveness of auger therapy by concentrating energy deposition near DNA.^{[ 2 ]} Auger electron emitters when localized in DNA are considered more effective than alpha particle emitters.^{[ 3 ]}Clinically, cancer metastasis often leads to recurrence and patient mortality.^{[ 4 ]} Targeted auger therapy is effective in managing micro metastases of disseminated cancers compared to chemotherapy with less side effects.^{[ 5 ]} With increasing understanding of intra and inter‐tumoral biological heterogeneity, along with insights into tumor resistance mechanisms against various monotherapies, it becomes apparent that combining two or more therapies is a rational approach for cancer management. Integrating multiple therapies at lower doses can help overcome resistance development, improve the therapeutic index, and reduce side effects.^{[ 6 ]}Nanoparticles represent promising carriers for the precise co‐delivery of multiple therapies to tumors, achieving optimal ratios and rates. They enable the customization of personalized treatments, fostering enhanced therapeutic synergy in addressing individual patient tumors.^{[ 7 ]} Nanoparticles offer several advantages viz. increased therapeutic load, controllable and targeted release profiles of drugs, improved drug pharmacokinetics with reduced side effects.^{[ 8 ]}

Herein, we report the formulation of galectin‐3 (Gal‐3) targeted tumor responsive core‐shell glyconanoparticles based on periodate oxidized citrus pectin (citrus pectin dialdehyde, CPDA) incorporating radionuclide Iodine‐125 and drug Doxorubicin (Dox) for auger therapy and chemotherapy of triple‐negative breast cancer (TNBC). CPDA nanoparticles (CPDANP) are shown to have an affinity for a unique lectin Gal‐3 which is upregulated in transformed and metastatic cells and in many human carcinomas. The upregulation of Gal‐3 correlates with immune suppression, progressive tumor stages, and metastasis. Gal‐3 is a potential therapeutic target and prognostic marker for TNBC.^{[ 9} , ^{10 ]} It also contributes to expression of epithelial‐mesenchymal transition‐related gene and its knockdown increases TNBC sensitivity to drugs.^{[ 11 ]} Koo and Jung showed expression of Gal‐3 at higher levels in triple‐negative compared to non‐triple negative cancers.^{[ 12 ]} CPDANP were prepared by previously reported protocol and later functionalized by incorporating radionuclide Iodine‐125.^{[ 13 ]} The detailed CPDA preparation, characterization, and affinity toward Gal‐3 (Kd = 160.90 µM) was ascertained in previous report.^{[ 13 ]}

Iodine‐125 is a reactor produced radioisotope which emits ∼21 Auger electrons of ∼20‐500 eV per atom decay suitable for therapy. It also emits 35 keV gamma rays and 27–32 keV X‐rays.^{[ 14 ] 125}I‐iododeoxyuridine (¹²⁵I‐IUDR) targeting DNA was first reported as a prototype for radionuclide therapy with auger emitters.^{[ 15 ] 125}I was incorporated in CPDANP by conjugating the NPs with radioiodinated Bolton‐Hunter reagent (BHR). One of the most potent Food and Drug Administration (FDA) approved chemotherapeutic drug is Doxorubicin (Dox) for the treatment of various cancers. Dox was conjugated to CPDANP via. pH sensitive imine linkages (CPDANP‐Dox) to facilitate its release in low tumor pH milieu.^{[ 16 ]} The CPDANP were made glucose responsive for Dox release in the tumor microenvironment specifically by the introduction of boronate ester between polymeric chains of NP.

The CPDANP and CPDANP‐Dox prepared were characterized by dynamic light scattering (DLS) and field emission gun transmission electron microscopy (FEG‐TEM) and evaluated for their tumor pH responsive and high‐glucose concentration dependent release of drug. After formulation of radiolabeled CPDANPs, detailed in vitro evaluation in TNBC cells, i.e., MDA‐MB‐231 and 4T1 was performed. [¹²⁵I] I‐CPDANP‐Dox was observed to significantly localize in the cell nucleus. Viability assay, cell cycle arrest analysis, and analysis of mode of cell death in response to different combination treatment of ¹²⁵I and Dox incorporated in CPDANP was performed. Formulations were evaluated for their therapeutic effect in animal model of tumor. Bioimaging and biodistribution studies after intravenous and intratumoral injection of ¹²⁵I‐CPDANP‐Dox in BALB/c mice bearing 4T1 tumor for evaluating pharmacokinetics and tumor uptake were done. Tumor regression studies for evaluating the in vivo combinatorial therapeutic effect of ¹²⁵I and Dox using CPDANP were performed. These studies confirmed the potential of [¹²⁵I] I‐CPDANP‐Dox as a promising therapeutic modality for Auger‐chemo therapy of TNBC.

2. Experimental Section

2.1. Materials

Citrus pectin, sodium metaperiodate, Bolton‐hunter reagent, chloramine‐T, sodium‐ metabisulfite, potassium iodide, lactose, Cellytic nuclear extraction kit, and dialysis tubing were procured from Sigma‐Aldrich; USA. Doxorubicin hydrochloride was obtained from RPG Life Sciences, India. Kits for viability, cell cycle, and apoptotic nexin assays were procured from Guava Technologies, Inc., Merck Millipore Corp., Germany. ¹²⁵Iproduced in Dhruva reactor, BARC was utilized for radiolabeling studies. TNBC cells 4T1 was obtained from ATCC, CRL‐2539, and MDA‐MB‐231 were from NCCS, Pune, India. For cell culture, Dulbecco's Modified Eagle's medium (DMEM), antibiotic/antimycotic solution, trypsin solution, and fetal bovine serum (FBS) were procured from Himedia Laboratories, India. Well‐type NaI (Tl) detector (ECIL, India) was used for measuring radioactivity. An HPLC system from JASCO (Tokyo, Japan) equipped with a C‐18 reversed phase HiQ Sil (5 µm, 4 × 250 mm) column coupled to a PU 1575 UV–visible detector was used for HPLC analyses. The chromatograms were evaluated using the GINA STAR software (Version 4, M/s. Raytest, GmBH, Germany). ITLC‐SG paper from Agilent technologies (Lakefront, CA USA) was used for estimation of radiochemical yield of the complexes. Guava EasyCyte Flow cytometer (Guava Technologies, Inc., Merck Millipore Corp. Germany) was used to acquire flow cytometry data, and analyses was performed by Cyflogic v. 1.2.1. Single photon emission computed tomography (SPECT) images were acquired using Bioemtech γ‐eye system.

3. Methods

3.1. Preparation and Characterization of CPDANP‐Dox

CPDANP were prepared and characterized as per protocol reported previously.^{[ 13 ]} In brief, citrus pectin (CP) was partially oxidized using periodate in heterogeneous medium to yield CP dialdehyde (CPDA) oligomers. For this, CP (5.00 g, 26.5 mmol) dispersed in ethanol (100 mL) was treated with 100 mL of aqueous solution of sodium periodate (2.83 g, 13.2 mmol) with stirring at room temperature under a dark environment to get CPDA. The degree of oxidation was analyzed by iodometry. The mixture was dialyzed against distilled water after 4 h of the reaction, until the dialysate was free of periodate and the dialysate was lyophilized. CPDANP were prepared by using CPDA treated with 0.1 M Borax to introduce glucose‐responsive boronate ester between the diols of CPDA and borax. Briefly, stearic acid (12.00 mg, 42.2 µmol) dissolved in methanol (3 mL) was added dropwise under sonication (Ultrasonic Processor, Sonics VCX 130) to solution of CPDA (≈12.00 mg) in 0.1% Tween 80 (20 mL) containing 0.1 M borax (100 µL). Methanol was subsequently removed under vacuum, and the aqueous solution was centrifuged at 20,000 g for 1 h at 4 °C. The pellet obtained was resuspended in MilliQ water (6 mL). Dox was conjugated to CPDANP via pH‐responsive imine linkages as per protocol previously reported and schematically shown in Figure 1 .

Figure 1.

Schematic representation of preparation of [¹²⁵I] I‐CPDANP‐Dox.

Briefly, for drug loading, 6 mg of nanoparticles were resuspended in MilliQ water (2 mg/mL) and treated with 1 mL of Dox (4.50 mg mL⁻¹, 7.8 µmol) for 1 h under stirring. This was followed by addition of 1 mL of CPDA (9 mg mL⁻¹ solution in 0.1 M borax) and leaving the solution overnight at room temperature under constant stirring. The suspension was then centrifuged at 20000 g for 1 h at 4 °C. The pellet was resuspended in 6 mL of MilliQ water and the supernatant was analyzed for the remaining amount of Dox by noting the absorbance at 485 nm by UV–visible spectroscopy. The CPDANP and CPDANP‐Dox were characterized by DLS and FEG‐TEM. The release of Dox in tumor mimicking environment (pH 6 + 25 mM glucose), pH 6, and in physiological pH 7.4 was evaluated using sink buffer of 0.1 M phosphate buffered saline (PBS) by dialysis bag method. At different time intervals, dox released was followed by monitoring absorbance at 485 nm using high‐performance liquid chromatography (HPLC) (C18 reverse phase column using 90% acetonitrile: 10% water with 0.05% trifluoroacetic acid (TFA) as mobile solvent). The stability of CPDANP and CPDANP‐Dox in plasma and saline was observed using DLS up to 144 h.

3.2. Radioiodination of CPDANP Using Bolton‐Hunter Reagent

The CPDANP‐Dox and CPDANP was radiolabeled with ¹²⁵I for auger therapy using Bolton‐Hunter reagent (BHR) using a two‐step procedure. First, BHR was radioiodinated using chloramine‐T method. For this, 2 µg BHR (1 mg mL⁻¹ in DMSO) was added to phosphate buffer (0.25 M, pH‐7) followed by ¹²⁵I(4‐5 mCi) and 50 µg chloramine‐T (5 mg mL⁻¹ in 0.25 M phosphate buffer, pH‐7) to initiate the radiolabeling. After 1 min, the reaction was terminated by the addition of 100 µg sodium‐metabisulfite (10 mg/mL in 0.05 M phosphate buffer, pH‐7) and 200 µg potassium iodide (20 mg mL⁻¹ in 0.05 phosphate buffer, pH‐7). Thereafter, the reaction mixture was transferred to a glass tube containing 5 µL DMF and radioiodinated BHR was extracted using benzene. Benzene was evaporated from radioiodinated BHR under a stream of nitrogen gas. In second step, 2 mg CPDANP‐Dox or CPDANP (4 mg mL⁻¹ in borate buffer 0.25 M, pH‐ 8.5) was added to radioiodinated BHR and incubated for 1 h at 4 °C with gentle shaking. The radiolabeling yield of BHR and CPDANP‐Dox and CPDANP was evaluated by thin layer chromatography (TLC) using ethyl acetate: toluene (1:1) and saline as mobile phase. The R_f value for radiolabeled CPDANP‐Dox or CPDANP and free ¹²⁵I is 0.0–0.2 and for radiolabeled BHR is 0.6–0.7 in ethylacetate: toluene (1:1) as mobile phase. In saline, the R_f of free ¹²⁵I is 0.9–1, and of radiolabeled CPDANP‐Dox or CPDANP is 0.0–0.2. The radiolabeled CPDANP was purified from unconjugated radiolabeled BHR by centrifugation at 14000 rpm for 10 min. The stability of radiolabeled CPDANP in plasma and saline at 4 and 37 °C for up to 144 h was evaluated.

3.3. Cell culture and Treatment Doses

TNBC cells, 4T1 and MDA‐MB‐231 were grown in DMEM media with 10% FBS in a humidified atmosphere at 37 °C, 5% CO₂ incubator. Cells were trypsinized using 0.1% trypsin and harvested in DMEM media containing 10% FBS. For all experiments until otherwise mentioned, Cells (≈1 × 10⁵) were seeded in 12 well tissue culture plates in triplicates and allowed to grow till 60–70% confluence overnight before giving treatment. All treatments doses for ¹²⁵I are in µCi and for Dox are in µM incorporated in CPDANP in a total volume of 1 mL DMEM with 10% FBS.

3.4. Gal‐3 Specific Uptake and Nuclear Localization Studies

For evaluating Gal‐3 specific uptake of [¹²⁵I] I‐CPDANP‐Dox by fluorescence microscopy, 4T1 and MDA‐MB‐231 cells (5 × 10⁴) cells were seeded on poly‐L‐Lysine coated cover slips in 24 well plates and incubated overnight in triplicates. Cells were then incubated with [¹²⁵I] I‐CPDANP‐Dox (5 µCi + 0.1 µM) for 3 h in serum free media at 37 °C. For observing Gal‐3 Specific uptake of [¹²⁵I] I‐CPDANP‐Dox, another set of cells were pre‐incubated with lactose (10 mM) for 1 h followed by treatment. Lactose is a known Gal‐3 binding ligand, and was therefore used to inhibit the Gal‐3 specific uptake of [¹²⁵I] I‐CPDANP‐Dox.^{[ 13 ]} After treatment, cells were washed twice with PBS (0.05 M, pH‐7.4) and fixed with ice chilled 4% paraformaldehyde followed by DAPI staining. The Dox uptake and its inhibition in the presence of lactose inside cells was observed by confocal microscope IX3SVR using an Olympus IX83 inverted microscope with the laser beams focused on the back focal plane of a 40 × 0.75 NA apochromatic objective lens (Olympus). The intensity of laser illumination at samples was adjusted by FLUOVIEW software. The images were processed and quantified for Dox uptake using Olympus cellSens software and adobe photoshop 7.0. The mean fluorescence intensity of Dox was plotted on different treatments using Graphpad prism 6.

Gal‐3‐specific cellular uptake of [¹²⁵I]I‐CPDANP‐Dox (5 µCi + 0.1 µM) was also assessed by measuring the radioactivity (¹²⁵I) associated with the cells. For this, cells were seeded in a 6‐well plate and incubated overnight (n = 3). Treatments were performed as previously described for [¹²⁵I]I‐CPDANP‐Dox uptake observed via fluorescence microscopy. After treatment, cells were washed twice with PBS (0.05 M, pH 7.4), harvested, and collected in vials for radioactivity measurement. The cellular uptake was quantified using a NaI (Tl) scintillation counter. The percentage uptake was calculated and plotted using GraphPad Prism 6.

For Auger therapy to be effective by depositing energy near DNA, nuclear localization of [¹²⁵I]I‐CPDANP‐Dox is a prerequisite. The localization of Dox incorporated in [¹²⁵I]I‐CPDANP‐Dox (5 µCi + 0.1 µM) within the nuclei of 4T1 and MDA‐MB‐231 cells was evaluated by observing Dox fluorescence alongside DAPI nuclear staining using fluorescence microscopy at 6 and 24 h post‐treatment (n = 3). Images were acquired at 20× magnification using an Axioplan imaging fluorescence microscope. The localization of ¹²⁵I from [¹²⁵I]I‐CPDANP‐Dox in the cell nucleus was evaluated by isolating nuclei after treatment with [¹²⁵I]I‐CPDANP‐Dox or [¹²⁵I]I‐CPDANP for 6 and 24 h in complete media. Nuclear isolation was performed using the CelLytic NuCLEAR Extraction Kit (Sigma), and ¹²⁵I activity was measured using a NaI (Tl) scintillation counter (n = 3).

3.5. Cytotoxicity Studies

For evaluating the combinatorial therapeutic effect of ¹²⁵I and Dox using CPDANP, cytotoxicity studies in TNBC cell lines, 4T1 and MDA‐MB‐231 after treatment with [¹²⁵I] I‐CPDANP‐Dox (50 µCi – 0.4 µCi + 1 µM‐0.008 µM), [¹²⁵I] I‐CPDANP (50 µCi −0.4 µCi), and CPDANP‐Dox (1 µM‐0.008 µM) were carried out (n = 3). The viability assay kit Guava ViaCount, Millipore was used for cytotoxicity studies. Briefly, 50 µL of treated and untreated 4T1 and MDA‐MB‐231 cells from 12 well plate were mixed with 450 µL of guava viacount reagent and incubated in dark for 5 min. Sample acquisition and data analysis was performed using the ViaCount software module in the Guava EasyCyte Flow cytometer. The % cell death in treated compared to untreated cells was calculated and plotted against different combination treatments.

3.6. Cell cycle Analysis

For ascertaining the cellular response of TNBC 4T1 cells on combinatorial treatments of [¹²⁵I] I‐CPDANP‐Dox, cell cycle analysis using Guava cell cycle reagent kit was performed. Cell cycle progression was analyzed at 24 h in 4T1 cells treated with different concentrations of [¹²⁵I] I‐CPDANP‐Dox (50 µCi + 1 µM, 25 µCi + 0.5 µM, 12.5 µCi + 0.25 µM, & 5 µCi + 0.1 µM), [¹²⁵I] I‐CPDANP (50 µCi, 25 µCi, 12.5 µCi, & 5 µCi), CPDANP‐Dox (1 µM, 0.5 µM, 0.25 µM & 0.1 µM). For cell cycle analysis, untreated and treated 4T1 cells after 24 h were harvested using trypsin and washed with 0.05 M PBS, pH 7.4 twice, and resuspended in PBS. The cells were fixed in ice cold 70% ethanol and kept at 4 °C for at least 1 h. For staining, ethanol was completely removed and washed with PBS, pH 7.4 twice. The cells were then resuspended in 200 µL Guava cell cycle reagent and incubated for 30 min at RT in dark. After staining, samples were acquired using the Cell Cycle software module in Guava EasyCyte Flow cytometer and processed using Cyflogic software. The percentage of cells at different stages of the cell cycle after different combination treatments and their corresponding monotherapies in 4T1 cells were calculated and plotted using Graphpad prism 6.

3.7. Mode of Cell Death Analysis

Apoptosis is programmed cell death that, unlike necrosis, does not trigger inflammation due to the absence of uncontrolled release of intracellular contents.^{[ 17 ]} To evaluate the apoptotic/necrotic mode of death in 4T1 cells after treatment with [¹²⁵I] I‐CPDANP‐Dox (50 µCi + 1 µM, 25 µCi + 0.5 µM, 12.5 µCi + 0.25 µM & 5 µCi + 0.1 µM), [¹²⁵I] I‐CPDANP (50 µCi, 25 µCi, 12.5 µCi & 5 µCi), CPDANP‐Dox (1 µM, 0.5 µM, 0.25 µM & 0.1 µM) at 24 h, Guava nexin kit was used. It utilizes Annexin V‐PE to detect phosphatidyl serine (PS) exposed on the external membrane of cells as a marker of apoptosis and 7‐AAD, dye as an indicator of cell membrane structural integrity. Annexin V‐PE (+) indicate apoptotic death whereas, 7‐AAD (+) and Annexin V‐PE (−) indicate necrotic death. After treatment for 24 h, cells were harvested using trypsin and ∼1 × 10⁵ cells were mixed with 100 µL of Guava nexin reagent. After incubation for 20 min in dark at room temperature, samples were acquired using the Nexin software module in Guava EasyCyte Flow cytometer. Apoptotic death and necrotic death were plotted in response to different combination treatments using Graphpad prism 6.

3.8. Bioimaging and Biodistribution Studies

To evaluate the pharmacokinetics and tumor uptake of radioiodinated CPDANP, 4T1 tumors were induced in right thigh of BALB/c mice by injecting ∼7000 4T1 cells in serum‐free DMEM. Tumors of size 60–100 mm³ were observed at 10–12 days after injection. ∼370–444 KBq/100 µL of [¹²⁵I] I‐CPDANP‐Dox or [¹²⁵I] I‐CPDANP were injected intravenously (i.v.) via tail vein of mice (n = 3). SPECT imaging and biodistribution studies were performed at 1 h, 4 h, 24 h, and 48 h post injection (p.i). Studies with radioiodinated CPDANP were also performed after intratumoral injection in 4T1 tumors induced in BALB/c mice at 24 h, 48 h, 72 h, and 144 h p.i. At different time points, animals were euthanized and sacrificed in chamber saturated with CO₂. Different organs of interest were then excised, weighed, and counted for associated activity on a flat‐bed NaI (Tl) gamma counter. The percentage of injected dose per gram (% ID/g) of radioformulations in different organs were calculated and plotted using Graphpad prism 6 software. The accumulation of ¹²⁵I activity in tumor after injecting [¹²⁵I] I‐CPDANP‐Dox was correlated with Dox accumulation in tumor. Dox was extracted from tumor using 2 mL of chloroform/methanol mixture (9/1, v/v) thrice. The organic layer was removed and Dox was dissolved in PBS for analysis using C18 reverse phase column HPLC. For determination of extraction efficiency of Dox from tumor, known concentration of Dox was injected into tumor tissue and extracted. Dox concentration in [¹²⁵I] I‐CPDANP‐Dox treated tissues were then calculated. Blood was collected by cardiac puncture using syringe, weighed, and counted for radioactivity.

3.9. Tumor Growth Inhibition Studies

For evaluating the in vivo combination therapeutic effect of [¹²⁵I] I‐CPDANP‐Dox (100 µCi + 3 mg k⁻¹g) compared to monotherapies of [¹²⁵I] I‐CPDANP (100µCi) and CPDANP‐Dox (3 mg), formulations were injected intratumorally (tumor size: 80–110 mm³) twice at 0 and 7 days (n = 4). The tumor size was measured using Vernier caliper till the tumor size reached 1500 mm³. The tumors were then excised and photographed. The tumor volume was calculated and relative tumor growth rate was plotted against number of days post treatment in response to different treatments. The animal and different organ weights were monitored and compared with buffer control to observe side effect of treatments.

3.10. Toxicity Studies of CPDANP Formulations

Toxicity studies of CPDANP‐Dox and CPDANP in normal BALB/c mice (n = 4) were performed to observe the detrimental effects of treatment on critical organs viz. heart, kidney, intestine, and liver. Control group were treated with buffer and the other group were treated with CPDANP‐Dox (Dox concentration 15 mg k⁻¹g) and CPDANP (75 mg k⁻¹g)) at 0 and 7 days. Animals were observed for physical activeness, change in volume of water uptake or any change in body weight of animals. At 9 days, animals were sacrificed and blood were collected by cardiac puncture in heparin‐coated blood collection tube. The CBC profiling, heart, kidney and liver function test were analyzed by different biochemical test with blood and values were plotted using Graphpad prism 6. The organs heart, kidney, intestine, and liver were harvested and preserved in 10% formalin for histopathological investigation to observe side effects of formulations.

3.11. Statistical Analysis

All experiments were performed in triplicate, and the data are presented as mean ± SEM. Statistically significant differences between the treatment groups were analyzed using one‐way analysis of variance (ANOVA), followed by Tukey's multiple comparison test, using GraphPad Prism 6. For comparisons between two groups, unpaired t‐test was performed using the same software.

3.12. Ethics Approval

The animal studies reported in the present article were approved by the Institutional Animal Ethics Committee (IAEC) of BARC, Mumbai, India with project approval number BAEC/19/22. All animal experiments were carried out in strict compliance with the institutional guidelines following the relevant national laws related to the conduct of animal experimentation.

3.13. Results

3.13.1. Preparation and Characterization of CPDANP‐Dox

CPDANP and CPDANP‐Dox were successfully prepared by nanoprecipitation method from CPDA. NPs were characterized by DLS and FEG‐TEM. DLS analysis showed a Z‐average value of 64.8 ± 3 nm and 0.17 ± 0.02 PI (polydispersity index) (Figure 2a,i ) value of CPDANP while Z‐average value of 71.3 ± 4 nm and 0.21 ± 0.03 PI (polydispersity index) (Figure 2b, i) value was observed for CPDANP‐Dox. FEG‐TEM analysis revealed a core‐shell structure of CPDANP with an average diameter of 31 ± 4 nm (Figure 2a,ii) and 39 ± 6 nm for CPDANP‐Dox [Figure 2b, ii)]. The drug release study showed significantly higher release of Dox from CPDANP in a tumor‐mimicking environment (pH 6 with 25 mM glucose), followed by pH 6 alone, and then physiological pH 7.4 (P < 0.05) (Figure S1).

Figure 2.

Dynamic light Scattering (DLS) and Transmission electron microscopy (TEM) analysis of CPDA nanoparticles.

3.14. Radioiodination of CPDANP‐Dox Using Bolton‐Hunter Reagent

CPDANP‐Dox and CPDANP were radiolabeled with ¹²⁵I using bifunctional chelator BHR. The Radiolabeling yield of BHR was 61 ± 3%. The radioiodinated BHR was conjugated to CPDANP‐Dox and CPDANP with a 93 ± 4% radiolabeling efficiency. The specific activity of radioiodinated CPDANP‐Dox and CPDANP was 1.8 ± 0.2 µCi / µg of nanoparticles. The purified [¹²⁵I] I‐CPDA‐NP‐Dox was analyzed by ITLC for purity. The radioiodinated [¹²⁵I] I‐CPDANP‐Dox was found to be stable in saline as well as in plasma (1:10) at 4 °C and 37 °C with RCP retaining > 95% up to 144 h (Figure S2).

3.15. Gal‐3 specific Uptake and Nuclear Localization Studies

Cell uptake and inhibition studies using lactose demonstrated Gal‐3‐specific uptake of [¹²⁵I]I‐CPDANP‐Dox in 4T1 and MDA‐MB‐231 cells, as observed by fluorescence microscopy using 470/590 nm excitation/emission wavelengths for Dox and 402/460 nm for DAPI (Figure 3a). The mean fluorescence intensity graph showed a significant decrease in Dox fluorescence intensity (P < 0.0001) following pre‐incubation with lactose, indicating Gal‐3‐specific uptake of [¹²⁵I]I‐CPDANP‐Dox (Figure 3b).

Figure 3.

a) Fluorescence images of 4T1 and MDA‐MB‐231 cells incubated with [¹²⁵I] I‐CPDANP‐Dox or [¹²⁵I] I‐CPDANP‐Dox + Lactose. b) Graph for mean fluorescence intensity of Dox inside cells (n = 200) on different treatments. The mean fluorescence intensity between 2 groups was statistically compared using unpaired T‐test (∗∗∗∗ represent p < 0.0001). c) Graph for % specific uptake of radiolabeled CPDANP‐Dox in cells when incubated with [¹²⁵I] I‐CPDANP‐Dox or [¹²⁵I] I‐CPDANP‐Dox + Lactose. The % specific uptake between 2 groups was statistically compared using unpaired T‐test (∗ represent p < 0.05). d) Fluorescence images of 4T1 and MDA‐MB‐231 cells incubated with [¹²⁵I] I‐CPDANP‐Dox for 6 h and 24 h stained with DAPI.

Cellular uptake studies based on ¹²⁵I measurement further validated the Gal‐3‐specific uptake of [¹²⁵I]I‐CPDANP‐Dox in 4T1 and MDA‐MB‐231 cells. The % specific uptake graph showed a significant decrease in [¹²⁵I]I‐CPDANP‐Dox uptake (P < 0.05) following pre‐incubation with lactose, consistent with the results of confocal microscopy (Figure 3c). The nuclear localization study demonstrated the presence of Dox in the nucleus of 4T1 and MDA‐MB‐231 cells at 6 h and 24 h post‐treatment with [¹²⁵I]I‐CPDANP‐Dox, as visualized by DAPI nuclear staining (Figure 3d). Upon nuclear isolation and ¹²⁵I activity measurement after treatment with [¹²⁵I]I‐CPDANP‐Dox or [¹²⁵I]I‐CPDANP, ∼23 ± 3% of the total radioactivity was localized in the nuclei of 4T1 and MDA‐MB‐231 cells.

3.16. Cytotoxicity Studies

Cytotoxicity studies in 4T1 and MDA‐MB‐231 cells showed enhanced combinatorial therapeutic effect at different treatment dose of Dox and ¹²⁵I. ∼50% cell death in 4T1 cells was observed at (6.25 µCi + 125 nM) combinatorial dose of [¹²⁵I] I‐CPDANP‐Dox compared to monotherapy doses of 50 µCi [¹²⁵I] I–CPDANP and 250 nM CPDANP‐Dox when treated for 24 h (Figure 4a). Similarly, in MDA‐MB‐231 cells, ∼50% cell death was observed at a combinatorial dose (12.5 µCi + 250 nM) of [¹²⁵I] I‐CPDANP‐Dox compared to monotherapy doses of 50µCi [¹²⁵I] I–CPDANP and 500 nM CPDANP‐Dox (Figure 4b). Dox control in toxicity studies showed lower cytotoxicity compared to CPDANP‐Dox.Vehicle control CPDANP did not show any toxicity in 4T1 or MDA‐MB‐231 cells (Results not shown).

Figure 4.

a,b) Scatter plot of the viability assay showing death (%) of TNBC cells in response to different doses of formulations at 24 h post treatment of 4T1 cells & MDA‐MB‐231 cells respectively. c) Cell cycle analysis in 4T1 cells at 24 h post treatment with different doses of formulations by Flow cytometry. % of cells in different stages of cell cycle on different treatments were statistically compared using one way analysis of variance followed by Tukey's multiple comparison tests (∗ represent p < 0.05; ∗∗ represent p < 0.01; ∗∗∗ represent p < 0.001; ∗∗∗∗ represent p < 0.0001).

3.17. Cell Cycle Analysis

Cell cycle analysis in response to different doses of ¹²⁵I and Dox incorporated in CPDANP showed G1 arrest at higher combinatorial dose (50 µCi + 1 µM) of [¹²⁵I] I‐CPDANP‐Dox compared to G2/M arrest after respective monotherapy treatments.

Other lower combinatorial doses (25 µCi + 0.5 µM, 12.5 µCi + 0.25 µM, & 5 µCi + 0.1 µM) of [¹²⁵I] I‐CPDANP‐Dox used caused G2/M arrest in combination or monotherapy treatment (Figure 4c). The % of arrested cells decreased with combination treatment compared with their monotherapy treatment. The % of arrested cells were found to be inversely correlated with the % death of cells after different treatments.

3.18. Mode of Cell‐Death Analysis

The combination treatment of different doses of [¹²⁵I] I‐CPDANP‐Dox (50 µCi + 1 µM, 25 µCi + 0.5 µM, 12.5 µCi + 0.25 µM, & 5 µCi + 0.1 µM) showed apoptotic as well necrotic death in 4T1 cells which increased with the higher doses of treatment. The monotherapy using [¹²⁵I] I‐CPDANP (50 µCi, 25 µCi, 12.5 µCi & 5 µCi) caused significant necrotic death compared to apoptotic death. CPDANP‐Dox (1 µM, 0.5 µM, 0.25 µM & 0.1 µM) treatment caused both necrotic as well as apoptotic death which increased with the higher doses of treatment (Figure 5 ).

Figure 5.

Flow cytometry analysis after Annexin‐V and 7‐AAD (7‐Aminoactinomycin D) staining in 4T1 cells at 24 h post‐treatment with different doses of formulations. % of cells undergoing apoptosis or necrosis on different treatments were compared using one‐way analysis of variance followed by Tukey's multiple comparison tests (ns represent non‐significant; ∗ represent p < 0.05; ∗∗ represent p < 0.01; ∗∗∗∗ represent p < 0.0001).

3.19. Bioimaging and Biodistribution Studies

The radioiodinated CPDANP and CPDANP‐Dox showed similar biodistribution pattern and % tumor uptake. SPECT images at different time points after i.v. injection of [¹²⁵I] I‐CPDA NP‐Dox are shown in Figure 6a. Significant uptake of the radioformulation in tumor and fast clearance of activity from circulation was observed. The tumor uptake of radioiodinated CPDANP‐Dox was 1.6 ± 0.4% ID/g at 1 h p.i. in 4T1 tumor‐bearing BALB/c mice (Figure 6b). The concentration of Dox, measured by HPLC after extraction from tumor tissue, correlated well with the % tumor uptake determined by radioactivity measurement. Maximum accumulation of the radioiodinated formulation was observed in the intestine, followed by the stomach and liver at 1 and 4 h p.i., with clearance through the hepatobiliary route by 24 and 48 h. A significant percentage of the injected activity was detected in urine and stool at 4 h, and almost all activity was cleared by 24 h (Figure 6b), indicating rapid pharmacokinetics and clearance via both hepatobiliary and renal routes.

Figure 6.

a) SPECT images of animal injected with [¹²⁵I] I‐CPDA NP‐Dox via i.v route after different time points. b) Biodistribution profile of [¹²⁵I] I‐CPDANP‐Dox formulation in BALB/c mice bearing 4T1 tumor after intravenous injection at different time points. c) SPECT images of animals injected with [¹²⁵I]I‐CPDANP‐Dox intratumorally after different timepoints. d) Biodistribution profile of the [¹²⁵I] I‐CPDANP‐Dox formulation in BALB/c mice bearing 4T1 tumor after intratumoral injection at different time point. e) Tumor growth inhibition studies in 4T1 tumor‐bearing BALB/c mice after treatment with different formulations at 0 and 7 days. Relative tumor volume growth rate between treatments at end point was compared using unpaired T‐test (∗ represent p < 0.05) b.4T1. f) Tumors from animals treated with different formulations (n = 4).

Tumor uptake of [¹²⁵I]I‐CPDANP‐Dox was not significantly high following i.v. injection; hence, intratumoral injection was preferred to evaluate the combinatorial therapeutic effect of Dox and ¹²⁵I incorporated into [¹²⁵I]I‐CPDANP‐Dox in 4T1 tumors in vivo. Percentage uptake values of [¹²⁵I]I‐CPDANP‐Dox in 4T1 tumors following intravenous and intratumoral administration in the animal tumor model are provided in Table S1. Accordingly, bioimaging and biodistribution studies were conducted following intratumoral injection of the radioiodinated CPDANP to assess tumor retention for therapeutic efficacy (Figure 6c,d). Substantial tumor retention of the formulation was observed, which decreased over time, with 9.5 ± 3.9% of the injected dose per gram of tumor remaining at 144 h post‐injection (Figure 6d). Thyroid uptake increased over time, indicating deiodination of CPDANP. Uptake in other vital organs was minimal following intratumoral injection (Figure 6d).

3.20. Tumor Growth Inhibition Studies

The combination treatment of [¹²⁵I] I‐CPDANP‐Dox (100 µCi + 3 mg kg⁻¹) showed significant inhibition of tumor growth compared to respective monotherapy treatment of [¹²⁵I] I‐CPDANP (100 µCi) and CPDANP‐Dox (3 mg) when followed till tumor size of animals injected with buffer control reached 1500 mm³ (P < 0.05) (Figure 6e). The images of tumors in response to different treatments at end point are shown in Figure 6f. CPDANP‐Dox (3 mg) significantly inhibited tumor growth compared to [¹²⁵I] I‐CPDANP (100µCi). This study indicates the enhanced therapeutic effect of Dox and ¹²⁵I in combination using CPDANP in vivo compared to monotherapy. The weight of animals and its different organs did not change during treatment compared to buffer control. To validate the therapeutic differences between the nano‐drug and free Dox, another group of animals was injected with the free Dox formulation. The results demonstrated significantly greater tumor regression with CPDA‐Dox compared to the Dox control group(Figure S3).

3.21. Toxicity Studies of CPDANP Formulations

All experimental animals in buffer, CPDANP‐Dox, and CPDANP treated group showed no difference in physical activeness, volume of water uptake, and body weight till 9 days of observation. The CBC profiling of blood showed no significant difference in various observed parameters between buffer treated and nanoparticles treated groups. There was no indicative increase of biochemical markers for kidney and liver toxicity between buffer and nanoparticles treated group. LDH observed for heart toxicity showed a statistical increase in CPDANP‐Dox treated group compared to buffer group (p < 0.05). Blood urea nitrogen also showed significant increase in CPDANP‐Dox treated group compared to other groups (p < 0.05). Though any abnormalities in the heart, kidney, liver, and intestine was not observed in histopathological analysis (Figure S4).

4. Discussion

The targeted auger therapy to the nucleus of tumor cells is envisaged as a potential anti‐tumor therapeutic modality with negligible side effects.^{[ 18 ]} Combining well‐established anti‐tumor therapeutic modalities using nanoparticles allows harnessing their therapeutic potential in combination with minimal side effects at lower doses. It also restricts the development of resistance in response to monotherapies. Advanced nanoparticles facilitate active targeting of tumors and can be customized to integrate various therapeutic modalities while enhancing pharmacokinetics. Tumor environment responsive nanoparticles allow controlled release of drugs into tumor sparring dose to other critical organs.^{[ 19 ]}

In the present work, previously reported Gal‐3 targeting tumor responsive CPDANP were utilized for auger‐chemo therapy by incorporating radioisotope, I‐125, and drug Doxorubicin.^{[ 13 ]} Citrus pectin (CP) has been in pharmaceutical use for antitumor activity due to its Gal‐3 inhibition property.^{[ 20 ]} Carbohydrates show low affinity for Gal‐3 but have strong interaction and specificity when present in modified nanoparticle form due to multivalent interactions also known as glycocluster effect.^{[ 21 ]} The CPDANP described herein, prepared from CPDA via nanoprecipitation, has been previously reported to exhibit strong binding affinity for Gal‐3.^{[ 13 ]} This binding was shown to inhibit Gal‐3 expression and reduce homotypic cellular aggregation, tumor cell adhesion to endothelial cells, and endothelial tube formation—key processes involved in cancer progression^{[ 13 ]}

CPDANP formulated using nanoprecipitation method were of size below 100 nm when characterized by DLS and FEG‐TEM. The particles below 100 nm (at least in 1D) display enhanced physio‐chemical properties suitable for different applications.^{[ 22 ]} The drug Dox was successfully conjugated to nanoparticles via. tumor pH responsive imine linkage. The borate‐diol complexation in CPDANP aids in loosening of NPs in the presence of higher glucose concentration. This causes more release of drugs in tumor mimicking environment (low pH and high glucose concentration). The CPDANP were successfully radioiodinated via. BHR with ¹²⁵I. The free radioiodinated BHR was easily separated from radioiodinated CPDANP by centrifugation. Gal‐3‐specific uptake of [¹²⁵I]I‐CPDANP‐Dox was confirmed by observing Dox fluorescence using microscopy and by measuring ¹²⁵I uptake in cells following pre‐incubation with lactose, a known Gal‐3 binding ligand. The nuclear localization study of [¹²⁵I] I‐CPDANP‐Dox showed significant accumulation of Dox and ¹²⁵I in cell nuclei for auger‐chemo therapy. The cytotoxicity studies after combinatorial treatments of [¹²⁵I] I‐CPDANP‐Dox showed 50% cell death at lower doses than individual therapies in TNBC cells. The cytotoxicity was higher with increase in the dose of combination treatments compared to individual therapies.

Similar enhanced combined therapeutic effect using liposomal formulation incorporating Dox and ¹²⁵I‐labeled PARP inhibitor‐01 (∼1 MBq/10⁶ cells) in TNBC was reported by Sankaranarayana et al., 2023. The study revealed [¹²⁵I]‐PARPi‐01 in combination with liposomal Dox caused significant DNA damage and increased therapeutic response in breast cancer cells.^{[ 23 ]} Another study using ¹²⁵I in combination with platinum nanoparticles for chemo‐auger electron therapy of hepatocellular carcinoma showed encouraging results.^{[ 5 ]}

In our study, mitotic arrest analysis showed a decrease in the percentage of arrested cells with increasing doses of individual [¹²⁵I]I‐CPDANP or CPDANP‐Dox, as well as with the combinatorial treatment of [¹²⁵I] I‐CPDANP‐Dox. Similar observations have been reported in the literature, where Cisplatin‐treated HL60 leukemia cells exhibited increased mitotic arrest in the G0/G1 phase and decreased cytotoxicity with lower effective treatment doses.^{[ 24 ]} Another study on human bone osteosarcoma epithelial cells (U2OS) reported a similar pattern of increasing cytotoxicity and decreasing cellular arrest with higher effective doses of Cisplatin.^{[ 25 ]}

G2/M arrest in response to individual treatment [¹²⁵I] I‐CPDANP (50 µCi), and CPDANP‐Dox (1 µM) shifted to G0/G1 arrest upon combination treatment [¹²⁵I] I‐CPDANP‐Dox (50 µCi ± 1 µM). Other lower dose combinations and individual treatments lead to G2/M arrest. Such combinations of drugs are reported to arrest cells at different phases of cell cycle. In a study by Pani et al., different individual drugs curcumin, ellagic acid, quercetin, and resveratrol treatment arrested HeLa cervical cancer cells at the S‐phase while combination of Curcumin with ellagic acid, quercetin, or resveratrol arrested HeLa cells at the G2/M phase.^{[ 26 ]}Another study reported biphasic dose‐dependent G0/G1 and G2/M mitotic arrest by Arylpyridylindole derivatives in A549 lung cancer cells at their lower and higher concentration respectively.^{[ 27 ]} This biphasic dose‐dependent mitotic arrest could be attributed to higher cytotoxicity on combination treatment compared to individual treatment.

The evaluation of mode of death in response to individual treatments [¹²⁵I] I‐CPDANP, CPDANP‐Dox, and combination treatment [¹²⁵I] I‐CPDANP‐Dox at different doses showed dose‐dependent increase in apoptotic or necrotic death. The [¹²⁵I] I‐CPDANP‐Dox and CPDANP‐Dox caused significant cell death by apoptosis while mode of cell death was necrosis with [¹²⁵I]I‐CPDANP at different doses used. Similar results with ¹²⁵I labeled antibody targeting cell membrane causing cell death in human colorectal carcinoma cell line, HCT116 without the involvement of apoptosis and p53 are reported.^{[ 28 ]} A recent review on auger electron emitters in targeted radionuclide therapy reported necrosis as the primary mechanism of cytotoxicity.^{[ 29 ]}

Bioimaging and biodistribution studies of radioiodinated CPDANP showed very fast pharmacokinetics and rapid clearance in 4T1 tumor‐bearing BALB/c mice. The low tumor uptake could be attributed to its very fast pharmacokinetics in tumor‐bearing animals. The tumor uptake could be improved by prolonging the blood circulation time of nanoparticles. The mononuclear phagocyte system (MPS) typically clears the nanoparticles from blood very fast even within less than 1 min for some nanoparticles. The blood circulation time of nanoparticles could be increased by evading MPS. Different methods for evading MPS are coating of nanoparticles with hydrophilic polymers such as poly‐ethylene glycol, allowing it to “hitchhike” on red blood cells and other cells, or by depletion of macrophages using compounds like clodronate liposomes.^{[ 30} , ^{31 ]} Maximum accumulation of CPDANP was observed in intestine followed by the stomach and liver which could be possibly due to high expression of Gal‐3 in mouse digestive tract as reported in the literature.^{[ 32} , ^{33 ]} Due to low tumor uptake and retention by intravenous administration, combinatorial therapeutic studies to evaluate the therapeutic efficacy of ¹²⁵I and Dox in CPDANP were performed after intratumoral injection of the formulations. Bioimaging and biodistribution studies after intratumoral injection were also carried out to confirm retention of the formulation in tumor and possible release and distribution to other non‐target organs. Increasing accumulation of ¹²⁵I activity was observed in thyroid when studied up to 144 h indicating de‐iodination from [¹²⁵I] I‐CPDANP with time. Dox loading in CPDANP did not affect the pharmacokinetics and biodistribution of CPDANP. In tumor inhibition studies, two doses of [¹²⁵I] I‐CPDANP‐Dox (100 µCi + 3 mg kg⁻¹), [¹²⁵I] I‐CPDANP (100 µCi) and CPDANP‐Dox (3 mg) were injected at 0 and 7 days in 4T1 tumor bearing BALB/c. Dox control was not included in the animal treatment group due to its significantly lower cytotoxicity compared to CPDANP‐Dox, as observed both in this study and our previous work (IC₅₀ of Dox: 1571 ± 149 nM versus 162 ± 10 nM for CPDANP‐Dox.^{[ 13 ]} A statistically significant enhancement in therapeutic effect was achieved with the combination treatment compared to monotherapies.

Elevated LDH levels were observed as a marker of toxicity; however, no abnormalities were noted in the histopathological analysis of the heart. LDH is a non‐specific biomarker of tissue turnover and commonly shows a general increase in various cancers. An increase in LDH does not specifically correlate with abnormalities in any critical organs. An elevated BUN level was also observed, while creatinine levels remained unchanged. Clinically, an increased BUN level with normal creatinine typically excludes renal damage and may indicate dehydration, effects of medication in cancer patients, a high‐protein diet, or gastrointestinal or cardiac conditions. Consistent with this, histopathological analysis of the kidneys in our study revealed no histological abnormalities

This study unleashes the therapeutic effect of Auger electron emitter ¹²⁵I and Dox using nucleus localizing CPDANP. Employing alternative promising auger emitters like ¹⁶¹Tb, with shorter half‐life, in conjunction with other anti‐tumor modalities through appropriate delivery systems could emerge as a highly effective strategy in the management of cancers and their micrometastases.^{[ 34 ]}

5. Conclusion

CPDA nanoparticles incorporating Auger electron emitter Iodine‐125 and drug Doxorubicin could be successfully formulated and characterized. The radionanoformulation could deliver radionuclide and drugs to the nucleus of cancer cells for auger‐chemo therapy of TNBC. Different combinatorial doses of [¹²⁵I] I‐CPDANP‐Dox induced biphasic cellular arrest in TNBC cells. [¹²⁵I] I‐CPDANP‐Dox showed enhanced therapeutic effect in vivo in tumor inhibition studies compared to their monotherapies. Further studies are warranted to assess the potential of CPDANP in auger‐chemotherapy for the clinical management of TNBC.

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

Conceptualization: SKS and BB; methodology, SKS and BB.; writing original draft preparation, SKS, AM, BB & MBM; writing review and editing, A.M.; supervision A.M. All authors have given approval to the final version of the manuscript. Research at the Bhabha Atomic Research Centre (BARC) is part of the ongoing activities of the Department of Atomic Energy, India, and is fully supported by government funding.

Supporting information

Supporting Information

Suman S. K., Balakrishnan B., Mallia M. B., Mukherjee A., Tumor‐Targeted Radioiodinated Glyconanoparticles for Doxorubicin Delivery and Auger‐Chemotherapy in Triple‐Negative Breast Cancer. Small 2025, 21, 2502419. 10.1002/smll.202502419

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.