Apoferritin-Encapsulated Jerantinine A for Transferrin Receptor Targeting and Enhanced Selectivity in Breast Cancer Therapy

The O-acetyl (or acetate) derivative of the Aspidosperma alkaloid Jerantinine A (JAa) elicits anti-tumor activity against cancer cell lines including mammary carcinoma cell lines irrespective of receptor status (0.14 < GI50 < 0.38 μM), targeting microtubule dynamics. By exploiting breast cancer cells’ upregulated transferrin receptor 1 (TfR1) expression and apoferritin (AFt) recognition, we sought to develop an AFt JAa-delivery vehicle to enhance tumor-targeting and reduce systemic toxicity. Optimizing pH-mediated reassembly, ∼120 JAa molecules were entrapped within AFt. Western blot and flow cytometry demonstrate TfR1 expression in cancer cells. Enhanced internalization of 5-carboxyfluorescein-conjugated human AFt in SKBR3 and MDA-MB-231 cancer cells is observed compared to MRC5 fibroblasts. Accordingly, AFt–JAa delivers significantly greater intracellular JAa levels to SKBR3 and MDA-MB-231 cells than naked JAa (0.2 μM) treatment alone. Compared to naked JAa (0.2 μM), AFt–JAa achieves enhanced growth inhibition (2.5–14-fold; <0.02 μM < GI50 < 0.15 μM) in breast cancer cells; AFt–JAa treatment results in significantly reduced clonal survival, more profound cell cycle perturbation including G2/M arrest, greater reduction in cell numbers, and increased apoptosis compared to the naked agent (p < 0.01). Decreased PLK1 and Mcl-1 expression, together with the appearance of cleaved poly (ADP-ribose)-polymerase, corroborate the augmented potency of AFt–JAa. Hence, we demonstrate that AFt represents a biocompatible vehicle for targeted delivery of JAa, offering potential to minimize toxicity and enhance JAa activity in TfR1-expressing tumors.


SI1. Measuring concentration of protein and jerantinine A acetate (JAa)
The concentrations of all proteins used in this study were calculated using a calibration curve obtained by Bradford analysis ( Figure S1a). The concentration of encapsulated JAa was assessed using UV-Vis spectroscopy of JAa solution ( Figure S1b) with different concentrations. Based on these results, the calibration curve ( Figure S1c) was generated and used to calculate the concentration of encapsulated JAa.

SI2. Assessment of protein recovery and encapsulation efficiency EE%
To optimize the encapsulation methods, the percentage of protein recovery was calculated using Equation (1): , (S1) % = 100 * * 0 * 0 where W c is the weight of the formulation after purification, C c is the concentration of the formulation after purification, W 0 is the weight of formulation before purification and C 0 is the concentration of the formulation before purification. Encapsulation efficiency (EE%) is defined as the ratio between the encapsulated and the original amount of the drug added to the formulation: SI3. Growth-inhibitory assessment of AFt-JAa Table S1 summarizes the information about the cell lines used.
where , A 1 is absorbance in control (untreated) wells; A 0 is absorbance 50 = 1 -0 2 + 0 at T0, A H and A L are absorbances higher and lower than AGI 50 , at concentrations C H and C L , respectively. In clonogenic assays, plating efficiency (PE%) and survival fractions (SF%) of colonies were calculated using Equations (S4) and (S5), respectively:

SI4. Release study of jerantinine A acetate (JAa) from apoferritin (AFt)
Release studies were performed using Slide A-Lyzer MINI Dialysis Device (Thermo Scientific, PC-8840, 0.5 mL) with 10 K MWCO); 100 mM sodium acetate buffer (NaOAc) adjusted to pH 5.3 by acetic acid; phosphate-buffered saline (PBS, pH 7.4). The formulation (n = 4) was placed into the dialysis device and kept at 37 °C either in NaOAc or PBS. Direct and indirect samples were collected at different time intervals of 0 h, 1 h, 3 h, 6 h, 24 h ( Figure   S2). JAa concentration was estimated using the calibration curve obtained after the liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis. Indirect samples: The JAa concentration was estimated after measuring JAa concentrations remaining in the dialysis device. First, samples were treated with 1:5 formic acid: methanol to precipitate and open the AFt cage. Samples were then centrifuged (13,000 rpm; 10 min) and the supernatant was measured using EQMS

SI5. Stability study
Stability of the encapsulated agent was assessed using DLS measurements and by monitoring the concentration of the agent ( Figure S4). Figure S4. Stability study of AFt-encapsulated JAa monitored at storage conditions (T = 4 °C; pH 7.4). Hydrodynamic size (a) and zeta-potential (b) were measured using Malvern zeta sizer Nano ZS. Measurements of AFt-JAa and empty reassembled AFt (Re-AFt) were statistically comparable to AFt stock over 10 months of storage. The stability of encapsulated JAa was assessed by measuring its concentrations by UV-Vis spectroscopy at 330 nm over 5 weeks (c). Stability study of free and AFt-encapsulating JAa (0.4 µM) monitored at treatment conditions (T = 37 °C; over 72 h) in different media using HR-LCMS. Measurements show better stability of JAa following the encapsulation with AFt in 7 independent trials (d).