Cancer-Targeted Controlled Delivery of Chemotherapeutic Anthracycline Derivatives Using Apoferritin Nanocage Carriers

The interactions of chemotherapeutic drugs with nanocage protein apoferritin (APO) are the key features in the effective encapsulation and release of highly toxic drugs in APO-based controlled drug delivery systems. The encapsulation enables mitigating the drugs’ side effects, collateral damage to healthy cells, and adverse immune reactions. Herein, the interactions of anthracycline drugs with APO were studied to assess the effect of drug lipophilicity on their encapsulation excess n and in vitro activity. Anthracycline drugs, including doxorubicin (DOX), epirubicin (EPI), daunorubicin (DAU), and idarubicin (IDA), with lipophilicity P from 0.8 to 15, were investigated. We have found that in addition to hydrogen-bonded supramolecular ensemble formation with n = 24, there are two other competing contributions that enable increasing n under strong polar interactions (APO(DOX)) or under strong hydrophobic interactions (APO(IDA) of the highest efficacy). The encapsulation/release processes were investigated using UV-Vis, fluorescence, circular dichroism, and FTIR spectroscopies. The in vitro cytotoxicity/growth inhibition tests and flow cytometry corroborate high apoptotic activity of APO(drugs) against targeted MDA-MB-231 adenocarcinoma and HeLa cells, and low activity against healthy MCF10A cells, demonstrating targeting ability of nanodrugs. A model for molecular interactions between anthracyclines and APO nanocarriers was developed, and the relationships derived compared with experimental results.


Chemicals
The anticancer drugs (epirubicin -EPI, idarubicin -IDA, and daunorubicin -DAU) were received from Selleckchem (Houston, TX, USA), and the anticancer drug, doxorubicin (DOX), was purchased All aqueous solutions were prepared with deionized water (resistivity of 18.2 MΩ cm) purified with a Milli-Q reagent grade water system (Merck Millipore, Billerica, MA, USA).

Apparatus
The fluorescence spectra were recorded using LS55 Spectrometer (Perkin Elmer, Waltham, MA, U.S.A.) equipped with 20 kW Xenon light source operating in 8 μs pulsing mode. Separate monochromators for the incident and detector beams enabled to use monochromatic radiation with wavelengths from 200 to 700 nm. The dual detector system consisted of a photomultiplier tube (PMT) and an avalanche photodiode. The UV-Vis spectra were recorded using a Varian Cary 50 Bio UV-Visible Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) in the range from 200 nm to 700 nm at room temperature. FTIR spectra of APO nanocages were obtained using Model Nicolet iS-10 FTIR instrument (Thermo Fisher Sci., USA) working in specular mode enabling the reflection from the sensor surface to be analyzed. The circular dichroism (CD) spectra were recorded on a J-715 spectropolarimeter (JASCO, Tokyo, Japan). Far-UV (190-240 nm) recordings were performed in a 0.5 cm pathlength quartz cuvette.

Incorporation of anthracycline drugs to apoferritin nanocages
The four anthracyclines studied have a similar structure and their main pKa values are close to 8.46 and 7.34, due to the amine group protonation and phenolic dissociation, respectively. Hence, the net charges carried by these anthracycline molecules do not differ significantly. However, the small structural differences, including additional -O-CH3 group at C4, as well as -CH2OH or -CH3 group at C13, result in considerable differences in drugs lipophilicity and supramolecular interactions with apoferritin protein, thus influencing the encapsulation efficiency. The measurements performed in this work were designed to evaluate the efficiency of encapsulation of anthracyclines under study in APO nanocages.
The encapsulation of anthracycline drugs in APO nanocages was carried out by the disassembly/reassembly protocols based on those developed recently [1][2][3][4] and with changes adopted for the drugs used in this study. Briefly, a 5 or 25 µL of 100 mg/mL horse spleen APO (final concentration of APO 1 or 5 mg/mL) was mixed with 10 or 50 µL of 10 mM anthracycline derivative (DOX, EPI, DAU and IDA) in a molar ratio of APO:drug of 1:100. The total volume of the mixtures was 500 µL which was obtained by adding appropriate amounts of Milli-Q water. A 4 μL aliquot of 1 M hydrochloric acid was added to decrease the solution pH to 2.0 and dissociate the APO. Then the pH value was maintained for 15 minutes. Afterward, the pH was slowly increased up to 7.4 using 4 µL of 1 M sodium hydroxide. The resulting solution was stirred under room temperature for 2 hours to encapsulate the anthracycline in the APO. Then, the mixture was rinsed five times with Milli-Q water using Amicon Ultra-0.5 mL 30K (Merck Millipore, Billerica, MA, USA) to remove unbound drug molecules. The obtained anthracycline-containing APO nanocages (APO(drug)) were stored at 4 °C.

Modification of APO(drug) nanocages with folic acid
To improve the effectiveness of APO(drug) nanocages in targeted drug delivery, we have applied a covalent binding of folic acid (FA) to an APO(drug) nanocage using a modification of the standard EDC/NHS reaction. 10 mM FA was dissolved in 5 % NaHCO3. To activate carboxyl groups of the folic acid, 10 µL of FA solution was added to 900 µL of EDC/NHS mixture containing 7 mM EDC and 7 mM NHS in water and the resulting solution was allowed to react at room temperature for 30 minutes.
The activated mixture was added to the APO(drug) nanocages, prepared earlier by centrifugation using Amicon Ultra-0.5 mL 3K to remove water, and were incubated overnight at room temperature. The APO(drug)@FA nanocarriers were purified through the Amicon Ultra-0.5 mL 30K and washed 3-times with Milli-Q water. The obtained product was dispersed in water for further characterization and application.

Encapsulation ratio drug:APO
The quantification of encapsulated anthracyclines was performed by determining the concentration of free drugs in solution, separated from APO(drug) NPs by filtration and the initial drug concentration in the encapsulation solution. The concentrations of free drugs were evaluated using fluorescence emission measurements, for ex = 480 nm, with calibration data detailed in Figure S3. All the data are expressed as the average of at least three determinations.

Releasing of anthracyclines form APO(drug) nanocages
The kinetics of drug release from APO(drug) was evaluated using PBS buffer pH 7.4 and McIlvain buffers with pH 3.6, 4.6 and 5.6. In brief, 300 µL of the appropriate buffer was added to the APO(drug) and samples were incubated for 6 h at 37 °C. At the pre-set time points (1 h apart), the solution with unbound drug was separated by filtration using Amicon-Ultra-0.5 mL 3K at RT. The container with APO(drug) nanocarriers was replenished with an equal volume of blank buffer (300 µL). The release study was carried out in triplicate for each pH.

Cell death assay
To determine the fraction of dead and apoptotic cells induced by targeted free anthracyclines (DOX, EPI, IDA) and APO nanocages uploaded with encapsulated anthracyclines (DOX, EPI, or IDA), the Annexin V method was used. MDA-MB-231 cells were exposed to the indicated concentrations of

Colony formation assay by crystal violet staining
Colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. HeLa, MDA-MB-231, and MCF10A cells were seeded onto six-well plates at a concentration of 150,000 cells/well such that they were ~40% confluent at 24 hours. The cells were then treated with IDA and APO(IDA) at concentrations: 0.0, 0.5 and 1.0 µM of the drug. After the treatment, cells were incubated in 5 % CO2 atmosphere at 37 °C for 72 h to allow for colony formation. After the 72-hour treatment, pictures of each well on the six-well plates were taken. The cells were then fixed in 3.7 % formaldehyde for 15 minutes. The fixed cells were rinsed with PBS and stained for 10 minutes using 0.1 % crystal violet. After staining, the plates were rinsed with water to remove the staining solution and allowed to air dry. To quantify, the cells were solubilized in 1 % SDS and the absorbance at 590 nm was measured. The percentage of cell survival (PCS) was calculated as follows: .