PEGylated versus non-PEGylated magnetic nanoparticles as camptothecin delivery system

Summary Camptothecin (CPT; (S)-(+)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione) is a highly cytotoxic natural alkaloid that has not yet found use as chemotherapeutic agent due to its poor water-solubility and chemical instability and, as a consequence, no effective administration means have been designed. In this work, camptothecin has been successfully loaded into iron oxide superparamagnetic nanoparticles with an average size of 14 nm. It was found that surface modification of the nanoparticles by polyethylene glycol enables loading a large amount of camptothecin. While the unloaded nanoparticles do not induce apoptosis in the H460 lung cancer cell line, the camptothecin-loaded nanoparticle formulations exhibit remarkable pro-apoptotic activity. These results indicate that camptothecin retains its biological activity after loading onto the magnetic nanoparticles. The proposed materials represent novel materials based on naturally occurring bioactive molecules loaded onto nanoparticles to be used as chemotherapeutic formulations. The procedure seems apt to be extended to other active molecules extracted from natural products. In addition, these materials offer the potential of being further implemented for combined imaging and therapeutics, as magnetic nanoparticles are known to be multifunctional tools for biomedicine.


Materials
All chemicals are of reagent grade and have been used without further purification: iron (II) chloride tetrahydrate (FeCl 2 •4H 2 O, ≥99%), iron (III) chloride hexahydrate (FeCl 3 •6H 2 O, 97%), ammonia (28-30%), succinic acid, and poly (ethylene glycol)-bis-(3-aminopropyl)-terminated (PEG; 1500 g/mol) were purchased from Sigma-Aldrich. N-(3-dimethylaminopropyl)-N´-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS) were purchased from Alfa Aesar. Camptothecin (CPT) was purchased from Alexis Biochemicals. As a reference material, magnetite (Fe 3 O 4 , 99.997%) was purchased from Alfa Aesar. Non-small lung cancer cell line, H-460 was a gift from Dr. P.J. Woll (CRC Department of Clinical Oncology, City Hospital, Nottingham, UK). Cells H460 were cultured in RPMI 1640 by Biowhittaker. Water was purified using a Milli-Q reagent-grade water system from Millipore. Buffers were prepared according to standard laboratory procedures. which turns the solution black, indicating the formation of USM nanoparticles. After addition, stirring is maintained for one hour at room temperature, and USM nanoparticles are recovered and purified from reaction by-products and excess S3 ammonia by means of three steps of magnetic separation, removal of the supernatant and washing with water to obtain USM as a black powder.

Synthesis
USM-Suc nanoparticles. Succinic acid (83 mg) was added to an aqueous solution of USM nanoparticles (12 mL, 10.6 mg) and the reaction was allowed to proceed for 24 hours under vigorous stirring. USM-Suc nanoparticles were recovered and purified by means of three steps of magnetic separation, removal of the supernatant and washing with water to obtain 15.5 mg of USM-Suc as a black powder.
USM-PEG nanoparticles. EDC (12 mg, 0.06 mmol) and NHS (18 mg, 0.15 mmol) were added to an aqueous solution of USM-Suc (2.5 mL, 6 mg) and the mixture was stirred for 30 minutes at room temperature. Afterwards, amino-terminated PEG (333 mg) was added to the reaction mixture and stirred for 24 hours at room temperature.
Finally, USM-PEG nanoparticles were recovered and purified by means of three steps of magnetic separation, removal of the supernatant and washing with water to obtain 7.5 mg of USM-PEG as a black powder.

CPT loading
Loading of CPT in USM and USM-PEG nanoparticles was done by direct incubation of the drug in aqueous dispersions of the nanoparticles. To determine the maximum loading capacity of USM and USM-PEG nanoparticles, increasing volumes (from 1 to 25 μL in 1 μL steps) of a solution of CPT in dimethylsulfoxide (DMSO, 28.7 mM) were slowly added to an aqueous solution of USM or USM-PEG nanoparticles (700 μL, 1 mg/mL). After addition, the sample was gently vortexed and the mixture was The isothermal hysteretic behaviour of the USM nanoparticles was investigated by recording the field dependence of the magnetization (hysteresis loop) up to ±50 kOe at 5 K.

Cell culture
Non-small lung cancer cell line H460 was cultured in RPMI medium supplemented with penicillin, streptomycin and 10% Fetal Bovine Serum (FBS) at 37 ºC until 80% confluence.  MRmFilter Configuration: Apotome, Software: AxioVision 4.7 was used to visualize S7 the apoptotic effect in cell lines after being incubated with nanoparticles and cell viability quantified. Image J software was used for image analysis and cell count.

Stock dispersions of USM[CPT] and USM-PEG
Statistical analyses. All results are expressed as mean ± standard deviation. Two-tail Student's t-tests (paired and unpaired, according to samples) or ANOVA variance analyses were used as appropriate for statistical analysis. Only P-values <0.05 were considered as statistically significant.