Two new types of drug-loaded nanoparticles (NPs), liposomes and nanocapsules, have been functionalized with a DNA aptamer which can be recognized by tumor cell receptors, allowing the targeted administration of the antitumoral model drug (5-Fluorouracil).

Nanocapsules based on poly (N-vinylpyrrolidone-alt-itaconic anhydride) – poly (NVPAI) and a mixture of chitosan (CS)/carboxylate CS functionalized with AS1411 aptamer (CCA) were prepared by an interfacial condensation method. The condensation reaction took place at the interface between the two solutions by opening the anhydride cycles from the copolymer, under the action of the NH₂ groups from mixture of CS/aptamer-functionalized CS carboxylate.

Two types of nanocapsules were prepared:

• nanocapsules based on poly- (NVPAI) and chitosan (NC)
• nanocapsules based on poly- (NVPAI) and a mixture of CS/CS carboxylate functionalized with aptamer (NCA)

The prepared nanocapsules were characterized from several points of view, such as: infrared spectroscopy (FTIR), dynamic light scattering (DLS), zeta-potential, scanning electron microscopy (SEM). Nanocapsules swelling degree (Q) in slightly alkaline aqueous medium – pH = 7.4 over a 24-hour period was determined using the gravimetric method. The nanocapsules (NC and NCA type) were loaded with 5-Fluorouracil (5-FU) and the 5-FU release efficiency in PBS solution (pH=7.4) has been studied. The in vitro cytotoxic effects of the aptamer- functionalized nanocapsules (with and without drug) were assessed on the human dermal fibroblasts adult cell line (HDFa).

The obtained results have revealed that:

• Approximately 55% of the aptamer (AS1411-NH₂) was conjugated to the carboxylate chitosan and approximately 34% of the aptamer is found in the nanocapsule composition.
• The mean diameter of the capsules was situated between 118 and 267 nm and granulometric distribution curves, of obtained nanocapsules, showed a monomodal distribution.
• Negative charge of the nanocapsules (with values between -11 mV and -19 mV) was attributed to the carboxylate anions formed by the carboxyl groups present either in CS/CCA or by opening the anhydride cycle (NVPAI). Therefore, as expected, the nanocapsule based on non-functionalized chitosan show a lower zeta potential than nanocapsules based on CCA, fact which confirms the theoretical considerations.
• The SEM results demonstrated that these nanocapsules have a very well defined spherical shape and the capsule diameter was in the same range as those determined by DLS analysis (ranging from 100 to 200 nanometers).
• The nanocapsules swelling degree was between 1000-1680% in slightly alkaline medium (pH 7.4), decreasing with the increase of CS amount in the nanocapsules system.
• 5-FU release efficiency in alkaline medium was found to be between 27% and 39 % for the NCA nanocapsules, and for NC nanocapsules was between 42%-55%. Increasing in the amount of chitosan, but also the conjugation of aptamer to nanocapsules, led to a decrease in the percentage of drug released in the studied aqueous medium.
• Fibroblast cells treated with different concentrations of nanocapsules (100µg/ml, 250 µg/ml, 500 µg/ml) exhibited a good viability ranging from 80% to 96%. These results clearly demonstrate that both nanocapsule types, without drug and nanocapsules containing 5-FU, do not show toxic effects at high concentrations. The obtained results reveal that cell viability decreases as the amount of nanocapsules exposed to fibroblast cells increases, and increases with increasing of the amount of mixture of chitosan and chitosan carboxylate functionalized with aptamer, from the initial polymer system.

Liposomes based on egg phosphatidylcholine (PC), cholesterol (CHOL) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (PEG-2,000)] DSPE-PEG-MAL were prepared by the lipid film hydration method followed by sequential extrusion. Functionalization of liposomes has been achieved by the thio-ether bond formation between the SH group (AS1411-SH) and the maleimide group. Mean diameter of the liposomes and polydispersity index were determined by the laser diffractometry technique. The zeta potential was evaluated by microelectrophoresis at 25 °C after dilution in the hydration medium (PBS, pH 7.4) at a suitable counting rate using a Malvern Zetasizer Nano ZS. The aptamer conjugation efficiency to liposomes was determined by UV-Vis spectrophotometry. The liposomes cytotoxicity (with and without encapsulated drug) was evaluated at 3 different lipid concentrations: 100 μg/ml, 250 μg/ml, 500 μg/ml.

The obtained results have revealed that:

• The efficiency of the AS1411-SH aptamer functionalization to the liposomes varied between 28% and 39%.
• The mean diameter of the liposomes was around 200 nm and the value of the polydispersity index demonstrated that the obtained system is monodisperse.
• The liposomes stability in a solution that mimics the biological environment has been evaluated over a period of 24 hours. The obtained result indicated that the liposomes are stable at a temperature of 37 °C.
• The amount of 5-FU released from the liposomes is directly proportional with the amount of 5-FU loaded into liposomes. The loading efficiency of the drug in liposomes varied between 6.83% -14.53%.
• Aptamer-functionalized liposomes (with and without encapsulated drug) did not show cytotoxic effects on fibroblast cells, except for the liposome samples loaded with 5-FU that presented a strong cytotoxic effect.