New acrylate terpolymer-based nanoparticles for the release of nucleic acid: a preliminary study

Preparation of nanospheres and nanocapsules

Terpolymer-based NSs were obtained by radical polymerization starting from a diluted solution of BMA, (PEG)MEMA and DMAEMA (percentage molar ratio 80/10/10, respectively) in the presence of trimethylpropane trimethacrylate (TRIM) as cross-linker (20% mol/mol). The polymerization was carried out in bidistilled water in the presence of the radical initiator (Na₂S₂O₅/(NH₄)₂S₂O₈, 10% mol/mol). The reaction was carried out for 3 h, ensuring a constant stirring at 600 rpm at 37°C. At the end of the polymerization process, the NSs were recovered, rinsed 3 times with bidistilled water in order to remove the unreacted monomers, and then freeze-dried overnight.

NCs were obtained by an innovative technique consisting in a first step during which the PMMA NPs to be used as the core were synthesized by emulsion polymerization of methyl methacrylate in the presence of SDS. Then the polymerization of the monomers around the preformed core (PMMA core/monomers weight ratio 1:5) was carried out using the procedure previously described and subsequently the core was extracted by dispersing the NCs in chloroform (Carlo Erba Reagenti, Rodan, Italy). Finally the NCs were rinsed with bidistilled water and freeze-dried overnight. All the reagents were purchased from Sigma-Aldrich (St Louis, MO, USA).

Absorption and release tests

DNA (deoxyribonucleic acid, low molecular weight from salmon sperm, Sigma-Aldrich) was dissolved in phosphate buffered solution (PBS) to obtain a concentration of 1 mg/ml. Subsequently, 10 mg of NPs were dispersed in 1 mL of DNA solution and gently stirred for 2 h. The NPs were separated from the absorption solution by ultracentrifugation (Mikro 200 Hettich Zentrifugen, Tuttlingen, Germany) at 14 000 rpm for 15 min. A solution of DNA in PBS (1 mg/ml) was centrifuged in the same conditions and subsequently analyzed by high performance liquid chromatography (HPLC; Series 200; Perkin Elmer, Waltham, MA, USA) in order to verify the possible presence of pelleted DNA. Release tests were carried out by dispersing 10 mg of DNA-loaded NPs into 1 mL of PBS at 37°C. An aliquot of the release medium was collected at predetermined intervals, and fresh PBS was reinstated. Absorption and release tests were performed in triplicate both on the NSs and NCs. The collected samples were subsequently analyzed by HPLC.

Cytotoxicity test

To evaluate the cytotoxic characteristics of the produced NPs, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenil tetrazolium bromide (MTT) assay was performed using 3T3 cells from a mouse fibroblast cell line (Cell bank Interlab Cell Line Collection; IST, Genoa, Italy). NPs were extracted in Dulbecco’s Eagle’s Modified Medium (DMEM, Sigma-Aldrich) (20 mg/ml) for 24 h at 37°C. The cells were seeded onto 48-well plates at 2 different densities: 1.2·10⁴ cells/cm² for the 24 h and 0.6·10⁴ cells/cm² for the 72 h-exposure period test. Then, 24 h after seeding, culture medium was removed from the wells and replaced with fresh culture medium (800 µL) added with 200 µL of sterile filtered NC and NS extract. For each extract 10 replicates were performed. No extracts were added to 12 wells used as negative controls (NC); 4 wells containing only cell culture medium were used as blank. The plates were incubated in a humidified atmosphere containing 5% CO₂ at 37°C. After 24 h and 72 h of incubation, respectively, cell viability was measured by MTT assay as follows. A total of 100 µL of MTT solution (10% v/v with respect to the culture medium volume) were added to each well. After 4 h incubation at 37°C, the medium was completely removed and replaced with 1 mL of DMSO. After 5 min of slow agitation the absorbance was read at 570 nm using a UV spectrophotometer (V-530; Jasco, Easton, MD, USA).

Characterization of the nanoparticles

The morphology of the NPs was examined by scanning electron microscope (SEM) (JSM 5600; JEOL, Tokyo, Japan). The samples were placed on the SEM sample stage and sputter coated with gold. The mean size and polydispersity index (PDI) of the NPs dispersed in different media (ethanol, PBS, buffered solution with pH 4.00 and 9.21) were determined by dynamic light scattering (DLS) using a Zetasizer nano ZS90 (Malvern Instruments, Malvern, UK). DLS analysis were performed in triplicate on both systems.

Fourier-transform infrared (FTIR) chemical imaging analysis was carried out in order to obtain information regarding the composition of the NPs, the removal of the PMMA core and the presence of DNA after absorption tests. HPLC analysis was carried out to evaluate monomer conversion, using an Alltech Alltima C18 5u (250 mm × 4.6 mm; Fisher Scientific, Waltham, MA, USA) as a column, and acetonitrile (ACN)/water (80/20 v/v), fluxed at 1 ml/min as the internal mobile phase. The injected volume was 50 µL and the UV wavelength was 210 nm.

The final monomer conversion was evaluated using the following equation:

x   ( t )   =   ( C o − C t ) C o

where C₀ and C_t are respectively the initial and final monomer concentration in the reactive mass.

HPLC analysis was also performed to investigate the amount of absorbed and released DNA. Samples were analyzed using a Synergi 4u Hydro-RP C18 (80 Å, 250 mm × 3.00 mm; Phenomenex, Torrance, CA, USA) as a column and ACN/water (7/93 v/v), fluxed at 1ml/min as the internal mobile phase. The injected volume was 50 µL, and the chromatograms collected at the UV wavelength of 260 nm were used for quantitative analysis. Released kinetics were analyzed using the semi empirical equation of the power low to describe drug release from polymeric systems:

M t M ∞ = k t n

where M_t and M_∞ are the absolute cumulative amounts of drug released at time t and infinite time respectively, k is a constant incorporating structural and geometric characteristics of the device and n is the release exponent, indicative of the drug release mechanism (20).

Monomer conversion

The radical polymerization of monomers in diluted condition allowed the formation of NPs directly in the reaction phase. Final conversion of the 3 monomers was greater than 95% both for the NSs and NCs.

Morphological and dimensional analysis

Morphological analysis revealed a spherical shape and a rather homogeneous distribution for both the NSs and NCs, with a modest state of aggregation and a high yield (Fig. 1). The core extraction process did not alter the morphology of the NCs as shown in Figures 1B and 1C.

Quantitative information regarding the dimensions of the NPs were supplied by DLS measurements on NS and NC samples. The analysis showed an average radius between 127.5 ± 12.1 and 218.9 ± 21.2 nm for NSs and a slight increase in NC dimensions with an average radius between 163.3 ± 13.6 and 214.7 ± 19.7 nm. Table I summarizes the average radius and PDI of the NSs and NCs dispersed in different solvents, highlighting the influence of the dispersant medium on the state of aggregation. In particular, a lower average radius was observed for the NP aggregates dispersed in buffered solution at pH 4.00, in accordance with the molecular composition of the samples that caused an electrostatic repulsion. The PDI was 0.4 for NSs and 0.52 NCs suggesting that although the dispersant had a positive effect on the state of aggregation of the samples, some NPs aggregates were still present.

Absorption test

The encapsulation efficiency was evaluated by HPLC analysis of the absorption medium at the end of the test. Both the systems showed good encapsulation efficiency, reaching a value of 67.95% for the NSs and 87.1% for the NCs. The presence of the DNA was also investigated by FTIR chemical imaging analysis. Spectra acquired from the chemical map of the samples after adsorption showed the presence of a small band that fits on the adsorption of the ester group (data not shown). The second derivative spectra of the DNA showed a diagnostic band around 1685 cm^-1, related to the vibration of the carbonyl group of the DNA bases. The second derivative spectra of NSs and NCs, respectively, before absorption did not reveal the presence of the diagnostic band of the DNA. The band around 1685 cm^-1 was evident once again in the second derivative spectra of the NPs after absorption, confirming the presence of the nucleic acid on the NPs.

DNA release test

The amount of released DNA from NPs was evaluated by HPLC analysis on the samples collected at predetermined intervals. The release kinetics are shown in Figures 4A and 4B. In both cases, a rapid release in the first hours (burst effect) was observed, which tended to decrease over the following days. Moreover, HPLC analysis carried out on a solution of DNA in PBS (initial concentration of 1 mg/ml) after ultracentrifugation procedure highlighted a concentration value similar to the theoretical one, with an error of ±0.005 that can be neglected, suggesting that the initial burst effect is due to the release of DNA absorbed onto NPs. DNA release from NCs was higher than the one from NSs and prolonged in time. The models shown in Figure 4 demonstrate a good correlation degree for both the NSs and the NCs with a coefficient n<0.50.

Cytotoxicity test

The cytotoxicity of the produced NPs was preliminarily tested using the MTT cytotoxicity assay. NS and NC extracts were added to 3T3 cells and the viability of the cells was evaluated after exposure periods of 24 h and 72 h, respectively. The obtained results are shown in Figure 5. It can be observed that at both 24 h and 72 h the mitochondrial activity of the cells was not affected by the extracts. No significant differences were found between treated and control wells (F-test, P>.05) with the exception of NCs extracts after 24 h of incubation (P<.05).