Poly(lactide-co-glycolide) nanoparticle assembly for highly efficient delivery of potent therapeutic agents from medical devices

Abstract

Controlled delivery of therapeutic agents from medical devices can improve their safety and effectiveness in vivo, by ameliorating the surrounding tissue responses and thus maintaining the functional integrity of the devices. Previously, we presented a new method for providing simultaneous controlled delivery from medical devices, by surface assembly of biodegradable polymer nanoparticles (NPs) encapsulating fluorescent dyes. Here, we continue our investigation with NPs loaded with therapeutic agents, dexamethasone (DEX) or plasmid DNA, and evaluated the bioactivity of the released molecules with macrophage cells associated with inflammation. Over a period of one week, NPs encapsulating DEX released 24.9+/-0.8ng from the probe surface and was successful at suppressing macrophage cell growth by 40+/-10%. This percentage of suppression corresponded to approximately 100% drug delivery efficiency, in comparison with the unencapsulated drug. DNA NP coatings, in contrast, released approximately 1ng of plasmid DNA and were effective at transfecting macrophage cells to express the luciferase gene at 300+/-200 relative luminescence/mg total protein. This amount of luciferase activity corresponded to 100% gene delivery efficiency. Thus, NP coatings were capable of providing continuous release of bioactive agents in sufficient quantities to induce relevant biological effects in cell culture studies. These coatings also remained intact, even after 14 days of incubation with phosphate buffered saline. Although the maximum loading for NP coatings is inherently lower than the more established matrix coating, our study suggests that the NP coatings are a more versatile and efficient approach toward drug delivery or gene delivery from a medical device surface and are perhaps best suited for continuous release of highly potent therapeutic agents.

Figure 1. Nanoparticles Loaded with Biomolecules

NPs encapsulating (A) DEX and (B) plasmid DNA were examined under SEM. Both types of NPs had the normal spherical morphology. NPs encapsulating plasmid DNA had larger mean particle diameter, but both types of NPs had similar magnitude in their zeta potentials. The mean particle size was 180 ± 60 nm for DEX NPs and 230 ± 90 nm for DNA NPs; the zeta potential values were -14 ± 2 mV and -16 ± 1 mV respectively. (Scale bar = 1 μm)

Figure 2. Surface Morphology of Nanoparticle Coatings

Before PBS exposure: (A) bare silicon oxide surface, (B) DEX NP coated surface, and (C) DNA NP coated surface. After 14 days of PBS exposure at 37 °C: (D) bare silicon oxide surface, (E) DEX NP coated surface, and (F) DNA NP coated surface. Surface morphology of NP coatings remained intact before and after two weeks of PBS exposure, demonstrating that the NPs were stable on probe surface. Surface coverage also remained the same throughout the two-week period, ∼13% for DEX NPs and ∼4% for DNA NPs. (Scale bar = 10 μm)

Figure 3. Biomolecule Release Profiles from Nanoparticle Coatings

Both DEX and plasmid DNA were continuously released from NPs for up to 14 days in PBS at 37 °C. Release kinetics of DEX NPs on surface (A) was identical to 10 mg of DEX NPs in 10 ml bulk PBS suspension (B). Due to the low density of DNA NPs on surface, plasmid DNA release kinetics from DNA NP coating was not directly measured. Plasmid DNA release was investigated only in bulk PBS suspension (C). (-○- 7 wt% DEX NP coatings, -▲- 7 wt% DEX NPs in bulk PBS suspension, -△- 0.2 wt% DNA NPs in bulk PBS suspension, n=3) For symbols without error bars, the standard deviation of the measurements was smaller than the size of the symbol.

Figure 4. Macrophage Cell Responses to Nanoparticle Coatings

(A) The anti-inflammatory drug was released directly into cell culture for 7 days from DEX NP coated probes. A dose response curve of the unencapsulated DEX (insert) was also performed for 7 days, to determine the drug delivery efficiency of the DEX NP coatings. 24.9 ± 0.8 ng of DEX was released from NP coatings, which corresponded to >100% delivery efficiency in comparison with unencapsulated DEX of same amount. (B) ∼1 ng of plasmid DNA was released from NP coatings on day 3 and complexed with Lipofectamine™ before introduction to cell culture. The transfection result from DNA NP coatings (300 ± 200 relative luminescence/ mg total protein) was equivalent to 100% gene delivery efficiency, in comparison with unencapsulated plasmid DNA of same amount. (Grey bars = 3 days, black bars = 7 days, n = 3) *denotes statistical difference between the test groups and the untreated RAW 264.7

Figure 5. Comparison of Biomolecule Release Profiles with Matrix Coatings

Continuous release studies were conducted for 14 days at 37 °C in PBS buffer. (A) Three loadings were investigated with the DEX matrix coatings: 10 wt%, 30 wt%, and 50 wt%. Note that the 50 wt% coating thickness was twice that of the lower loadings, and that the total mass released has not yet been adjusted. (B) The highest 50 wt% matrix coating was compared to DEX NP coating in their release kinetics. (C) Only 20 wt% and 50 wt% loadings were tested for DNA matrix coatings. (D) Due to the low density of DNA NPs on surface, release from DNA NP coatings was not measured and was substituted with release profile from DNA NPs in bulk PBS suspension. In general, matrix coatings had higher loading capabilities than NP coatings. (-■- 50 wt% DEX matrix, -○- 30 wt% DEX matrix, -△- 10 wt% DEX matrix, n=6; -□- 7 wt% DEX NPs, n=3; -●- 50 wt% DNA matrix, -◇- 20 wt% DNA matrix, n=6; -X-0.2 wt% DNA NPs, n=3) For symbols without error bars, the standard deviation of the measurements was smaller than the size of the symbol.

Figure 6. Comparison of Macrophage Cell Response with Matrix Coatings

(A) The anti-inflammatory drug was released directly into cell culture for 7 days from DEX matrix coated probes. 15.3 ± 0.1 μg and 154.6 ± 6.3 μg of DEX was released from 10 wt% and 50 wt% matrix coatings respectively, which corresponded to near 90% drug delivery efficiency for both in comparison with unencapsulated DEX of same amount. (B) This value was compared with the % drug delivery efficiency obtained by DEX NP coating. (C) 28.5 ± 0.3 μg of plasmid DNA released from 50 wt% DNA matrix coatings on day 3 was diluted to 1 μg per well and was complexed with Lipofectamine™ before introduction to cell culture. The transfection result of DNA matrix coatings (13,000 ± 5,000 relative luminescence/mg total protein) was equivalent to 60% gene delivery efficiency, in comparison with unencapsulated, stock plasmid DNA of same amount. (D) This value was compared with the % gene delivery efficiency obtained by DNA NP coating. (Grey bars = 3 days, black bars = 7 days, n = 3) *denotes statistical difference between the test groups and the untreated RAW 264.7