Poly(omega-pentadecalactone-co-butylene-co-succinate) nanoparticles as biodegradable carriers for camptothecin delivery

Abstract

In this study, we show that degradable particles of a hydrophobic polymer can effectively deliver drugs to tumors after i.v. administration. Free-standing nanoparticles with diameters of 100-300 nm were successfully fabricated from highly hydrophobic, biodegradable poly(omega-pentadecalactone-co-butylene-co-succinate) (PPBS) copolyesters. PPBS copolymers with various compositions (20-80 mol% PDL unit contents) were synthesized via copolymerization of omega-pentadecalactone (PDL), diethyl succinate (DES), and 1,4-butanediol (BD) using Candida antarctica lipase B (CALB) as the catalyst. Camptothecin (CPT, 12-22%) was loaded into PPBS nanoparticles with high encapsulation efficiency (up to 96%) using a modified oil-in-water single emulsion technique. The CPT-loaded nanoparticles had a zeta potential of about -10 mV. PPBS particles were non-toxic in cell culture. Upon encapsulation, the active lactone form of CPT was remarkably stabilized and no lactone-to-carboxylate structural conversion was observed for CPT-loaded PPBS nanoparticles incubated in both phosphate-buffered saline (PBS, pH=7.4) and DMEM medium for at least 24 h. In PBS at 37 degrees C, CPT-loaded PPBS nanoparticles showed a low burst CPT release (20-30%) within the first 24 h followed by a sustained, essentially complete, release of the remaining drug over the subsequent 40 days. Compared to free CPT, CPT-loaded PPBS nanoparticles showed a significant enhancement of cellular uptake, higher cytotoxicity against Lewis lung carcinoma and 9L cell lines in vitro, a longer circulation time, and substantially better antitumor efficacy in vivo. These results demonstrate the potential of PPBS nanoparticles as long-term stable and effective drug delivery systems in cancer therapy.

Fig. 1

SEM images and particle size distributions of unhydrated 20% PDL-PPBS NP (A, C) and 50% PDL-PPBS NP (B, D).

Fig. 2

Effect of sugars on mean size stability of particles subjected to lyophilization (□) and resuspension (▪). Size distributions were determined by dynamic light scattering (mean ± SD; n = 10).

Fig. 3

In vitro cumulative release of CPT from different PPBS nanoparticles. Data were given as mean ± SD (n = 3).

Fig. 4

The stability of free CPT and CPT-loaded PPBS nanoparticles under different conditions. (A) HPLC chromatograms of carboxylate form and lactone form of CPT in different pH buffers. (B) Stability of CPT lactone form in different solutions (pH 7.4, 37°C): (▪) Free CPT in PBS; (○) Free CPT in DMEM; (Δ) CPT nanoparticle in PBS; (*) CPT nanoparticle in DMEM.

Fig. 5

In vitro cytotoxicity of CPT-loaded 20% PDL PPBS nanoparticles (○), 50% PDL PPBS nanoparticles (Δ) and free CPT (▪) against (i) LLC cell line after 24 (A) and 72 hr (B); (ii) 9L cell line after 24 (C) and 72 hr (D). Data were given as mean ± SD (n = 4).

Fig. 6

Uptake of CPT-loaded PPBS nanoparticles and free CPT by LLC cells: (i) flow cytometric histogram profiles of fluorescence intensity for cells treated with 50 µM (A) and 100 µM (B) of CPT; (ii) quantitative uptake of CPT-loaded nanoparticles and free CPT in different drug concentrations (C). Data were given as mean ± SD (n = 3). * p <0.05 and ** p<0.01 compared with free CPT.

Fig. 7

Concentrations of total CPT, carboxylate form, and lactone form of CPT in LLC cells after incubation with free CPT or CPT-loaded PPBS nanoparticles.

Fig. 8

Confocal microscopic images of LLC cells after 2 hr incubation with (A) free CPT and (B) CPT-loaded 50%-PDL PPBS nanoparticles. Cells and CPT are visualized in the red and blue channels, respectively.

Fig. 9

Antitumor effects of CPT/50% PDL-PPBS nanoparticles, free CPT, blank PPBS nanoparticles, and PBS on C57BL/6 mice bearing LLC. Data were given as mean ± SEM (n = 8). Compared to the free CPT group, both CPT/PPBS NP treatment groups showed substantially smaller tumor volume (p < 0.01). ^a Multiple injections of CPT/PPBS nanoparticles: initial injection at 10 mg/kg followed by two separate injections at 5 mg/kg; arrows indicate the days of drug injections.

Fig. 10

Biodistribution of CPT at 2 hr (A), 24 hr (B), and 48 hr (C) after i.v. injection of CPT-loaded 50% PDL-PPBS nanoparticles or free CPT into mice bearing LLC. Data were given as mean ± SD (n=3). * p<0.05 and ** p<0.01 compared with free CPT results.

Fig. 11

CPT concentration change in tumor (A) and plasma (B) at various time points after injection of 50% PDL-PPBS nanoparticles (○) or free CPT (▪). Data were given as mean ± SD (n = 3). * p <0.05 compared with free CPT.

Scheme 1

pH-dependent equilibrium of camptothecin.

Scheme 2

Two-Stage Process for Copolymerization of PDL, DES, and BD.