Poly(lactic acid-glycolic acid) nanoparticles markedly improve immunological protection provided by peptide P10 against murine paracoccidioidomycosis

Abstract

Background and purpose: The present study reports on the preparation and testing of a sustained delivery system for the immunomodulatory peptide P10 aimed at reducing the in vivo degradation of the peptide and the amount required to elicit a protective immune response against paracoccidioidomycosis.

Experimental approach: BALB/c mice were infected with the yeast Paracoccidioides brasiliensis to mimic the chronic form of paracoccidioidomycosis. The animals were treated daily with sulfamethoxazole/trimethoprim alone or combined with peptide P10, either emulsified in Freund's adjuvant or entrapped in poly(lactic acid-glycolic acid) (PLGA) nanoparticles at different concentrations (1 microg, 5 microg, 10 microg, 20 microg or 40 microg.50 microL(-1)). Therapeutic efficacy was assessed as fungal burden in tissues and the immune response by quantitative determination of cytokines.

Key results: Animals given combined chemotherapy and P10 nanotherapy presented a marked reduction of fungal load in the lungs, compared with the non-treated animals. After 30 days of treatment, P10 entrapped within PLGA (1 microg.50 microL(-1)) was more effective than 'free' P10 emulsified in Freund's adjuvant (20 microg.50 microL(-1)), as an adjuvant to chemotherapy. After treatment for 90 days, the higher doses of P10 entrapped within PLGA (5 or 10 microg.50 microL(-1)) were most effective. Treatment with P10 emulsified in Freund's adjuvant (20 microg.50 microL(-1)) or P10 entrapped within PLGA (1 microg.50 microL(-1)) were accompanied by high levels of interferon-gamma in lung.

Conclusions and implications: Combination of sulfamethoxazole/trimethoprim with the P10 peptide entrapped within PLGA demonstrated increased therapeutic efficacy against paracoccidioidomycosis. P10 incorporation into PLGA nanoparticles dramatically reduced the peptide amount necessary to elicit a protective effect.

Figure 1

Fungal burden recovery assessed by colony-forming units [CFU (g·lung·tissue)⁻¹] in mice infected with P. brasiliensis Pb18 and subjected to a combined therapy of sulfamethoxazole/trimethoprim (Sulfa + Trim; 15 mg·kg⁻¹ and 3 mg·kg⁻¹ respectively) and either 20 µg·50 µL⁻¹ P10 peptide solubilized in Freund's adjuvant (‘free’) or P10 peptide (1, 5 or 10 µg·50 µL⁻¹) entrapped within PLGA. Each bar represents the average CFU (g·tissue)⁻¹ with standard deviations. After 30 days of treatment, 1 µg·50 µL⁻¹ of P10-PLGA/Sulfa + Trim yielded the best response (lowest fungal CFU recovery) of all groups (+P < 0.05). After 90 days of treatment, no significant differences were found between the responses of the ‘free’ P10 (20 µg·50 µL⁻¹) and the P10-PLGA (5–10 µg·50 µL⁻¹)-treated groups (marked with #). At day 90, a significantly lower number of fungal cells were recovered from mice treated with 1 µg·50 µL⁻¹ P10-PLGA compared with the PLGA alone treated group (*P < 0.05). PLGA, poly(lactic acid-glycolic acid).