Synthesis and Characterization of NPs

An emulsion-solvent evaporation technique (12) was used to synthesize RIF loaded PLGA (PLGA(RIF)) and Glu-PLGA NPs (Glu-PLGA(RIF)). To synthesize PLGA(RIF) NPs, a 20 mg/ml PLGA solution in ethyl acetate (containing 2 mg/ml RIF) was added drop-wise to 10 mg/ml PVA solution in de-ionised water under stirring at 1000 rpm. Upon complete addition, the suspension on ice was subjected to high speed homogenization at 8000 rpm for 10 min using a Silverson L4R mixer homogenizer (Silverson, MA). Ethyl acetate was subsequently removed in vacuo at 40°C for 1 h and NPs collected through centrifugation at 10000 x g for 5 min, followed by washing thrice with de-ionized water, and lyophilization over 72 h in the presence of sucrose as a cryoprotectant. To synthesize C-PLGA(RIF) NPs, the procedure described above was followed, however, a dimethylformamide: chloroform mixture (20:80) was used as solvent. Rh-PLGA and Glu-PLGA-Rh NPs were also synthesized as above, except that the organic phase did not contain RIF.

Suspensions of NPs in de-ionized water (1 mg/ml) were characterized for size, polydispersity index (PDI) and zeta potential using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd, UK). Measurements were conducted at 25°C in triplicate. The amount of RIF encapsulated in the NPs was determined using a validated HPLC-UV method, following destruction of the NPs through sonication in acetonitrile.