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For low protein concentrations containing biological samples (in proteomics) and for non proteinaceous compound assays (in bioanalysis), there is a critical need for a simple, fast, and cost-effective protein enrichment or precipitation method. However, 2,2,2-trichloroacetic acid (TCA) is traditionally used for protein precipitation at ineffective concentrations for very low protein containing samples. It is hypothesized that response surface methodology, can be used to systematically identify the optimal TCA concentration for protein precipitation in a wider concentration range. To test this hypothesis, a central composite design is used to assess the effects of two factors (X1 = volume of aqueous solution of protein, and X2 = volume of TCA solution 6.1N) on the optical absorbance of the supernatant (Y1), and the percentage of protein precipitated (Y2). Using either bovine serum albumin (BSA) as a model protein or human urine (with 20 ppm protein content), 4% w/v (a saddle point) is the optimal concentration of the TCA solution for protein precipitation that is visualized by SDS-PAGE analysis. At this optimal concentration, the Y2-values range from 76.26 to 92.67% w/w for 0.016 to 2 mg/mL of BSA solution. It is also useful for protein enrichment and xenobiotic analysis in protein-free supernatant as applied to tenofovir (a model HIV microbicide). In these conditions, the limit of detection and limit of quantitation of tenofovir are respectively 0.0014 mg/mL and 0.0042 mg/mL. This optimal concentration of TCA provides optimal condition for protein purification and analysis of any xenobiotic compound like tenofovir.

Purpose It is hypothesized that sodium acetate (SA) can be used for in situ coating of drug loaded chitosan NPs for improved physico-chemical properties. Methods Tenofovir (TFV) is used as a model drug. Uncoated chitosan NPs are prepared by ionic gelation. SA is generated in situ from half neutralization of acetic acid with sodium hydroxide, and coats chitosan NPs during freeze-drying. The NPs physico-chemical properties [e.g. particle mean diameters (PMD) and zeta potential (ζ), EE%, drug release profile, morphology] are characterized by dynamic light scattering, spectrophotometry, Korsmeyer-Peppas model, transmission electron microscopy (TEM), respectively. Melting point (MP), non-aqueous titration, Fourier transform infrared (FTIR) analysis, and X-ray powder diffractometry (XRD) pattern evaluated the SA coated chitosan NPs. The NPs cytotoxicity on macrophages Raw 264.7 is assessed by neutral red, resazurin, nitrite oxide (NO) and cytokines assay. Results Collectively, FTIR, ζ, XRD, MP, and TEM data confirmed that SA coats chitosan NPs. The PMD range is 136–348 nm (uncoated) and 171–379 nm (coated) NPs. The ζ values range is +24.3–28.5 mV (uncoated) and 0.1–3.1 mV (coated). The EE% ranges from 5.5–11.7 % (uncoated NPs) and increased up to 8–17 fold (86.3–92.7% after coating). The SA also prevents NPs aggregation’s during the freeze-drying. The core-shell NPs exhibited a sustain release of TFV following anomalous transport mechanism (R2~0.99). Coated NPs are non-cytotoxic (cell viability ~100%) and without any proinflammatory response. Conclusions These SA coated chitosan NPs may be useful for (i) efficient encapsulation, (ii) masking tastes, (iii) controlling the release and improving solubility of drug.

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