Model uv 1601 pc

The direct technique was conducted for the estimation of the EE%, DL% of ZT in the terpesomal dispersions [29 (link)]. Briefly, terpesomal dispersions (1 mL) were ultra-centrifuged, at 22,000 rpm (Sigma 3–30 KS, Sigma Laborzentrifugen GmbH, Germany) for 1 h at 4 °C. The residues, composed of the ZT-loaded terpesomes, were lysed and solubilized by sonication with ethanol (Crest ultrasonics corp., Trenton, USA). The entrapped ZT concentrations were assessed spectrophotometrically after appropriate dilution with ethanol (Shimadzu, model UV-1601 PC, Kyoto, Japan) at λ_max 285.6 nm [7 (link)]. In a parallel line, drug-free terpesomes were used as controls [22 (link)].
ZT EE% was calculated as follows; $$\mathrm{EE}\%=\frac{\mathrm{Entrapped}\mathrm{amount}\mathrm{of}\mathrm{ZT}(\mathrm{mg})}{\mathrm{Total}\mathrm{amount}\mathrm{of}\mathrm{ZT}(\mathrm{mg})}\times 100$$
ZT DL% was calculated as follows; $$\mathrm{DL}\%=\frac{\mathrm{Entrapped}\mathrm{amount}\mathrm{of}\mathrm{ZT}(\mathrm{mg})}{\mathrm{Total}\mathrm{weight}\mathrm{of}\mathrm{terpesomes}(\mathrm{mg})}\times 100$$

In vitro drug release experiment was performed for SPR-BS and free SPR suspension (control) using the bag diffusion technique [29 (link)]. Dialysis bags (Visking^®, MWCO 12,000–14,000, Serva, Denver, CO, USA) containing F3, F4, and SPR were immersed in PBS (pH 6.8) ensuring sink conditions [87 (link)]. Bags were placed in a shaking water bath (100 rpm) (Memmert GmbH, Schwabach, Germany) at 32 ± 0.5 °C [29 (link)]. At predetermined time intervals, samples were withdrawn and compensated with fresh medium. The amount of SPR released was determined using UV spectroscopy (Shimadzu, model UV-1601 PC, Kyoto, Japan) at 620 nm [85 (link)]. The experiment was conducted in triplicates and data were expressed as the mean ± SD. The SPR release mechanism from the developed nanocarrier was investigated using DD Solver 1.0 Software (Microsoft Excel add in program, Microsoft Corporation, Redmond, WA, USA) [88 (link)].

The encapsulation efficiency (EE %) of both siRNA or Dox was quantified indirectly by measuring the difference between the total amount of siRNA or Dox added during the preparation of SNALPs and the quantity of non-entrapped amounts remaining in the filtrate after the SNALPs were buffer exchanged. The siRNA amount was determined using gel red assay technique using previously constructed calibration curve. In brief: to construct the calibration curve, different volumes of siRNA were mixed with gel red (3 µl, 1:1000) then the volume was adjusted to 100 µl with Tris buffer pH 7. The coefficient of determination (R²) of the siRNA calibration curve in Tris buffer pH 7 in the concentration range of 0.1-1 nM, was 0.9977 (Figure S1A). The amount of siRNA was determined using UV-vis spectrofluorometer at excitation and emission of 300 and 590 nm respectively (Model UV-1601 PC; Shimadzu, Kyoto, Japan). The amount of Dox was determined using previously constructed standard plot of serial concentrations of Dox in PBS pH 7.4 by measuring the absorbance at 480 nm as reported in the literature 26
(Figure S1B). The EE % of either siRNA or Dox was calculated as follows:

Encapsulation efficiency (EE%) for quercetin loaded niosomes was calculated by measuring the free quercetin concentration in the niosomal dispersion. The unencapsulated drug was separated by centrifugal filters (Nanosep^® centrifuge tube) with Mw cutoff 100 kDa. Then, 0.5 mL samples of the prepared niosomes were transferred to the upper chamber of the Nanosep^® and then centrifuged for 120 min, at 6000 rpm. The amount of unencapsulated quercetin in the collecting chamber liquid was measured by measuring the absorbance at 370 nm, using UV–vis spectrophotometer (Model UV–1601PC; Shimadzu, Kyoto, Japan), as previously described [33 (link)].
The EE (%) was calculated by using the following equation: $\mathrm{EE}(\%)=\frac{{\left[Drug\right]}_{total}-{\left[Drug\right]}_{free}}{{\left[Drug\right]}_{total}} \times 100$
where [Drug]_total is the total amount of added drug, and [Drug]_free is the amount of unencapsulated free drug.