(C) AFM image of the (MTX + PEG)-CS-NPs Scale bars = 500 nm Ins

(C) AFM image of the (MTX + PEG)-CS-NPs. Scale bars = 500 nm. Inset: TEM image of the (MTX + PEG)-CS-NPs. Scale bars = 50 nm.

(D) Particle size distribution of the (MTX + PEG)-CS-NPs. (E) Zeta potential distribution of the (MTX + PEG)-CS-NPs. (F) In vitro stability tests of the (MTX + PEG)-CS-NPs in PBS (mean ± SD, n = 3). (G) In vitro stability tests of the (MTX + PEG)-CS-NPs in 10% PD-0332991 ic50 plasma in PBS (mean ± SD, n = 3). Drug-loading CAL-101 clinical trial content. CS-NPs possessing peripheral amino groups provided us great opportunities to easy surface biofunctionalization. In our study, the γ-carboxyl groups of MTX were conjugated to the residual amino groups of the PEGylated CS-NPs. The drug-loading content of the (MTX + PEG)-CS-NPs was calculated as 7.23 ± 0.11%. The simple conjugation chemistry and appropriate drug-loading content could favor the dual-acting role of Janus-like MTX. In vitro stability tests No significant variation of the particle size was observed in the (MTX + PEG)-CS-NPs even after incubation with PBS for a long period of time (Figure 4F). Notably, the CS-NPs (without

PEGylation) could precipitate after 48 h in the presence of salts. It was implied that PEG could protect the see more (MTX + PEG)-CS-NP against ionic strength. No significant change of the particle size was also shown in the (MTX + PEG)-CS-NPs after incubation with 10% plasma for 120 h (Figure 4G). It should be inferred that PEG could reduce the plasma proteins adsorption, and more importantly, preserve the targeting potential of MTX. All of the results suggested that the (MTX + PEG)-CS-NPs were sufficiently stable to sustain physiological conditions for extended blood circulation. In vitro drug release profiles In vitro drug release profiles of the Protirelin free MTX and (MTX + PEG)-CS-NPs were presented in Figure 5. To mimic the physiological conditions of the bloodstream, the (MTX + PEG)-CS-NPs were incubated with 10% plasma at pH 7.4. In sharp contrast to the free MTX with accumulated release amounts of almost 90% within 6 h,

a more sustained release of the NPs was clearly observed due to the slow hydrolysis of amide bonds. Nevertheless, within 48 h, only no more than 10% of MTX from NPs was released at pH 7.4. Once intravenously administrated, the NPs could ensure minimal premature release of MTX during the circulation, and thereby greatly reduces the systemic toxicity. It was expected that the NPs will accumulate at the tumor site by the EPR effect. Once inside the tumor tissue, these MTX-targeted PEG-CS-NPs will be internalized by the tumor cells, largely via FA receptor-mediated endocytosis (discussed below). Figure 5 In vitro drug release profiles of the (MTX + PEG)-CS-NPs in different physiological media (mean ± SD, n  = 3). It was well established that the amide bonds could be selectively cleaved at acidic pH by proteases (also called proteolytic enzymes) overexpressed in the tumor cells [33–36].

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