Dual-layered nanogel-coated hollow lipid/polypeptide conjugate assemblies for potential pH-triggered intracellular drug release

Abstract

To achieve effective intracellular anticancer drug delivery, the polymeric vesicles supplemented with the pH-responsive outlayered gels as a delivery system of doxorubicin (DOX) were developed from self-assembly of the lipid/polypeptide adduct, distearin grafted poly(γ-glutamic acid) (poly(γ-GA)), followed by sequential deposition of chitosan and poly(γ-GA-co-γ-glutamyl oxysuccinimide)-g-monomethoxy poly(ethylene glycol) in combination with in situ covalent cross-linking on assembly surfaces. The resultant gel-caged polymeric vesicles (GCPVs) showed superior performance in regulating drug release in response to the external pH change. Under typical physiological conditions (pH 7.4 and 37 °C) at which the γ-GA/DOX ionic pairings remained mostly undisturbed, the dense outlayered gels of GCPVs significantly reduced the premature leakage of the uncomplexed payload. With the environmental pH being reduced from pH 7.4 to 4.7, the drug liberation was appreciably promoted by the massive disruption of the ionic γ-GA/DOX complexes along with the significant swelling of nanogel layers upon the increased protonation of chitosan chain segments. After being internalized by HeLa cells via endocytosis, GCPVs exhibited cytotoxic effect comparable to free DOX achieved by rapidly releasing the payload in intracellular acidic endosomes and lysosomes. This strongly implies the great promise of such unique GCPVs as an intracellular drug delivery carrier for potential anticancer treatment.

Figure 1. Synthetic route of (a) poly(γ-GA-co-γ-DSGA) and (b) poly(γ-GA-co-γ-GAOSu)-g-mPEG.

Figure 2. ¹H-NMR spectra of (a) poly(γ-GA-co-γ-DSGA) and (b) poly(γ-GA-co-γ-GAOSu)-g-mPEG in DMSO-d₆ at 25°C.

Figure 3. Angle-dependent DLS/SLS data and TEM images of lipid/polypeptide conjugate vesicles (a, b) and DOX-loaded GCPVs (c, d).

Figure 4. Cumulative drug release profiles of DOX-loaded vesicles and DOX-loaded GCPVs in aqueous solutions of pH 7.4 and 4.7, respectively, at 37°C.

Figure 5. pH-dependent characteristics of DOX-loaded GCPVs and DOX-loaded chitosan/ poly(γ-GA)-deposited polymeric vesicles.

(a) DLS colloidal particle size distribution profiles of DOX-loaded GCPVs in aqueous media of various pH. (b) Zeta potentials of DOX-loaded GCPVs and DOX-loaded chitosan/ poly(γ-GA)-deposited polymeric vesicles in different pH aqueous media.

Figure 6. Schematic illustration of the development of DOX-loaded GCPVs and their pH-triggered drug release.

Figure 7. Flow cytometric histogram profiles of HeLa cells incubated with free DOX, DOX-loaded vesicles and DOX-loaded GCPV.

DOX fluorescence intensity of HeLa cells incubated with free DOX (red), DOX-loaded vesicles (green) and DOX-loaded GCPV (blue) at 37°C for 1 and 2 h, respectively. Untreated cells (black) were used as a control.

Figure 8. CLSM images of HeLa cells incubated with free DOX and DOX-loaded GCPVs at 37°C for 2 h (DOX concentration  =  10 μM).

Cell nuclei were stained with Hoechst 33258.

Figure 9. In vitro cytotoxicity of free DOX and DOX-loaded GCPVs against HeLa cells with an incubation time of 48 h.

The data presented herein represent an average of at least triplicate experiments.