Layer-by-layer pH-sensitive nanoparticles for drug delivery and controlled release with improved therapeutic efficacy in vivo

Abstract

In this work, a pH-sensitive liposome-polymer nanoparticle (NP) composed of lipid, hyaluronic acid (HA) and poly(β-amino ester) (PBAE) was prepared using layer-by-layer (LbL) method for doxorubicin (DOX) targeted delivery and controlled release to enhance the cancer treatment efficacy. The NP with pH-sensitivity and targeting effect was successfully prepared by validation of charge reversal and increase of hydrodynamic diameter after each deposition of functional layer. We further showed the DOX-loaded NP had higher drug loading capacity, suitable particle size, spherical morphology, good uniformity, and high serum stability for drug delivery. We confirmed that the drug release profile was triggered by low pH with sustained release manner in vitro. Confocal microscopy research demonstrated that the NP was able to effectively target and deliver DOX into human non-small cell lung carcinoma (A549) cells in comparison to free DOX. Moreover, the blank NP showed negligible cytotoxicity, and the DOX-loaded NP could efficiently induce the apoptosis of A549 cells as well as free DOX. Notably, in vivo experiment results showed that the DOX-loaded NPs effectively inhibited the growth of tumor, enhanced the survival of tumor-bearing mice and improved the therapeutic efficacy with reduced side-effect comparing with free drug. Therefore, the NP could be a potential intelligent anticancer drug delivery carrier for cancer chemotherapy, and the LbL method might be a useful strategy to prepare multi-functional platform for drug delivery.

Keywords: Drug delivery; HA-targeting; cancer therapy; controlled release; layer-by-layer; nanoparticle; pH-sensitivity.

Figure 1.

Schematic illustration of preparation process of LbL DOX-loaded NPs and targeted drug delivery for anticancer.

Figure 2.

Preparation and characterization of DOX-loaded NPs. Hydrodynamic diameters (A), PDI (B), and charge reversal in zeta-potential (C) of DOX-loaded liposomes and DOX-loaded NPs. (D) TEM image of DOX-loaded NPs (scale bar: 1 μm).

Figure 3.

Confirmation of pH-sensitivity and stability of DOX-loaded NPs. (A) The potentiometric titration of the PBAE solutions dependent on the volume of NaOH solution. The particle size (B), PDI (C), and zeta-potential (D) of DOX-loaded NPs dependent on the different pH. Hydrodynamic diameters (E) and PDI (F) of DOX-loaded NPs after different incubation time in PBS with 20% FBS at 37 °C.

Figure 4.

In vitro pH-triggered drug release profiles of multi-layered DOX-loaded NPs at pH 7.4 and 5.0.

Figure 5.

Cellular uptake of free DOX and DOX-loaded NPs. Confocal microscopy image of cells incubated with free DOX (A) and LbL DOX-loaded NPs (B) for different time intervals (upper: 1 h, bottom: 8 h, scale bars are 20 μm).

Figure 6.

In vitro cytotoxicity of liposome and NPs (A), free DOX and DOX-loaded NPs (B) at different concentrations against A549 cells for 24 h.

Figure 7.

Therapeutic efficacy of A549 tumor-bearing mice. (A) Tumor volume growth curves of different groups of mice after various treatments; (B) average weights of tumors collected from the mice at the end of therapy; (C) the weight of A549 tumor-bearing mice (n = 10, mean ± SD). *p< .05, ***p< .001. (D) Survival rates of mice after treatment. Statistical analysis was done using Kaplan–Meier’s method (n = 10).