Doxorubicin-loaded quaternary ammonium palmitoyl glycol chitosan polymeric nanoformulation: uptake by cells and organs

Abstract

Purpose: This study was aimed to develop doxorubicin-loaded quaternary ammonium palmitoyl glycol chitosan (DOX-GCPQ) nanoformulation that could enable DOX delivery and noninvasive monitoring of drug accumulation and biodistribution at tumor site utilizing self-florescent property of doxorubicin.

Materials and methods: DOX-GCPQ amphiphilic polymeric nanoformulations were prepared and optimized using artificial neural network (ANN) and characterized for surface morphology by atomic force microscopy, particle size with polydispersity index (PDI), and zeta potential by dynamic light scattering. Fourier transformed infrared (FTIR) and X-ray diffractometer studies were performed to examine drug polymer interaction. The ANN-optimized nanoformulation was investigated for in vitro release, cellular, tumor, and tissue uptake.

Results: The optimized DOX-GCPQ nanoformulation was anionic spherical micelles with the hydrodynamic particle size of 97.8±1.5 nm, the PDI of <0.3, the zeta potential of 28±2 mV, and the encapsulation efficiency of 80%±1.5%. Nanoformulation demonstrated a sustained release pattern over 48 h, assuming Weibull model. Fluorescence microscopy revealed higher uptake of DOX-GCPQ in human rhabdomyosarcoma (RD) cells as compared to free DOX. In vitro cytotoxicity assay indicated a significant cytotoxicity of DOX-GCPQ against RD cells as compared to DOX and blank GCPQ (P<0.05). DOX-GCPQ exhibited low IC₅₀ (1.7±0.404 µmol) when compared to that of DOX (3.0±0.968 µmol). In skin tumor xenografts, optical imaging revealed significantly lower DOX-GCPQ in heart and liver (P<0.05) and accumulated mainly in tumor (P<0.05) as compared to other tissues.

Conclusion: The features of nanoformulation, ie, small particle size, sustained drug release, and enhanced cellular uptake, potential to target tumor passively coupled with the possibility of monitoring of tumor localization by optical imaging may make DOX-GCPQ an efficient nanotheranostic system.

Keywords: artificial neural network; biodistribution; doxorubicin; nanotheranostic; optical imaging; quaternary ammonium palmitoyl glycol chitosan.

Figure 1

Architect of artificial network employed for the optimization of DOX formulation using GCPQ. Abbreviations: DOX, doxorubicin; GCPQ, quaternary ammonium palmitoyl glycol chitosan.

Figure 2

NMR spectrum of (a) degraded glycol chitosan and (b) GCPQ. Abbreviation: GCPQ, quaternary ammonium palmitoyl glycol chitosan.

Figure 3

Percentage effect of input factors on DOX–GCPQ formulation. Abbreviations: DOX, doxorubicin; GCPQ, quaternary ammonium palmitoyl glycol chitosan.

Figure 4

Response surface plots of the combined effect of (A) amplitude of sonication and drug concentration on size, zeta potential, PDI, and loading along with SD and (B) amplitude of sonication and time on size, zeta potential, PDI, and loading along with SD. Abbreviation: PDI, polydispersity index.

Figure 4

Response surface plots of the combined effect of (A) amplitude of sonication and drug concentration on size, zeta potential, PDI, and loading along with SD and (B) amplitude of sonication and time on size, zeta potential, PDI, and loading along with SD. Abbreviation: PDI, polydispersity index.

Figure 5

DOX–GCPQ. Notes: (A) DLS plots. (B) AFM image. Abbreviations: AFM, atomic force microscopy; DLS, dynamic light scattering; DOX, doxorubicin; GCPQ, quaternary ammonium palmitoyl glycol chitosan.

Figure 6

FTIR spectra of GCPQ, DOX and DOX–GCPQ. Abbreviations: DOX, doxorubicin; FTIR, Fourier transformed infrared; GCPQ, quaternary ammonium palmitoyl glycol chitosan.

Figure 7

X-Ray diffractometer spectra of DOX and DOX–GCPQ formulation. Abbreviations: DOX, doxorubicin; GCPQ, quaternary ammonium palmitoyl glycol chitosan.

Figure 8

In vitro drug release of DOX and DOX–GCPQ nanoformulation in phosphate buffer (pH 7.4) at 37°C. Abbreviations: DOX, doxorubicin; GCPQ, quaternary ammonium palmitoyl glycol chitosan.

Figure 9

Cellular uptake and intracellular localization of DOX and DOX–GCPQ nanoformulations in human RD cells observed by fluorescence microscopy of (A) untreated cells, (B) fluorescence of internalized DOX inside RD cells, and (C) fluorescence of internalized DOX–GCPQ inside RD cells. Note: Magnification 40×. Abbreviations: DOX, doxorubicin; GCPQ, quaternary ammonium palmitoyl glycol chitosan; RD, rhabdomyosarcoma.

Figure 10

In vitro cytotoxicity DOX and DOX–GCPQ nanoformulation in human RD cells observed by inverted microscope after 24 h of incubation. Notes: (A) Untreated RD cells. (B) Cell viability with DOX. (C) Cell viability with DOX–GCPQ. Magnification 40×. Abbreviations: DOX, doxorubicin; GCPQ, quaternary ammonium palmitoyl glycol chitosan; RD, rhabdomyosarcoma.

Figure 11

Cell viability of DOX and DOX–GCPQ against human RD cells. Note: All cells were incubated for 24 h, and cell viability was determined by MTT assay (P<0.05). Abbreviations: DOX, doxorubicin; GCPQ, quaternary ammonium palmitoyl glycol chitosan; RD, rhabdomyosarcoma.

Figure 12

Biodistribution of intravenously injected DOX and DOX–GCPQ in skin tumor bearing mice. Notes: (A) Representative fluorescent images of tumor and excised organs 24 h after injection. (B) Quantitative fluorescent intensity of excised organ and tumor (P<0.05). ***P<0.0001. Abbreviations: DOX, doxorubicin; GCPQ, quaternary ammonium palmitoyl glycol chitosan.