In vitro cellular localization and efficient accumulation of fluorescently tagged biomaterials from monodispersed chitosan nanoparticles for elucidation of controlled release pathways for drug delivery systems

Abstract

Background: Inefficient cellular delivery and poor intracellular accumulation are major drawbacks towards achieving favorable therapeutic responses from many therapeutic drugs and biomolecules. To tackle this issue, nanoparticle-mediated delivery vectors have been aptly explored as a promising delivery strategy capable of enhancing the cellular localization of biomolecules and improve their therapeutic efficacies. However, the dynamics of intracellular biomolecule release and accumulation from such nanoparticle systems has currently remained scarcely studied.

Objectives: The objective of this study was to utilize a chitosan-based nanoparticle system as the delivery carrier for glutamic acid, a model for encapsulated biomolecules to visualize the in vitro release and accumulation of the encapsulated glutamic acid from chitosan nanoparticle (CNP) systems.

Methods: CNP was synthesized via ionic gelation routes utilizing tripolyphosphate (TPP) as a cross-linker. In order to track glutamic acid release, the glutamic acid was fluorescently-labeled with fluorescein isothiocyanate prior encapsulation into CNP.

Results: Light Scattering data concluded the successful formation of small-sized and mono-dispersed CNP at a specific volume ratio of chitosan to TPP. Encapsulation of glutamic acid as a model cargo into CNP led to an increase in particle size to >100 nm. The synthesized CNP exhibited spherical shape under Electron Microscopy. The formation of CNP was reflected by the reduction in free amine groups of chitosan following ionic crosslinking reactions. The encapsulation of glutamic acid was further confirmed by Fourier Transform Infrared (FTIR) analysis. Cell viability assay showed 70% cell viability at the maximum concentration of 0.5 mg/mL CS and 0.7 mg/mL TPP used, indicating the low inherent toxicity property of this system. In vitro release study using fluorescently-tagged glutamic acids demonstrated the release and accumulation of the encapsulated glutamic acids at 6 hours post treatment. A significant accumulation was observed at 24 hours and 48 hours later. Flow cytometry data demonstrated a gradual increase in intracellular fluorescence signal from 30 minutes to 48 hours post treatment with fluorescently-labeled glutamic acids encapsulated CNP.

Conclusion: These results therefore suggested the potential of CNP system towards enhancing the intracellular delivery and release of the encapsulated glutamic acids. This CNP system thus may serves as a potential candidate vector capable to improve the therapeutic efficacy for drugs and biomolecules in medical as well as pharmaceutical applications through the enhanced intracellular release and accumulation of the encapsulated cargo.

Keywords: FITC; chitosan; drug delivery; glutamic acid; nanotechnology.

Figure 1

Influence of TPP volume on size and PDI value of CNP-F₁. Notes: A volume of 20–300 µL TPP was added into 600 µL CS. Bar graph represents particle size and line graph represents PDI. The particle size and PDI value decreased with increasing TPP volume. The smallest particle size and PDI value were produced at 250 µL TPP volume. Error bars represent SEM from triplicate independent experiments, where n=3. ^aSignificant difference from 20 µL TPP addition at P<0.05. ^bSignificant difference from 20 µL TPP addition at P<0.01. ^cHighly significant difference from 20 µL TPP addition at P<0.001. ^dSignificant difference from 50 µL TPP addition at P<0.05. ^eSignificant difference from 50 µL. TPP addition at P<0.01. Abbreviations: CNP, chitosan nanoparticle; CS, chitosan solution; PDI, polydispersity index; TPP, tripolyphosphate.

Figure 2

Influence of TPP volume on size and PDI value of CNP-F₂. Notes: A volume of 20–300 µL TPP was added into 600 µL CS. Bar graph represents particle size and line graph represents PDI. The particle size and PDI value decreased with increasing TPP volume. The smallest particle size and PDI value were produced at 250 µL TPP volume. Error bars represent SEM from triplicate independent experiments, where n=3. ^aSignificant difference from 20 µL TPP addition at P<0.05. ^bSignificant difference from 20 µL TPP addition at P<0.01. ^cHighly significant difference from 20 µL TPP addition at P<0.001. ^dHighly significant difference from 100 µL TPP addition at P<0.001. ^eSignificant difference from 150 µL TPP addition at P<0.01. Abbreviations: CNP, chitosan nanoparticle; CS, chitosan solution; PDI, polydispersity index; TPP, tripolyphosphate.

Figure 3

Influence of TPP volume on size and PDI value of CNP-F₃. Notes: A volume of 20–300 µL TPP was added into 600 µL CS. Bar graph represents particle size and line graph represents PDI. The particle size and PDI value decreased with increasing TPP volume. The smallest particle size and PDI value were produced at 250 µL TPP volume. Error bars represent SEM from triplicate independent experiments, where n=3. ^aSignificant difference from 20 µL TPP addition at P<0.05. ^bSignificant difference from 20 µL TPP addition at P<0.01. ^cSignificant difference from 250 µL TPP addition at P<0.05. Abbreviations: CNP, chitosan nanoparticle; CS, chitosan solution; PDI, polydispersity index; TPP, tripolyphosphate.

Figure 4

Size and PDI value of particles before and following encapsulation of GA into CNP-F₃. Notes: Bar graph represents particle size and line graph represents PDI. The particle size increased to >100 nm along with the increase in GA molarity. Error bars represent SEM from triplicate independent experiments, where n=3. ***Highly significant difference from CNP, 0.05 M GA-CNP, and 0.1 M GA-CNP at P<0.001. Abbreviations: CNP, chitosan nanoparticle; GA, glutamic acid; PDI, polydispersity index.

Figure 5

Size and PDI value of particles before and following encapsulation of fGA into CNP-F₃. Notes: Three different molarities of GA were used (0.05, 0.1, and 0.2 M). Bar graph represents particle size and line graph represents PDI. The particle size increased to >100 nm along with the increase in GA molarity. Error bars represent SEM from triplicate independent experiments, where n=3. ^aHighly significant difference from CNP-F₃ at P<0.001. ^bSignificant difference from 0.05 M GA-CNP at P<0.01. ^cHighly significant difference from 0.05 M fGA-CNP at P<0.001. Abbreviations: CNP, chitosan nanoparticle; fGA, fluorescently labeled glutamic acid; PDI, polydispersity index.

Figure 6

Fraction of free amine groups of CNP-F₁, CNP-F₂, and CNP-F₃. Notes: A volume of 50–300 µL TPP was added into 600 µL CS. Percentage of free amine groups for all CNP formulations reduced by addition of more TPP volume. Error bars represent SEM from triplicate independent experiments, where n=3. Abbreviations: CNP, chitosan nanoparticle; CS, chitosan solution; TPP, tripolyphosphate.

Figure 7

Morphology of CS, TPP, CNP, GA, GA-CNP, and fGA-CNP under field emission scanning electron microscopy. Notes: CNP exhibited a discrete and spherical shape with diameters less than 100 nm. Following encapsulation of GA and fGA molecules, particle sizes increased above 100 nm. Abbreviations: CNP, chitosan nanoparticle; CS, chitosan solution; fGA, fluorescently labeled glutamic acid; TPP, tripolyphosphate.

Figure 8

Transmission electron microscope images of CNP, GA-CNP, and fGA-CNP. Notes: CNPs appeared as spherical-shaped particles ranging from 30 to 50 nm with a dark and dense internal structure indicating the formation of compact particle structure. The particle size expanded to over 100 nm following addition of GA and fGA molecules, suggesting the successful encapsulation of these molecules within CNPs. Abbreviations: CNP, chitosan nanoparticle; fGA, fluorescently labeled glutamic acid.

Figure 9

Infrared spectra of (A) CS, TPP, and CNP, (B) CNP, GA, and GA-CNP, (C) FITC, GA-CNP, and fGA-CNP. Abbreviations: CNP, chitosan nanoparticle; CS, chitosan solution; fGA, fluorescently labeled glutamic acid; TPP, tripolyphosphate; FITC, fluorescein 5(6) isothiocyanate.

Figure 10

Cell viability of 786-O cells following 24 hours of treatment with CNP-F₃. Notes: Error bars represent SEM from a triplicate independent experiment, where n=3. *Significant difference from control at P<0.05. The colored lines represent the percentage of cell viability following 24 hours treatment with CNP-F₃. Abbreviation: CNP, chitosan nanoparticle.

Figure 11

In vitro cellular release of fGA (green) from CNPs into 786-O cells. Notes: DAPI was used to stain the cell nucleus (blue). Gradual increase in fluorescence was observed from 30 minutes to 48 hours treatment point with fGA-CNP. No green fluorescence was observed in cells treated with CNP, fluorescein 5(6)-isothiocyanate, and fGA. (Images were acquired at 100× magnification with 50 µm scale bar). Abbreviations: CNP, chitosan nanoparticle; DAPI, 4,6-diamidino-2-phenylindole dihydrochloride; fGA, fluorescently labeled glutamic acid.

Figure 11

In vitro cellular release of fGA (green) from CNPs into 786-O cells. Notes: DAPI was used to stain the cell nucleus (blue). Gradual increase in fluorescence was observed from 30 minutes to 48 hours treatment point with fGA-CNP. No green fluorescence was observed in cells treated with CNP, fluorescein 5(6)-isothiocyanate, and fGA. (Images were acquired at 100× magnification with 50 µm scale bar). Abbreviations: CNP, chitosan nanoparticle; DAPI, 4,6-diamidino-2-phenylindole dihydrochloride; fGA, fluorescently labeled glutamic acid.

Figure 12

Detection of fluorescence signal of fGA in 786-O cells through flow cytometry analysis. Notes: Fluorescence profile of cells treated with fGA-CNP for 30 minutes, 6 hours, 24 hours, and 48 hours. No FITC signal was detected in nontreated cells (control) and cells treated with CNP FITC and fGA. The fluorescence peaks shifted to the right (higher fluorescence intensity) from 30 minutes to 48 hours treatment time point (full overlay). Abbreviations: CNP, chitosan nanoparticle; fGA, fluorescently labeled glutamic acid; FITC, fluorescein 5(6)-isothiocyanate.