Decitabine enclosed biotin-zein conjugated nanoparticles: synthesis, characterization, in vitro and in vivo evaluation

Abstract

Aim: This study focuses on biotinylated nanocarriers designed to encapsulate amphiphilic molecules with self-biodegradable properties for enhanced drug delivery.Methods: Biotin-zein conjugated nanoparticles were synthesized and tested in C6 cell lines to evaluate their viability and cellular uptake. Optimization was achieved using a a central composite design. The nanoparticles underwent thermogravimetric analysis, and their pharmacokinetics and biodistribution were also studied.Results: The optimized nanoparticles displayed 96.31% drug encapsulation efficiency, a particle size of 95.29 nm and a zeta potential of -17.7 mV. These nanoparticles showed increased cytotoxicity and improved cellular uptake compared with free drugs. Thermogravimetric analysis revealed that the drug-loaded nanocarriers provided better protection against drug degradation. Pharmacokinetic and biodistribution studies indicated that the formulation had an extended brain residence time, highlighting its effectiveness.Conclusion: The biotin-zein conjugated nanoparticles developed in this study offer a promising nano-vehicle for in vivo biodistribution and pharmacokinetic applications. Their high drug encapsulation efficiency, stability and extended brain residence time suggest they are effective for targeted drug delivery and therapeutic uses.

Keywords: C6 cell line; biodistribution; central composite design; decitabine; in vivo bioimaging; nanoparticle; zein.

Graphical abstract

Figure 1.

(A) NMR spectra of biotin, zein and biotin-zein conjugate. The NMR spectra reveals that the biotin shows -COOH peak whereas the zein shows characteristic peak suggest the purity of excipients. The formation of amide proton upon conjugation with biotin-COOH and zein active sites. This confirms the formation of biotin-zein conjugate. (B) FTIR spectra of decitabine, biotin, zein, biotin-zein conjugate and decitabine loaded lyophilized nanoformulation. In zein spectra presence of free amine groups suggest the available amine binding sites. Whereas upon conjugation with biotin the diminishing extent of these free amine groups suggest binding at these sites and formation of amide functional group in FTIR spectra. (C) Circular dichroism spectra of all crude compounds along with mixture and conjugate. The secondary structural elucidation suggests decrease in the extent of α-helix and increase in formation of β-sheets. (D) DSC thermogram of decitabine, biotin, zein, biotin-zein conjugate, excipient blend and drug loaded lyophilized nanoformulation. This suggests successful encapsulation of drug within polymeric conjugate.

Figure 2.

(A & B) SEM analysis of prepared nanoparticle. (C & D) TEM analysis of prepared nanoparticle. The prepared nanoparticles reveal spherical shape on higher, magnification. The SEM images exhibits clear external morphology of prepared nanoparticles showed smooth particles within the predefined range i.e. <200 nm. The TEM micrographs suggest a circular shaped, smooth particle within the range of <200 nm.

Figure 3.

Nasal ciliotoxicity study (A) Positive control (B) negative control (C) DEC-Z-NPs (D) DEC-BZ-NPs. Under nasal ciliotoxicity we have evaluated the biosafety of prepared formulation. The Figure 3A suggest impact of positive control on the tissue. Whereas both the prepared nanoformulation reveals maintenance of tissues structural integrity and showed less toxicity compared with positive control.

Figure 4.

(A) % Cell viability of C6 cells observed under different concentrations of Decitabine post 6-h incubation (B) The cytotoxic potential of nanoparticles (C) Cellular uptake of FITC conjugated formulations captured by confocal microscope in C6 cell line at 0H, 3H and 6H incubation time points where (1.1), (2.1), (3.1) is free FITC at 0H, 3H and 6H respectively; (1.2), (2.2), (3.2) is conjugated formulation at 0H, 3H and 6H respectively; (1.3), (2.3), (3.3) is unconjugated formulation at 0H, 3H and 6H respectively. All images were captured at 40×. (D) Cellular uptake of FITC conjugated formulations captured by confocal microscope in C6 cell line at 0H, 3H and 6H incubation time points where (1.1), (2.1), (3.1) is free FITC at 0H, 3H and 6H respectively; (1.2), (2.2), (3.2) is conjugated formulation at 0H, 3H and 6H respectively; (1.3), (2.3), (3.3) is unconjugated formulation at 0H, 3H and 6H respectively. All images were captured at 63×. The cellular uptake analysis with FITC tagged nanocarriers were evaluated in C6 cell line at 3 different time points. The data suggest a time dependent cellular uptake within cells. Highest amount of green fluorescence is seen at 6H treatment group followed by 3H and 0H. Data are expressed as mean ± SD; statistical analysis was carried out using one-way ANOVA with Dunnett's multiple comparison test (n = 3).

Figure 5.

Migration analysis of C6 cells upon treatment with NPs, Decitabine and Blank NPs the scratch was observed for 0 hr, 24 hr and 48 hr respectively. The scratch closure was micrographed with inverted microscope (Leica Microsystems) and analyzed by image J software. The inhibition percentage was expressed by normalizing with control wells at 100%. The scratch assay suggested that the prepared nanoformulation maintains the inhibitory percentage compared with the control and drug solution group. This suggested that formulation provides extended-release dependent enhanced cell migration ability in time dependent fashion. This reveals the efficacy of nanoparticle intervention in impeding cancer cell migration, a pivotal characteristic in cancer progression.