OBJECTIVE To investigate the preparation and characterization of α-Hederin-loaded galactosylated chitosan nanoparticle (α-Hederin-GC-NP) and to study its anti-proliferative activity in HepG2 cell.METHODS The emulsion solvent diffusion technique was used to prepare α-Hederin-loaded galactosylated chitosan nanoparticle (α-Hederin-GC-NP).The entrapment efficiency of α-Hederin-GC-NP was determined through indirect method by using the supernatant obtained after ultracentrifugation of the prepared NP.Morphological characteristics of the NP were examined using a high resolution Transmission Electron Microscope (TEM) machine.The Fourier transform infrared (FTIR) spectra were obtained for characterizing the chemical integrity of α-Hederin inside galactosylated chitosan NP.Differential scanning calorimetry(DSC) analysis was conducted to determine the phase peak of drug and its formulations to demonstrate the change of phase.XRD analysis was carried out to know the crystalline peak of the drug and confirm its physical state inside the NP.The dialytic method was used to investigate in vitro release of α-Hederin-GC-NP.Cellular binding and eventual uptake studied of NP by HepG2 cell were done using Olympus inverted fluorescence microscope, which were also quantified using flow cytometer.The cytotoxic effect of void GC-NP, free α-Hederin and its formulations were analyzed in HepG2 cell by MTT assay.Changes in PS asymmetry, analyzed by measuring Annexin V-FITC binding to the cell membrane in flow cytometry was detected after treatment of HepG2 with free α-Hederin, void GC-NP, α-Hederin-CS-NP and α-Hederin-GC-NP.RESULTS The improved preparation technique was well practical for hydrophobic α-Hederin.The entrapment efficiency of prepared nanoparticles was nearly (78.53±4.39) %.TEM analysis revealed the smooth, distinct spherical shape of the NP with a solid dense structure.As shown in particle size distribution picture, particle-size analysis reveals that the formulated α-Hederin-GC-NP had an average diameter of (88.9±2.1) nm.FTIR curves, as is shown in figure2(Ⅰ), briefly displayed the FTIR spectra of GC (a), free α-Hederin (b), α-Hederin/GC physical mixture (c) and α-Hederin-GC-NP (d), respectively.The FTIR spectra of α-Hederin-GC-NP shows the characteristic peak 2956.2 cm-1 similar to that of α-Hederin at 2958.8 cm-1,along with the presence of peaks at 3466.1 cm-1v(-OH), 1640 cm-1v(O=C<), 1612.5 cm-1 δ(-NH2), 1101.3 cm-1v(C-O-C)for GC.The peak at 3489.2 cm-1 is slightly displaced to 3294.4 cm-1due to some chemical interaction between α-Hederin and galactosylated chitosan.As is revealed in figure 2 (Ⅱ), the new endothermic peaks of α-Hederin-GC-NP at the T value of 149.76 and 158.97 ℃ has, for the most part, masked or impaired the original heat absorption peak of GC (58.72 ℃), α-Hederin (49.84,91.93,260.12 ℃) and α-Hederin/GC physical mixture (57.65 ℃), suggesting that a new phase might have been generated.Characteristic peaks observed in XRD atlas at 20 value of 10.8,12.7,13.2, 14.39, 15.75 and 17.86 degrees indicated the crystalline nature of α-Hederin, which were absent in GC, α-Hederin-GC-NP.However, crystal peaks of α-Hederin were present in case of physical mixture of α-Hederin and GC, indicating that the drug is present in crystalline form due to the absence of any interaction with GC.Therefore, it can be anticipated that α-Hederin is present in an amorphous state in the nanoparticulate formulations.The in vitro release of α-Hederin-GC-NP in figure2(Ⅳ)showed that pH might have an impact on release rate and extent, and acid condition was beneficial to the in vitro ralease.HepG2 cell displayed higher cytotoxicity when treated with α-Hederin-GC-NP (IC50 ~26.09 μg/mL) in comparison with free α-Hederin treatment(IC50 ~48.87 μg/mL) and α-Hederin-CS-NP treatment(IC50 ~31.08 μg/mL).Cells exposed to FITC-labeled α-Hederin-GC-NP, as is shown in figure3, demonstrated increased fluorescence activity in comparison with α-Hederin-CS-NP.Moreover, it was also observed that NP uptake after binding was also dependent on the incubation time.Internalization of NPs after binding increased gradually with incubation time, which was reflected by the increase in the fluorescence intensity (maximum in case of α-Hederin-GC-NP in both periods).In addition, cellular uptake of α-Hederin-GC-NP was 1.47 times higher and in the case of α-Hederin-CS-NP, and the average fluorescence intensity had been obviously increased.Cell apoptosis,as is exhibited in figure5,showed enhanced apoptosis in the case of HepG2 cells treated with α-Hederin-GC-NP followed by higher cell death by α-Hederin-CS-NP in comparison with free α-Hederin at 24 hours, α-Hederin-GC-NP were capable of inducing apoptosis in 14.58% cells, and α-Hederin-CS-NP showed 13.26% cell death, additionally, free α-Hederin caused 9.98% cell death and void GC-NP caused only 3.03%, indicating its nontoxic nature.CONCLUSION α-Hederin-GC-NP was nearly round and spherical, and characterized with high entrapment efficiency, homogeneous size distribution.The characteristic of the in vitro slow-release of α-Hederin-GC-NP was significant and partly pH-dependent.FTIR, DSC and XRD demonstrated that α-Hederin had been entrapped into GC-NP or embedded into the surface of GC-NP.α-Hederin-GC-NP had a higher cytotoxic effect on HepG2 cell.Moreover, the cellular binding and eventual uptake studied of α-Hederin-GC-NP by HepG2 cell was much higher than α-Hederin-CS-NP ,and its probable that the higher cellular binding with eventual uptake observed in the case of α-Hederin-GC-NP is presumably due to their greater intracellular delivery by asialoglycoprotein receptor mediated endocytosis.