Fig 1.
Illustration showing the functionalization or conjugation reaction steps for DSPE-PEG-NH2 conjugation with Lactobionic acid (LA).
Fig 2.
Size distribution analysis (DLS) on control (CL) and lactobionic acid-conjugated (LL) DOX liposomes.
Left Y-axis represents the hydrodynamic radius (nm) and right Y-axis indicates the polydispersity index.
Fig 3.
FTIR confirmation of DSPE-PEG-LA conjugate formation.
FTIR spectra comparing DSPE-PEG-NH2 (unreacted lipid), LA, a physical mixture of DSPE and LA (DSPE-LA mix), and the chemically conjugated product (DSPE-LA conjugate). The appearance of new peaks and characteristic shifts is highlighted by dashed lines, indicating successful covalent conjugation.
Fig 4.
Absorbance and Fluorescence Emission profiles of DOX-loaded LL.
A) Absorbance spectrum (Left Y-axis) and fluorescence emission spectra (Right Y-axis) of LL. B) The emission spectra of LL before and after the addition of Triton X-100 (Ex: 490 nm, Em: 500–900 nm) confirm significant DOX loading.
Fig 5.
Stability and release studies on liposomes.
A) Stability studies at 37 °C. Liposomes (CL and LL) were incubated at 37 °C, with fluorescence measurements taken for each sample over 60 s after a specified incubation period (3 and 24 hours). The results represent the average of three liposome batches. B) Normalized-averaged DOX release profiles of CL and LL at different US-intensities. Experiments were conducted at two power densities [6.2 mW/cm2 (20%) and 10 mW/cm2 (30%)].
Fig 6.
Cytotoxicity analysis A) Cytotoxicity analysis on HepG2 cell line with different levels of free DOX.
B) MTT assay on HepG2 cell line with conjugated (LL) and non-conjugated liposomes (CL) with and without LFUS. Liposomes with DOX concentration of 8 µM (LL and CL) were used for the assays. The data illustrates the enhanced cytotoxicity in ASGPR-positive HepG2 cells, particularly with LL and US treatment. The results represent the mean value of 3 different experiment data and error bar indicates ±SD.
Fig 7.
Cellular uptake of DOX by Flow cytometry analysis.
Flow cytometry analyses on different cell lines (HepG2, Jurkat, and 3T3) to evaluate the effects of CL and LL, both with and without ultrasound treatment.
Fig 8.
Phase contrast microscopic morphological examinations.
Phase contrast microscopy images of HepG2 cells treated with CL and LL, both with and without LFUS. Images were captured to assess morphological changes upon treatments.
Fig 9.
Annexin V-FITC/PI double staining analysis revealing apoptosis in HepG2 cells. Each quadrant description is as follows: Upper right quadrant (Q2)-dead cells in late stage of apoptosis; lower right quadrant (Q3)-cells undergoing apoptosis (early-stage apoptotic cells); lower left quadrant (Q4) – healthy viable cells and upper left (Q1) represents non-viable necrotic stage. The cells treated with free DOX and targeted liposome (LL) with US exposure showed (LL + US) significantly (p < 0.0001) high late apoptotic cells as compared to control cells (insert). The data show mean ± SD percentages of late apoptosis in HepG2 cells, treated with different conditions in terms of liposome or US treatment.