HER-2 Receptor and αvβ3 Integrin Dual-Ligand Surface-Functionalized Liposome for Metastatic Breast Cancer Therapy
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Methods
2.2.1. Synthesis of TPGS-COOH
2.2.2. Synthesis of Trast-TPGS-COOH Hybrid Conjugate
2.2.3. iRGD-TPGS-COOH Hybrid Conjugate
2.2.4. Experimental Design and Optimization of GFB and LCH Dual Drug-Loaded Liposome
2.2.5. Surface Functionalization of Optimized Liposome by iRGD and Trast
3. Characterization of DSDLs
3.1. Zeta Analysis
3.2. Scanning Electron Microscopy (SEM) Analysis
3.3. Transmission Electron Microscopy (TEM) Study
3.4. X-ray Diffraction (XRD) Study
3.5. Fourier Transform Infrared Spectrophotometer (FTIR) Analysis
3.6. Determination of Encapsulation Efficiency (EE%) and Drug Loading (DL%)
3.7. Stability Studies
3.8. In-Vitro Drug Release Study
3.9. Blood Compatibility Study
3.10. In-Vitro Cell Study
3.10.1. In-Vitro Cytotoxicity (MTT Assay)
3.10.2. Cellular Uptake by Confocal Laser Scanning Microscopy (CLSM)
3.10.3. Wound-Healing Assay
3.11. Statistical Analysis
4. Results
4.1. Experimental Design and Optimization of Liposomes
4.1.1. BBD 3 Factor-3 Level Approach
Effect of Formulation Parameters (E-80, Cholesterol, and TPGS) on PS
Effect of Formulation Parameters (E-80, Cholesterol, and TPGS) on PDI
Effect of Formulation Parameters (E-80, Cholesterol, and TPGS) on ZP
4.1.2. Optimization of Formulation by Numerical Analysis and Point Prediction
4.2. Zeta Analysis
4.3. Surface Morphology by SEM and TEM Analysis
4.4. Characterization of Hybrid Conjugates FTIR Analysis
4.4.1. Characterization of TPGS-COOH by FTIR
4.4.2. Characterization of iRGD-TPGS-COOH by FTIR
4.4.3. Characterization of Trast-TPGS-COOH by FTIR
4.4.4. Characterization of DSDL Formulation
4.5. EE (%) and DL (%)
4.6. In-Vitro Drug Release Profile
4.7. XRD Analysis
4.8. Stability Studies
4.9. Biocompatibility Study
4.10. In-Vitro Cell Line
4.10.1. Cytotoxicity Studies
4.10.2. Cellular Uptake of SDLs and DSDLs on SK-BR-3 and MDA-MB-231
4.10.3. Wound-Healing Assay
5. Discussion
6. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
HER-2 | Human epidermal growth factor receptor-2 |
Trast | Trastuzumab |
GFB | Gefitinib |
LCH | Lycorine hydrochloride |
TPGS-COOH | Activated D-Alpha-Tocopheryl polyethylene glycol 1000 succinate |
DSDL | Dual surface-decorated liposomes |
PS | Particle size |
PDI | Polydispersity index |
ZP | Zeta potential |
IC-50 | Half-maximum inhibitory concentration |
SDL | Single decorated liposomes |
CLSM | Confocal laser scanning microscopy |
ErbB-2 | Erythroblastic leukemia viral oncogene homolog-2 |
RGDK | Cryptic CendR motif |
NRP-1 | Neuropilin-1 |
TCRP1 | Tongue cancer resistance-associated protein |
Akt | Protein kinase B |
mTOR | Mammalian target of rapamycin |
BCS | Biopharmaceutical classification system |
MAPK/PI3K | Mitogen-activated protein kinase/phosphatidylinositol 3-kinase |
TME | Tumor microenvironment |
TPGS | D-Alpha-Tocopheryl polyethylene glycol 1000 succinate |
HLB | Hydrophilic and lipophilic balance |
FDA | Food and drug administration |
DSPE-PEG2000 | 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] |
CAFs | Cancer-associated fibroblasts |
ECM | Extracellular matrix |
TAMs | Tumor-associated macrophages |
MDSCs | Myeloid-derived suppressor cells |
TGF-β | Transforming growth factor beta |
VEGF | Vascular endothelial growth factor |
TILs | Tumor-infiltrating lymphocytes |
HIFs | Hypoxia-inducible factors |
PD-L1 | Programmed-death ligand |
CTLA-4 | Cytotoxic T-lymphocyte-associated protein-4 |
NKG-2A | Natural killer group 2A |
EDC | 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride |
NHS | N-hydroxy succinimide |
DMAP | 4-(Dimethyl amino) pyridine |
MTT | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
DAPI | 4′,6-diamidino-2-phenylindole dihydrochloride |
DMEM | Dulbecco’s Modified Eagle Medium |
FBS | Fetal bovine serum |
DCM | Dichloromethane |
PBS | Phosphate buffered saline |
RSM | Response surface methodology |
DOE | Design of experiments |
BBD | Box–Behnken design |
R2 | Correlation coefficient |
RMSE | Root mean square error |
SEM | Scanning electron microscopy |
TEM | Transmission electron microscopy |
XRD | X-ray diffraction |
FTIR | Fourier transform infrared spectroscopy |
EE | Entrapment efficiency |
DL | Drug loading |
RP-HPLC | Reverse-phase high-performance liquid chromatography |
ICH | International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use |
ACN | Acetonitrile |
TFA | Trifluoroacetic acid |
NCCS | National Centre for Cell Science |
DMSO | Dimethyl sulfoxide |
CLSM | Confocal laser scanning microscopy |
MFI | Mean fluorescence intensity |
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Variables | Levels | ||
---|---|---|---|
−1 | 0 | +1 | |
Independent variables | |||
X1 = E-80 (mg) | 30 | 45 | 60 |
X2 = Cholesterol (mg) | 10 | 15 | 20 |
X3 = TPGS (mg) | 5 | 7.5 | 10 |
Dependent variables | Desired response | ||
Y1 = PS (nm) | Minimum | ||
Y2 = PDI | Minimum Maximum | ||
Y3 = ZP (±) |
Independent Variable (Factors) | Dependent Variable (Response) | |||||
---|---|---|---|---|---|---|
Formulation Code | Factor 1 (X1) | Factor 2 (X2) | Factor 3 (X3) | PS (nm) Y1 | PDI Y2 | ZP (±mV) Y3 |
LP1 | 45 | 15 | 7.5 | 152.8 ± 1.54 | 0.375 ± 0.046 | −7.36 |
LP 2 | 60 | 15 | 5 | 189.4 ± 2.06 | 0.423 ± 0.068 | −11.32 |
LP 3 | 30 | 15 | 5 | 135.5 ± 1.36 | 0.355 ± 0.034 | −9.1 |
LP 4 | 30 | 15 | 10 | 143.5 ± 1.48 | 0.362 ± 0.081 | −6.31 |
LP 5 | 30 | 20 | 7.5 | 149.2 ± 1.59 | 0.348 ± 0.059 | −8.26 |
LP 6 | 45 | 20 | 5 | 164.3 ± 1.85 | 0.318 ± 0.047 | −9.69 |
LP 7 | 60 | 15 | 10 | 194.6 ± 2.18 | 0.463 ± 0.065 | −9.2 |
LP 8 | 60 | 10 | 7.5 | 180.6 ± 2.03 | 0.494 ± 0.072 | −10.3 |
LP 9 | 45 | 10 | 5 | 147.9 ± 1.47 | 0.395 ± 0.061 | −8.19 |
LP 10 | 45 | 15 | 7.5 | 152.8 ± 1.54 | 0.375 ± 0.058 | −7.669 |
LP 11 | 30 | 10 | 7.5 | 114.7 ± 1.16 | 0.385 ± 0.055 | −8.88 |
LP 12 | 45 | 10 | 10 | 151.1 ± 1.54 | 0.378 ± 0.053 | −6.8 |
LP 13 | 45 | 15 | 7.5 | 152.8 ± 1.54 | 0.375 ± 0.062 | −7.36 |
LP 14 | 45 | 20 | 10 | 183.6 ± 1.97 | 0.325 ± 0.048 | −9.16 |
LP 15 | 60 | 20 | 7.5 | 198.4 ± 2.38 | 0.338 ± 0.056 | −15.62 |
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Arya, D.K.; Deshpande, H.; Kumar, A.; Chidambaram, K.; Pandey, P.; Anjum, S.; Deepak, P.; Kumar, V.; Kumar, S.; Pandey, G.; et al. HER-2 Receptor and αvβ3 Integrin Dual-Ligand Surface-Functionalized Liposome for Metastatic Breast Cancer Therapy. Pharmaceutics 2024, 16, 1128. https://doi.org/10.3390/pharmaceutics16091128
Arya DK, Deshpande H, Kumar A, Chidambaram K, Pandey P, Anjum S, Deepak P, Kumar V, Kumar S, Pandey G, et al. HER-2 Receptor and αvβ3 Integrin Dual-Ligand Surface-Functionalized Liposome for Metastatic Breast Cancer Therapy. Pharmaceutics. 2024; 16(9):1128. https://doi.org/10.3390/pharmaceutics16091128
Chicago/Turabian StyleArya, Dilip Kumar, Hemali Deshpande, Ashish Kumar, Kumarappan Chidambaram, Prashant Pandey, Shabnam Anjum, Payal Deepak, Vikas Kumar, Santosh Kumar, Giriraj Pandey, and et al. 2024. "HER-2 Receptor and αvβ3 Integrin Dual-Ligand Surface-Functionalized Liposome for Metastatic Breast Cancer Therapy" Pharmaceutics 16, no. 9: 1128. https://doi.org/10.3390/pharmaceutics16091128