الفهرس 
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Abstract
In this study, we presented the fabricationand the co-delivery of exemestane (EXE) and methotrexate (MTX) in breast cancer targeted self-assembled liquid crystalline nanoparticles(LCNPs).
Cationic Lfwas attached at the surface of anionic LCNPs as a targeting ligand via electrostatic interactions.
The combinational therapy of EXE and MTX demonstrated a successful synergistic combination.
First, blank LCNPs F1weresynthesized by ’’Emulsification technique’’ as reported previously with modification.
EXE-loaded LCNPs dispersion F2was also prepared by the same methodwhile the hydrophobic drug was incorporated intothe lipid phase.
MTX-Lf conjugate F3 was prepared using carbodiimide coupling, where a stable amide bond between the carboxylic group in MTX and the amino group in Lf is formed.
The amide bond prevents MTXrelease into systemic circulation and permit its release specifically in cancer cells upon cleavage of bond by lysosomal enzymes.
Finally, targeted dual loadedLCNPsF4was prepared by utilizing the electrostatic interaction between the anionic EXE-loaded LCNPs and the cationic MTX-Lf conjugate.
MTX-Lf conjugate F3was added drop-wisely ontoEXE-loaded LCNPs F2 under mild magnetic stirring atroom temperature to deposit a layer of the MTX-Lf conjugate on the surface of the LCNPs.Different physicochemical characterizations were doneto ensure the optimization of the formulations.
Particle size and polydispersity index were measured using dynamic light scattering technique of Malvern Zetasizer for blank LCNPsF1 (124±4.57nm, 0.145), EXE-loaded LCNPsF2(137.3±4.06nm, 0.184) and targeted dual loaded LCNPsF4(143.6±3.24nm, 0.180).
Zeta potential was also measured for blank LCNPsF1 (-28.5 mV), EXE-loaded LCNPsF2(-28.4 mV) and targeted dual loaded LCNPsF4(+5.64 mV)to ensure the physical stability of the formulations and to ensure the complete uniform deposition of a layer of MTX-Lf conjugate F3 on the surface of EXE-loaded LCNPs F2to obtain targeted dual drug loaded LCNPsF4.
Physical stability of the targeted dual loaded LCNPsF4was tested as well and demonstrated particle size of170±4.16nmand zeta potential of +3.5 mVcompared to the initial NPs of particlesize 143.6±3.24 nm and zeta potential of +5.64 mVafter 3 months in 4±1ºC.Moreover, morphological analyses of blank LCNPsF1 and the dual loaded targeted LCNPs F4werepresented where the formulations showed uniform particles with smooth surface and without aggregation.
Fourier Transform Infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC)were performed on blank LCNPs F1, EXE-loaded LCNPs F2, MTX, Lf, MTX-Lf F3, targeted dual drug-loaded LCNPs F4for the characterization of possible interactions
92between EXE and LCNPs, MTX and Lf, as well as, F2 andF3.In addition, Nuclear Magnetic Resonance (1H-NMR)was done to confirm the formation of MTX-Lf conjugate F3.
HPLC was used to develop and validate methods used to quantify EXE and MTX.
EXE-loaded LCNPs F2showed encapsulation efficiency of 95% and MTX-Lf conjugate F3 showed conjugation efficiency of 33.33%.
In-vitro release of EXEfrom the different prepared formulationsat physiological pH(pH = 7.4) was carried out.
In-vitrorelease profiles of free EXE and free MTX were recorded and compared to their release profiles from the loaded formulationsF2 and F4.
Release profiles showed sustained release of the drugs from the NPs in comparison to the free drugs.
Free EXE was released by 75% after only 2 hours while about 35% of EXE was released from F2 and F4 at the same time.
On the other hand, free MTXwas released by 85% after 6 hours while MTX was not releasedfromF4.
This proves the stability of the amide bond in the physiological pH.
Additionally, in-vitro serum stability was tested in 10% fetal bovine serum over 6 hour period.The size of targeted dual drug loaded LCNPs F4reachedfrom143.6±3.24 nmto163.62±3.01nm after 6 hours.
In-vitrocytotoxicity assay was performedon MCF-7 cell line.
It showed synergistic action between EXE and MTX.
It also provided a proof that targeted dual drug loaded LCNPs F4 improved the free drugs combination potency to give IC50value with 0.38 fold reduction in comparison to theIC50of the free combined drug solution.
Moreover, in-vitro cellular uptake study and flow cytometry study were done using MCF-7 cell line, as well.
These tests provided results that showed the superiority of the targeted formulation against the untargeted formulation and the free dye(Coumarin-6).
Finally, it can be concluded that the targeted dual drug loaded LCNPs F4 showed optimum size, narrow PDI, appropriate zeta potential, excellent encapsulation efficiency of EXE and high conjugation efficiency of MTX with a sustained release profile.
The finalformulationF4 was the excellent candidate for further investigation for breast cancer therapy as an injectable formulation