targeted delivery and controlled release of doxorubicin to
TRANSCRIPT
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TARGETED DELIVERY AND CONTROLLED RELEASE OF DOXORUBICIN TO CANCER CELLS
USINGMODIFIED SINGLE WALL CARBON NANOTUBES
By,Laksgmi Indira Vadlamani
2020H146042
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INTRODUCTION
Doxorubicin is a cytotoxic anthracycline antibiotic. Doxorubicin binds to nucleic acids, by specific intercalation of the planar anthracycline nucleus with the DNA double helix.
The anthracycline ring is lipophilic, but the saturated end of the ring system contains abundant hydroxyl groups adjacent to the amino sugar, producing a hydrophilic center.
The molecule is amphoteric, containing acidic functions in the ring phenolic groups and a basic function in the sugar amino group. It binds to cell membranes as well as plasma proteins.
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PROPERTIES OF INTEREST IN CNT’S Ability to carry a high cargo loading,
their intrinsic stability and structural flexibility, which could prolong the circulation time and hence the bioavailability of the carried drug molecules
Ability to enter mammalian cells. Potential delivery vehicles for
intracellular transport of nucleic acids , proteins and drug molecules.
SWCNTs have been functionalized with antibodies and low molecular weight targeting agents providing a high efficiency for nanotube internalization into cells
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RATIONALE A targeted drug delivery system that is triggered by changes in pH based on
single wall carbon nanotubes (SWCNTs). Derivatized with carboxylate groups and coated with a polysaccharide
material, can be loaded with the anticancer drug doxorubicin (DOX). The drug binds at physiological pH (pH 7.4) and is only released at a lower
pH, for example, lysosomal pH and the pH characteristic of certain tumor environments.
By manipulating the surface potentials of the modified nanotubes through modification of the polysaccharide coating, both the loading efficiency and release rate of the associated DOX can be controlled.
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MATERIALS
The SWCNTs (purity, >90%, length, >50 mm, diameter, 1–2 nm,
Sodium Alginate (ALG) Chitosan (CHI ) Folic acid (FA ) Doxorubicin hydrochloride (DOX ) N,N-(3-dimethylaminopropyl)-N’-ethyl carbodiimide
hydrochloride EDC-HCl Porous poly (vinylidene chloride) (PVDC, 0.22 mm pore size) Fetal bovine serum (FBS), High glucose Dulbecco’s Modified Eagle’s medium (DMEM) WST-1 reagent
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METHODS
Preparation of the SWCNTs
Cutting and purification of the SWCNTs was carried out as follows. The
SWCNTs (500 mg) were added to a mixture of 98% H2SO4 and 65%
HNO3 (V:V¼ 3:1, 200 mL) and exposed to sonic irradiation at 0 C for 24
h.
The cut SWCNTs were thoroughly washed with ultrapure water (18.2 MU)
and filtered through a micro-porous filtration membrane (F 0.22 mm).
They were redispersed in HNO3 (2.6 M, 200 mL) and refluxed for 24 h,
collected by filtration and washed with ultrapure water to neutrality. The
product was then dried under vacuum at 50 C for 24 h.
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Preparation of ALG-SWCNTs
Cut SWCNTs (20 mg) were sonicated in sodium ALG solution (40 mg in 0.1
M aqueous NaCl, 40 mL) for 20 min and then stirred at room temperature for
16 h. The modified SWCNTs were collected and washed with ultrapure water
by ultracentrifugation to remove unbound ALG, then collected and dried at
room temperature to obtain ALG-SWCNTs.
Preparation of CHI-SWCNTs CHI-SWCNTs were prepared in the same way as that described above for the
ALG-SWCNTs by replacing the ALG solution for CHI solution.
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Preparation of CHI/ALG-SWCNTs
The ALG-SWCNTs (10 mg) were sonicated for 20 min and then a CHI
solution (20 mg in 0.1 M aqueous NaCl) was added. The mixture was
stirred for 16 h at room temperature to give the product following
ultracentrifugation, washing and drying as described above.
Preparation of FA-CHI/ALG-SWCNTs
The CHI/ALG-SWCNTs (4 mg) were suspended with FA (6 mg) in a pH
7.4 PBS buffer solution (8 mL) and then EDC-HCl (5 mg) was added.
After stirring the reaction mixture at room temperature for 16 h, the
product was washed with ultrapure water several times by repeated
ultracentrifugation to remove unreacted reagents and then dried at room
temperature.
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DOX LOADING ONTO THE NANOTUBES
DOX hydrochloride (9 mg) was stirred with the modified nanotubes (3 mg)
dispersed in a pH 7.4 PBS buffered solution (6 mL) and stirred for 16 h at
room temperature.
The products (denoted as DOX-SWCNTs, DOX-ALG-SWCNTs, DOX-
CHISWCNTs, DOX-CHI/ALG-SWCNTs and DOX-FA-CHI/ALG-
SWCNTs) were collected by repeated ultracentrifugation with PBS until
the supernatant became colour free.
The amount of unbound DOX was determined by measuring the
absorbance at 490 nm (the characteristic absorbance of DOX) relative to a
calibration curve recorded under the same conditions, allowing the drug
loading efficiency to be estimated.
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PREPARATION
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TEM IMAGES OF MODIFIED SWCNTS
(a) Cut SWCNTs, (b) ALG-SWCNTs, (c) CHI-SWCNTs, (d) CHI/ALG-SWCNTs, (e) DOX-SWCNTs, (f) DOX-ALG-SWCNTs, (g) DOX-CHI-SWCNTs and (h) DOX-CHI/ALG-SWCNTs
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DOX RELEASE FROM THE NANOTUBES
Suspensions of the DOX loaded
SWCNTs (1 mg) were allowed to
stand at 37 C in pH 7.4 and pH 5.5
PBS buffered solutions (5 mL).
After different time intervals, the
nanotubes were separated from the
buffer by ultracentrifugation, and the
concentration of released DOX in the
supernatant was estimated by UV-Vis
spectroscopy.
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INCUBATION OF HELA CELLS WITH DOX LOADED NANOTUBES
HeLa cells were cultured in DMEM supplemented with 10% FBS in a humidified
Incubator at 37 C in which the CO2 level was maintained at 5%.
For cells incubated with the DOX loaded nanotubes or free DOX, the cells were
cultured overnight to allow attachment, washed with FBS-free DMEM and then
incubated with 20 mg/mL DOX-FA-CHI/ALG-SWCNTs, 20 mg/mL
DOX-CHI/ALGSWCNTs or 50 mg/mL DOX at 37 C for 1 h in FBS-free medium.
After incubation, the cells were washed repeatedly with sterilized PBS before
further analysis.
To further evaluate the role of FA in the cellular uptake of
DOX-FA-CHI/ALGSWCNTs, the cells were pretreated with free FA (0.5 mg/mL)
for 2 h, DOX-FACHI/ALG-SWCNTs (20 mg/mL) was then added and the cells
were cultured for another 1 h, washed with sterilized PBS and analyzed by
fluorescence microscopy.
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Fluorescence images of cells incubated with (a) DOX-FA-CHI/ALG-SWCNTs (20 mg/mL), (b) DOX-CHI/ALG-SWCNTs (20 mg/mL), (c) FA for 2 h followed by DOX-FA-CHI/ALG-SWCNTs (20 mg/mL), and (d) free DOX (50 mg/mL) at 370C for 1 h.
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CELL VIABILITY TEST
The WST-1 assay was used to measure cell viability. In brief, HeLa cells
were seeded into a 24-well flat culture plate. After culturing overnight the
cells were washed with FBS-free DMEM and incubated with a specific
concentration of DOX-FA-CHI/ALG-SWCNTs (10, 25, 50 mg/mL), FA-
CHI/ALG-SWCNTs (50 mg/mL) and free DOX (100 mg/mL) in FBS-free
culture medium at 37 C for 1 h.
After incubation for 2 h at 37 C, the absorbance was measured at 450 nm
using a microplate reader. The background absorbance was measured at
450 nm before adding the WST-1 reagent and the cells cultured in the
absence of a drug were used as controls.
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Fluorescence images of HeLa cells treated with DOX-FA-CHI/ALG-SWCNTs (20 mg/mL)for (b) 1 h and (c) the treated cells continued culturing in fresh media (10% FBS) after 72 h.
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CONCLUSION
The complete system displays excellent stability under physiological
conditions, but at reduced pH typical of the tumor environment and
intracellular lysosomes and endosomes, the DOX is efficiently released and
enters the cell nucleus and induces cell death.
Based on a number of pertinent control experiments it is possible to
conclude that the overall nanoscale drug system is more selective and
effective than the free drug and it should result in reduced general toxicity,
and hence reduced side-effects in patents, and also allow a lower amount
of the drug to be applied.
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REFERENCES Xiaoke Zhang a , Lingjie Meng a , Qinghua Lu a,b,*, Zhaofu Feic, Paul J.
Dyson c “Targeted delivery and controlled release of doxorubicin to cancer
cells using modified single wall carbon nanotubes” Biomaterials 30 (2009)
6041–6047.
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Queries????