TARGETED DELIVERY AND CONTROLLED RELEASE OF DOXORUBICIN TO CANCER CELLS

USINGMODIFIED SINGLE WALL CARBON NANOTUBES

By,Laksgmi Indira Vadlamani

2020H146042

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.

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

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.

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

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.

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.

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.

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.

PREPARATION

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

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.

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.

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.

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.

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.

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.

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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