A Novel System for Drug Delivery Using Nanoparticle

 

J. Madhusudhanan1*, M. Indumathi2, S. Ammu2

1Associate Professor, Department of Biotechnology, Shri Andal Alagar College of Engineering (SAACE), Mamandur-603 111.

2FinalYear, B.Tech, Department of Biotechnology, Shri Andal Alagar College of Engineering (SAACE), Mamandur-603 111.

*Corresponding Author E-mail:- jmadhuj2008@gmail.com

 

 

ABSTRACT:

Several biological drugs such as proteins and nucleic acids require novel delivery technologies that will minimize side effects and lead to better patient compliance. Hence, various research using nanoparticle as a carrier has been done for the delivery of gene drug. In the present work, delivery of proteases and plasmid DNA using nanoparticle as a carrier on E.colis was studied. Proteases, a proteolytic enzyme which is capable of cleaving the peptides are made inactive by inhibitors present inside the cell. But proteases can be made resistive by coating with nanoparticles. The action of protease has been analysed on a specific enzyme i.e. alkaline phosphatase. Along with the protease delivery, plasmid delivery is also done to develop a carrier capable of delivering drug and gene complex. Similarly, carrier system for drug and plasmid was studied and developed for cancer cells which were capable of delivering drugs as well as short polynucleotide complex. It was found that the cancer cells lines SW480 and A549 showed cell death due to the action of drugs. This novel carrier showed effective delivery of drug and high efficacy in action. From this study it was confirmed that a protein or drug can be conjugated with nanoparticles along with gene carrier to import both functions of drugs and gene on a targeted cell using single carrier. Such system can be developed specific genetic disorders such as Osteogenesis imperfecta, cancer.

 

KEYWORDS: Plasmid, Protease, Cancer cell, Osteogenesis imperfecta.

 


INTRODUCTION:

Nanotechnology involves the development and use of materials and devices to manipulate matter at the level of molecules and atoms. Nanotechnology is defined as the study and use of structures between 1 nanometre and 100 nanometers in size. One of the most promising societal impacts of nanotechnology is in the area of nanomedicine.

 

Each nanoparticle can carry hundreds of thousands of tiny molecules on its surface. Medical researchers now have the ability to attach molecules that home in on cells within the body having complementary molecules on their surfaces. A nanoparticle’s ability to carry therapeutic agents – actual medicine – means drugs specifically designed to kill cancer cells can be included.

 

Efficiency is important because many diseases depend upon processes within the cell and can only be impeded by drugs that make their way into the cell. Triggered response is one way for drug molecules to be used more efficiently. Drugs are placed in the body and only activate on encountering a particular signal. For example, a drug with poor solubility will be replaced by a drug delivery system where both hydrophilic and hydrophobic environments exist, improving the solubility. Also, a drug may cause tissue damage, but with drug delivery, regulated drug release can eliminate the problem.

 

Floating drug delivery systems were prepared with the objective of increasing overall gastric retention time in order to prolong the release. The floating tablets were prepared by using Hydroxy Propyl Methyl Cellulose (HPMC) polymers at different drug to polymer ratio with gas generating agents like sodium bicarbonate and citric acid10.

 

If a drug is cleared too quickly from the body, this could force a patient to use high doses, but with drug delivery systems clearance can be reduced by altering the pharmacokinetics of the drug.

 

MATERIALS AND METHODS:

Materials:

Nupore filter paper, Auric chloride, Trisodium citrate, Sodium  chloride, Modified poly-A-nucleotide, Distilled water, Ethanol,  Ethylenediaminetetraacetic acid, Agarose, Doxorubicin, Ethidium bromide, Hydrochloric acid, Bovine Serum Albumin, Trypsin, Glycine, pUC19-plasmid DNA, Boric acid, Sodium hydroxide, Mueller Hinton  agar, Luria Bertani medium,  HB101 and DH5-alpha strain of   E. Coli, CaCl2, Ampicillin, Petri plates, 5-bromo-4-chloro-indolyl-β-D-galactopyranoside (X-gal), MEDOX (MX-1181-02) Plasmid extraction kit, sucrose, filter paper, p-nitro phenyl phosphate, cancer cell lines, DMEM, Penicillin, streptomycin, FBS, lysis buffer, EDTA, SDS, Protein kinase, isoamyl alcohol, chloroform, phenol, Sodium acetate, Isopropanol, Orange-G dye, Trypan blue, DMSO.

 

Instruments:

Laminar Air Flow, CO2 Incubator, Light Microscope, Electronic Balance, Autoclave, Cooling Centrifuge, Nitrogen Cylinder, Water Bath, Hot Plate, Magnetic Stirrer, Micropipette, Biospectrometer, UV Transilluminator, UV-Vis Spectrophotometer, Deep Freezer, Electrophoresis Tank, Hot Air Oven, Incubator, FTIR Spectrometer, TEM and SEM.

 

Gold nanoparticle synthesis:

HAuCl4 was added to a beaker with a magnetic stir bar on a stirring hot plate and brought the solution to a rolling boil, To the rapidly-stirred boiling solution, quickly add trisodium citrate dehydrate solution. A small amount of the gold nanoparticle solution was transferred into two test tubes. One tube was used for colour reference and drops of NaCl solution was added to the other tube, the colour of the solution changed as the addition of chloride makes the nanoparticles closer together. Gold nanoparticle was characterized under UV-VIS spectrum at different wavelength. Maximum peak was obtained. It was further characterized by TEM.

 

Coating with Polynucleotide:

Modified poly-A-nucleotide was dissolved in double distilled water. The reaction mixture was then added with NaCl solution and then incubated for 24hrs. Salt concentration was gradually increased and each incubated for 8hrs. 0.8% agarose gel electrophoresis was performed to confirm the binding to the AuNP’s. 0.8% agarose was prepared in TE buffer by heating the agarose containing solution until clear solution was obtained. Gel casting tray was prepared by giving diluted ethanol wash to the tray and its comb. Once the solution heat was palm bearable, 20µl of Ethidium bromide was added. Then the solution was poured into gel tray and left undisturbed to solidify. Comb was removed without disturbing the gel, and placed in agarose chamber followed by connecting the power card.Sample was loaded and electrophoresis was carried out at 50V with tracking dye. After 3/4th run, gel was viewed under UV transilluminator.

 

Coating with Doxorubicin:

Standard graph was plotted for Doxorubicin and the solutions were measured using Biospectrometer which showed maximum absorbance. The curve was plotted for the measured value.

 

DRUG LOADING EFFICIENCY:

Drug loading efficiency was determined using the formula,

 

                   OD510 of DOX-OD510 value of DOX in supernatant

% Loading efficiency=  --------------------------------------------------x 100

                                                        OD510 of DOX

 

 

DRUG RELEASE BEHAVIOUR

The drug release, was determined with the formula,

 

                                  OD510 value of DOX in supernatant

% Drug Release=  --------------------------------------------------x 100

                                     OD510 of DOX in drug loaded complex

 

Agarose gel electrophoresis was performed and the plasmid presence was confirmed. Competent cell was prepared with the usual procedure followed by transformation.

 

Invitro studies in Cancer Cell Lines:

For sub culturing adherent mammalian cells, DMEM and L-15 medium was used for A549 and SW480 cell lines respectively.

 

After several passaging the cells were analysed for their morphology on initial and for confluent cell lines, and DNA was extracted from the cancer cells. Agarose gel electrophoresis was performed.

 

Cell viability is calculated as the number of viable cells divided by the total number of cells within the grids on the Hemocytometer.  If cells take up Tryphan blue, they are considered as non-viable.

 

% Viable cells

= [1.00 – (Number of blue cells / Number of total cells)]  ×100

 

RESULTS AND DISCUSSIONS:

Gold nanoparticle synthesis:

Gold nanoparticles were synthesized by reduction method using tri sodium citrate as reducing agent and the size of AuNP’s were confirmed by plasma resonance effect which gave maximum peak. This showed that the size to be around 20±2nm. The gold nanoparticle was further characterized by TEM images to determine the grain size and particle size distribution.

 

Figure 1: UV-Vis absorption, AuNP - max. peak

 

The optical spectra of colloidal nanoparticle were acquired on UV-vis-spectrophotometer, (Figure1). TEM image for gold nanoparticle was obtained, which confirmed the size of gold nanoparticle as 20±2nm, (Figure 2).

 

Figure 2: TEM image of Gold nanoparticles confirming the size

TEM analysis

Figure 3: TEM image for DOX coated gold nanoparticles

 

The TEM image of DOX coated gold nanoparticles.

FTIR analysis

Figure 4: FTIR graph for trypsin

 

From the above FTIR graph, an interaction is found between gold nanoparticles and trypsin which can be confirmed by sudden shift in CH2 and NH2, C=O and C-S bonds.

 

DRUG LOADING EFFICIENCY:

 

                                          UV-Vis Value of drug in supernatant

Loading efficiency=  1-   ------------------------------------------------x 100

                                             UV-Vis Value of DOX

 

Table 1: Drug loading efficiency for Doxorubicin

TIME

DRUG LOADING EFFICIENCY

1hr

26.8%

24hr

86.12%

 

Figure 5: Drug loading efficiency for AuNP-DNA-DOX

 

IN VITRO STUDIES IN E. coli:

Transformation of E.coli DH5α:

The E. coli DH5α cells were transformed into Ampicillin resistant which was confirmed by antibiotic plating.


  

Ampicillin resistant DH5α colonies after transformation

 

Figure 6: Transformed DH5α:  Ampicillin plating


 

 


In vitro studies in Cancer Cell Lines:

Morphology analysis:

The morphology of A549 and SW480 was analysed for day 1 cell lines and also confluent cell lines.

 

In morphological assay, the mature cell will adhere to the bottom of flask. The cells took several for maturation, and after the maturation of young cells, it adhered to the bottom of the T-flask. The young cells allowed the light to pass through it, but the dead cells didn’t allow the light to pass.


 

 


Figure 7

 

Genomic DNA extraction from Cancer cell.

The cancer cell DNA was isolated and determined by agarose gel electrophoresis. The bands for the genomic DNA of A549 and SW480 cancer cell lines can been seen in the lanes.

 

Lane 1 to 4 = A549 Genomic DNA; Lane 5 to 8 = SW480 Genomic DNA; Lane 9 = Ladder

Figure 8: Genomic Cancer cell DNA

 

It is well presented in the figure 8 that the cancer cell DNA was extracted successfully and the band confirms the presence of Cancer cell’s Genomic DNA.

 

CONCLUSION:

The nanocarriers developed for delivering Doxorubicin was also found to be effective in Cancer cell’s DNA. This showed more effective even in low dosage of almost 1/10th of the drug administered to patients; this indicates that the nanocarriers are of great advantage in delivering drugs.

 

Thus the nanocarriers developed for drug is one of the most useful tool in treatment which will subside the present mode of treatment and will prove to be useful in eliminating genetic disorder associated with defective protein effects.

 

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Received on 20.09.2013          Accepted on 01.10.2013        

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Asian J. Pharm. Tech.  2013; Vol. 3: Issue 4, Pg  137-141