INTRODUCTION:

Nanotechnology involves the development and use of materials and devices to manipulate matter at the level of molecules and atoms. One of the most promising societal impacts of nanotechnology is in the area of nanomedicine.

Nanomedicine is a promising field of research related to nanotechnology. It uses nanoparticles – extremely small, bead-shaped carriers of medicinal agents – to locate, diagnose and treat disease. Injected into the bloodstream, these tiny spheres travel deep into the body to identify and highlight tumors undetectable by methods typically used today. The nanoparticles also can deliver therapeutic agents to destroy the tumor^5.

Drug delivery systems, lipid or polymer-based nanoparticles¹, can be designed to improve the pharmacological and therapeutic properties of drugs¹.

The strength of drug delivery systems is their ability to alter the pharmacokinetics and biodistribution of the drug. When designed to avoid the body's defense mechanisms², nanoparticles have beneficial properties that can be used to improve drug delivery. Where larger particles would have been cleared from the body, cells take up these nanoparticles because of their size. Complex drug delivery mechanisms are being developed, including the ability to get drugs through cell membranes and into cell cytoplasm.

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 solubility⁷. Also, a drug may cause tissue damage, but with drug delivery, regulated drug release can eliminate the problem. 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. Poor bio-distribution is a problem that can affect normal tissues through widespread distribution, but the particulates from drug delivery systems lower the volume of distribution and reduce the effect on non-target tissue. Potential nanodrugs will work by very specific and well-understood mechanisms; one of the major impacts of nanotechnology and nanoscience will be in leading development of completely new drugs with more useful behavior and fewer side effects⁶.

The basic point to use drug delivery is based upon three facts: a) efficient encapsulation of the drugs, b) successful delivery of said drugs to the targeted region of the body, and c) successful release of that drug there.

Chemotherapy Drug have lower tendency to reach the target site of the cell, as it get denatured or taken up by non-target cell on its path to target cell. To increase stability and specificity of drug, Nanoparticles are used as carrier. These Nanoparticles are found to be effective in delivering the drug even in low dosage form by protecting activity of drug from denaturants. Its specificity is increased by attaching suitable ligand which will direct its path to the target cell. Many research work based on drug delivery using Nanoparticles has been increased mainly for overcoming the side effects of drugs due to high dosage &aggressive action leading to various side effects such as Addiction, Birth defects, Bleeding of the intestine, Cardiovascular disease, Deafness, kidney failure, Depression or hepatic injury, Diabetes, Diarrhea, Erectile dysfunction, Fever, Glaucoma, Hair loss, anemia, Headache, Hypertension, Insomnia, Lactic acidosis, Mania, Liver damage, thrombosis, Seizures, Drowsiness or increase in appetite, Stroke or heart attack.

Most drugs have a large list of non-severe or mild adverse effects which do not rule out continued usage. These effects, which have a widely variable incidence according to individual sensitivity, include nausea, dizziness, diarrhea, malaise, vomiting, headache, dermatitis, dry mouth, etc. These can be considered a form of pseudo-allergic reaction, as not all users experience these effects; many users experience none at all.

The various application of nanotechnology in nanomedicine involves, Drug delivery¹, Gene delivery, Molecular manufacturing, Biosensing device and it can be broadly classified into following categories, Nanobiopharmaceuticals, Oncology Imaging⁹, Photodynamic Therapy, Surgical flesh welder⁴, Visualization of drugs, Medical Imaging, Diagnostic Sensors¹, Neuroelectronic interfacing⁸, Molecular nanotechnology, nanorobots³, Nanonephrology.

The main objective is to produce model stable delivery system for Drug, and to study its stability in bacterial system such as E.coli and study its activity inside the cytoplasm.

MATERIALS AND METHODS:

Materials and instrumentation:

Tetrachloroauric acid (HAuCl4) (Sigma Aldrich, Product no: HT1004), Trisodium citrate (Na3C6O7), Bovine Serum Albumin, Luria Bertani broth (Himedia, M1245), E.coliDH5_(Medox) and other chemicals used were of high purity and grade from Himedia.Biospectrometer – Basic (Eppendorf), Cooling Centrifuge- C24BL (Remi), PAGE set up (Medox), Magnetic stirrer – 2MLH (Remi)

Methods:

Synthesis of gold nanoparticle:

The preparation of 15±2nm diameter GNPs was done by reduction method. An aqueous solution of HAuCl4 (1mM, 250mL) was brought to a vigorous boiling followed by stirring and then tri sodium citrate (38.8mM, 25mL) was added rapidly. The solution was boiled till the color of the solution changes from pale yellow to deep red. The solution was cooled to room temperature with continued stirring. BSA was used as substrate to check the activity of trypsin. The tryptic digest was assayed by Bradford method and the trypsin activity was found using standard curve.

Coating of trypsin to gold nanoparticle:

The gold nanoparticle solution prepared above was separated into two tubes and one tube was diluted by a factor of 1 with the glycine buffer (75mM, pH 7) and the other tube was diluted by a factor of 1 with the glycine buffer (75mM, pH 10.1). Trypsin (1mg/ml, 100μL) was added with stirring to a portion of the dilute solution containing GNPs. The solution was incubated, before being centrifuged, to remove the uncoordinated trypsin remaining in solution. The precipitate obtained subjected to 2 repeated wash cycles involving rinsing with 50mM glycine buffer (50) and centrifuged. Finally the GNP-trypsin was suspended in the glycine buffer for further experiments and then freeze dried.

% Bound Trypsin =

{[OD595 of 100μl of trypsin in 1mg/ml of glycine buffer – OD595 of supernatant]} *100

OD595 of 100μl of trypsin in 1ml of glycine buffer

Characterization of nanoparticles:

Gold nanoparticle synthesized by above method was characterized using UV absorption spectroscopy at defined wavelength to determine the size of the gold nanoparticles. And the maximum was obtained at 522nm which confirms 15±2nm size of synthesized gold nanoparticles. It was further confirmed by TEM images obtained from Madras Veterinary College, Veppery.

In Vitro test:

In vitro analysis was carried out in E. coli cells. Nanoparticles are easily taken up by bacterial cells. The analysis for studying the variation in uptake of Bio functionalized nanoparticles two different conditions and a control were used. The two different conditions were competent cells and normal cells. Competent cells were prepared by calcium chloride method and stored for further studies.

Intake of AuNP-Drug complex by Transformation for competent cells:

Transformation process was carried out for uptake of Bio functionalized nanoparticles in competent cells and the control was also kept in this process. Then the cells were incubated for 24hrs and then screening process was carried out.

Screening by protein extraction:

Cells incubated for 24hrs were centrifuged and the supernatant was analyzed by Biospectrometer to determine the uptake of Bio functionalized nanoparticles. Further screening process was carried out by protein extraction using alkaline lysis method and the amount of protein was determined by Bradford assay and measuring absorbance at 280nm.

Alkaline phosphatase assay:

The protein extraction carried out by alkaline lysis method was further subjected to ammonium sulphate precipitation to precipitate alkaline phosphatase. The precipitate was centrifuged at high rpm and the pellet obtained was dissolved in buffer A. ONPP was used as substrate to test the presence of ALPase enzyme and to determine the effect of Biofunctionalized nanoparticles i.e. trypsin with AuNP on bacterial cytoplasmic proteins by alkaline phosphatase assay at 405nm. SDS-PAGE & Native PAGE was carried out for visualizing the alkaline phosphatase band and to confirm AuNP bound trypsin action inside the bacterial cells.

RESULTS AND DISCUSSION:

AuNP’s of 15±2 were synthesized by reduction method which was confirmed by the changing of the colour from pale yellow to deep red. The trypsin was found active on the substrate – BSA, and the BSA standard curve was plotted which was measured using the BioSpectrometer.

Fig 1: Standard plot for BSA using Bradford assay.

GNP’s coated with the active trypsin were characterized using UV absorption spectroscopy which showed a maximum absorbance at 522nm confirming 15±2nm size of synthesized gold nanoparticles. It was further confirmed by TEM images obtained from Veppari institute.

Fig 2: a) TEM image of AuNP of 15±2nm b) TEM image of trypsin bound AuNP

The activity of trypsin coated in GNP’s was confirmed by testing with BSA and the value was found out using the BSA standard curve. And the amount of trypsin bound to AuNP was determined by Trypsin standard curve which was plotted by Bradford assay.

Fig 3: Trypsin Standard graph by Bradford assay

Then the GNP’s coated with trypsin were inserted into the normal E. coli cells and the competent E. coli cells. After incubation, the cell free supernatant obtained from the cultures showed no corresponding peaks for the GNP’s thus proving the absorbance of the Bio functionalized nanoparticles into the cells. The competent E. coli cells showed less absorbance at 522 nm showing more intake of Bio functionalized AuNP’s in comparison to the normal E. coli cells.

Further screening with protein estimation by Bradford assay showed very lesser proteins in competent E.coli cells and comparatively lesser proteins in normal E.coli cells and higher protein content in control cells. Similarly, the alkaline phosphatase assay showed decreased value in competent E. coli cells whereas it appeared a little high with normal E. coli. The SDS and Native PAGE showed cleaved thinner bands for alkaline phosphatase instead of those obtained in the normal control proving the trypsin-AuNP’s had entered and acted upon the cytoplasmic proteins.

CONCLUSIONS:

As the above results say, the AuNPs coated with trypsin showed good activity in E.coli cells which we took as microbial models. Trypsin is unable to cleave the proteins inside the cells due to protease inhibitors inside the cells which reduce activity or completely denature trypsin. From this study, it is proved that trypsin can be easily carried inside the bacterial cell and its activity is maintained by combining with nanoparticles.

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Received on 25.08.2013 Accepted on 01.09.2013

Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 260-263