INTRODUCTION:

Nanomedicine, a branch of Nanotechnology involves the use of drugs and carriers to manipulate at the level of molecules and atoms. It’s a promising field of research – to locate, diagnose and treat disease¹. It involves the use of particles at nano scale to carry the medicinal agents to identify and highlight tumors which are undetectable by current diagnosis techniques. It is now majorly researched in delivering therapeutic agents to the target site².

Delivery systems based on lipid or bio-polymer nanoparticles³ is manipulated to alter the important properties of drugs such as pharmacological and therapeutic effects⁴. The pharmacokinetics and biodistribution of the drug is majorly regulated by the delivery systems. Its most advantageous factor is to avoid the body’s defence mechanisms⁵, as the cells take up these nanoparticles. This has led to the development of complex systems which has the ability to get drugs into cytoplasm via cell membranes.

Many diseases are associated with the malfunctioning processes within the cell, and only those drugs can act which make their way into the cell for better efficiency. In order to increase the efficiency, various techniques are adopted to deliver drugs such as Triggered response, in which the drugs are in inactive form within the body and gets activated on sensing a particular signal. This technique is found to be more efficient as it acts only on the targeted site. Similarly in certain cases where drug has poor solubility, carriers containing both hydrophilic and hydrophobic environments are provided⁶. Also, tissue damage caused by the drug can be eliminated by regulating the drug release properties. Delivery systems can also be used to alter the pharmacokinetic of the drug to prevent patient from consuming high doses of drug due to drug clearance from body at quick phase.

Such drug delivery systems will reduce the effect on non-target tissue, thus solving the problem of poor distribution. Nanodrugs will have high potential to have its action in very specific mechanisms which will lead to new generation of drugs with more efficient in its action without side effects due to usage of low doses of drugs.

Drug delivery should have the following advantages: a) Efficient capping of the drugs, b) Delivery of drug to targeted site, and c) Release of drug without losing its property.

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 cell²⁰.

Many research work based on drug delivery using Nanoparticles has been increased mainly for overcoming the side effects of drugs due to HIGH dosage and Aggressive action leading to various side effects such as Cardiovascular disease, kidney failure, intestinal disorder, and genetic disorders. Each drug has list of adverse effects on continued usage.

Simple UV and first derivative spectrophotometric methods have been developed for the determination of dosage form. In simple UV method, spectrum of a drug showed absorbance maximum, where as in first derivative spectrum it shows maxima and minima. Beer’s law is obeyed over for various concentration²⁶.

The various application of nanotechnology in nanomedicine involves, Drug delivery^1,4, Gene delivery, Molecular manufacturing¹⁴, Biosensing device and it can be broadly classified into following categories, Nanobiopharma- ceuticals, Oncology Imaging⁷, Photodynamic Therapy⁸, Surgical flesh welder⁹, Visualization of drugs, Medical Imaging, Diagnostic Sensors, Neuro-electronic interfacing¹⁰, Molecular nanotechnology¹¹, nanorobots¹², Nanonephrology.

The main objective is to study stable model for delivery of protease’s and study its stability in bacterial system such as E.coli and its activity inside the cytoplasm¹⁹.

EXPERIMENTAL:

Materials:

Tetrachloroauric acid, Trisodium citrate, Bovine Serum Albumin, Luria Bertani broth, E.coli HB101and other chemicals used were of high purity and grade from Himedia.

Instrumentation:

Biospectrometer, Cooling Centrifuge, Magnetic stirrer, TEM, Spectrometer

Methods:

Synthesis of gold nanoparticle

The AuNPs was synthesized by citrate reduction method. An aqueous solution of HAuCl₄was brought to a vigorous boiling with continuous stirring in a round bottom flask, fitted with a reflux condenser and then trisodium citrate was added. The solution was boiled till the color of the solution changes. The solution was cooled to room temperature.

Coating of Trypsin to gold nanoparticle

A standard graph was plotted for Trypsin to determine the saturation point for Trypsin on gold nanoparticles¹⁶. The saturation point of Trypsin on AuNP was determined and the solution was incubated, before being centrifuged, to remove the unbound Trypsin remaining in solution. The precipitate obtained was subjected to wash cycles involving rinsing with glycine buffer and centrifuged. Finally the GNP-Trypsin was suspended and then freeze dried.

Characterisation of nanoparticles:

Gold nanoparticle synthesized was confirmed using UV absorption spectroscopy. To determine the size, it was further characterized by using TEM and FTIR analysis.

Activity test for Trypsin coated on nanoparticles

The formation of Trypsin bounded AuNP’s was confirmed by determining the amount of unbound Trypsin in the supernatant using a standard graph for Trypsin by Bradford assay. The activity of the bound Trypsin was determined using BSA as a substrate⁸.

In Vitro test

In vitro analysis was carried out in E.coli cells. Nanoparticles are easily taken up by bacterial cells¹³ yet analyses were carried out with competent cells. In order to analyze the variation in uptake of nanoparticles, two different conditions along with a control was used.

Screening by protein extraction

Screening process was carried out by protein extraction using alkaline lysis method. Cultures obtained from the above were centrifuged to remove any trace of LB medium. The Pellet obtained was resuspended in shock buffer and incubated at room temperature for 10min. Protein present in the filtrate was determined by measuring the absorbance at 290nm.

Alkaline phosphatase assay:

The protein extraction carried out by alkaline lysis method^22,
23 was further subjected to Ammonium Sulphate precipitation to precipitate alkaline phosphatase. The precipitate was centrifuged at high speed and the pellet obtained was dissolved in buffer. The amount of ALPase extracted was determined by ONPP substrate assay. SDS-PAGE was performed for the alkaline phosphatase extracted by ammonium sulphate precipitation^21,24.

RESULTS AND DISCUSSIONS:

Synthesis of gold nanoparticle:

Gold nanoparticles were synthesized and the size of AuNP’s were confirmed by plasma resonance effect which gave maximum peak at 528nm and the size was found to be around 11-18nm¹⁵ using TEM analysis.

Coating of Trypsin to gold nanoparticle

A standard graph was plotted for Trypsin using Bradford assay in order to regulate Trypsin concentration left in the supernatant after adsorption on gold nanoparticle. The adsorption of protein on nanoparticle is carried out around the pI of the protein but the activity of

The activity of Trypsin bounded to AuNP’s was confirmed by proteolytic action on BSA and the value was found using the BSA standard curve. The amount of Trypsin bound to AuNP was determined by Trypsin standard curve by Bradford assay,

% Bound Trypsin = {[A–B] / C} *100

Where,

A = OD at 595nm of 100µl of Trypsin in 1mg/ml of glycine buffer

B = OD at 595nm of supernatant

C = OD at 595nm of 100µl of Trypsin in 1mg/ml of glycine buffer

Characterisation of nanoparticles:

1)UV Visible spectrum:

Fig. 1: Shows the UV-Vis absorption for AuNP with Trypsin

Figure 1, displays the UV-visible spectra for citrate capped gold nanoparticles.

2) Transmission Electrons microscopy (TEM):

Fig. 2-Transmission Electron Microscope image of AuNP’s(a) and Trypsin bounded AuNP’s(b)

TEM images of Gold Nanoparticle as given in Figure 5,

(Fig 2a), Gold Nanoparticle size was found using Computer aided TEM

(Fig 2b), Gold Nanoparticle was added with protease to interact and the TEM analysis confirmed the size of Trypsin bounded AuNP’s

3) FTIR analysis:

From the below 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. The alcohol (OH) group present in amino acids of Trypsin shows peaks.

Fig. 3: FTIR analysis of Trypsin and AuNP with Trypsin showing the shift in bond due to binding of Trypsin on Gold Nanoparticles.

In Vitro Studies:

Nanoparticle incorporation within bacterial cells:

Cells incubated with AuNP’s and AuNP-Trypsin for 24hrs were centrifuged to determine the uptake of nanoparticles using Biospectrometer, and the values were as shown in below Table-1.

Table 1: Percentage Uptake of Au nanoparticle bound to Trypsin by E.coli cells

Sample	O.D. @ 531nm after 5 min	O.D. @ 528nm	O.D. @ 531/528nm after 24h (Supernatant)	% Uptake of AuNP-Trypsin/ AuNP
A	0.320	-	0.145	42.08
B	0.321	-	0.201	32.04
C (Control)	-	0.746	0.342	49.05

A = E.coli cells with AuNP-Trypsin; B = Competent E.coli cells with AuNP-Trypsin; C = E.coli cells with AuNP alone.

The result shows that almost AuNP-Trypsin was taken up by cells, as plotted in figure 4.

Further protein extraction was carried out and the absorbance was measured at 280nm to determine the amount of protein.

Fig. 4: Percentage Uptake of Gold Nanoparticles bound to Trypsin by E.coli cells

Protein extraction:

Protein extraction was carried out by alkaline lysis methods, and the amount of protein extracted was measured using Biospectrometer. The concentration of protein determined is shown in Table-2,

Table 2: Estimation of protein by Absorbance method @ 260nm

Sample	O.D. @ 280nm	Protein concentration (mg/ml)	% of Protein
A	1.43	0.62	65.93
B	1.54	0.66	60.44
C (control)	2.12	1.02	100

Alkaline phosphatase assay:

Alkaline phosphatase, a periplasmic enzyme¹⁷, of Escherichia coli is encoded by the PhoA gene consisting of two identical subunits as a zinc-containing protein. Intramolecular disulfide bridges are present between the two subunits. The complete protein of PhoA’ is converted into protease resistant and enzymatically active conformation only after entering into the periplasmic space; its precursor is an N-terminal 20-residue signal sequence. But those that lack functional signal sequence and synthesized in the dsbA-deficient cell are prone to be reduced and degraded by the protease action¹⁸.

Fig. 5: Percentage of protein extracted from E.coli cells after incubation with Au nanoparticles bound to Trypsin.

CONCLUSIONS:

AuNP’s as suitable carrier has been proved and are being studied as carrier for various drug for different disorders. Even gene therapy has evolved for various genetic disorders; still we need to improve for better treatment²⁵. In Certain Cancer, Gene therapy can only impart changes to the defective gene but can’t overcome the metabolic problems imposed by the defective protein synthesized by the same defective gene. These defective proteins remain in the affected cells and hinder the normal metabolic and building process as seen in Osteogenesis imperfecta where defective pro-collagen disturbs and results in bone fragility and other symptoms. For such disorder’s, various research are carried out to deliver drugs which are toxic to normal healthy cells. Hence, it requires thorough understanding of carrier such as Au nanoparticles to deliver these toxic drugs to the target cells and result in better treatment with better patient compliance.

In this paper, we have studied the delivery of protein using Au nanoparticle as carrier for a serine protease which showed positive results in carrying the Trypsin in less active form and protects Trypsin from protease inhibitors present inside the cell. Proteolytic enzyme’s can be designed to cleave specifically the defective proteins, which can be carried by Nanoparticles. It’s an alternative to the above mentioned treatment, where the toxic drug is replaced by proteolytic enzyme’s which are specific in its action. Thus, such treatment will be found to be more reliable and not fatal. The present research is a model to develop and study the factors associated in using Nanoparticles as protease carriers.

It shows that controlling such parameter’s in formation of Nanoparticles bounded biological compounds can lead to stable carrier formation. Such carriers would enroot the treatment for Genetic disorders associated with defective Proteins. This type of treatment can be of patience compliance and won’t kill neither the Healthy cells nor the Defective cells but only the defective Proteins.

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Received on 04.09.2013 Accepted on 01.10.2013

Asian J. Pharm. Tech. 2013; Vol. 3: Issue 4, Pg 165-169