Drug Delivery using Nanoparticle along with ssDNA

J. Madhusudhanan¹*, P. Monika², K. Monica²

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:

Recently nanoparticles have been developed for the photo acoustic imaging, delivery of genes and laser induced photo thermal therapy. In this study, we have developed ssDNA conjugated nanoparticles as the carrier for simultaneous DNA and anti-cancer nucleoside delivery. The ssDNA nanoparticle complex presented higher capacity in carrying anti-cancer drug compounds than the original nanoparticles. The hydrodynamic size of the nanoparticles increased from 25 to 35 nm with an increase in the negative surface charge from -9.58 to 21.66 mV after ssDNA conjugation and drug loading. A positive association between environmental pH and drug release was observed in PBS and Tris/HCl, which implied their potential use in the controlled localized drug release in the lower GI tract. The Tryphan Blue, Ethidium Bromide assay revealed dose dependent cytotoxicity to oral and lung cancer cell line than free compounds. These results suggest the potential use of this new ssDNA nanoparticles complex as the environmental controlled anti-cancer nanocapsule, especially suitable for oral and lung cancer chemotherapy.

KEYWORDS: Anticancer, Nanocapsule, Drug carrier, Cytotoxicity.

INTRODUCTION:

Nanotechnology is the understanding and control of matter generally in the 1–100 nm dimension range. The application of nanotechnology to medicine is known as nanomedicine. The use of engineered materials at this length scale to develop novel therapeutic¹. Metal nanoparticles, especially nanoparticles, have attracted much attention because of their extraordinary electrical and optical properties.5 Such opticalproperties are termed surface plasmon induced by the collective oscillation of electron density. The efficient absorbance and scattering of light of the metal nanoparticles may be utilized in electronic and photonic devices. However, metal nanoparticles in solution are susceptible to aggregation themselves. The synthesis of stabilized nanoparticles, a lot of effort has been given to the preparation of well-dispersed, stable protected metal clusters. Using polymeric stabilizing species for metal nanoparticles has inspired the studies of various synthetic routes to link polymers to metal particles².A major problem of the conventional cancer drugs relates to their toxicity which causes the death of healthy cells as well as cancerous cells.

Moreover, their life time in the body is short. Among the cancer chemotherpeutics, chemotherapy is a widely used for anti-cancer treatment⁴. Although it kills the cancerous cells by inhibiting the synthesis of nucleic acids within cells, three main problems are encountered with drug, first , high toxicity and large volume of distribution , second short life time in the body and third low solubility. To overcome the non-specificity and high toxicity of drug, many researchers have proposed conjugation to hydrophilic polymers to reduce the toxicity level while sustaining the therapeutic efficacy⁵.

The Carbopol polymer family is based on cross linked acrylic acid chemistry. The products are cross linked at different levels providing a portfolio of functionally diverse performance options. Carbopol polymers are efficient and effective rheology modifiers. They provide excellent thickening, suspension and stabilization benefits⁸.

The polymer which was completely water soluble, non-toxic and uncharged thus encountering less electrostatic interactions with nanoparticle.

MATERIALS AND METHODS:

Chemical synthesis of nanoparticles:

Auric chloride was added into distilled water, after stirring for one minute, then the NaBH4/ Tri sodium citrate solution was added drop by drop until the mixture turned red with vigorous stirring, and kept stirring for five minutes, then stored at 4ºC.The optical spectra if colloidal nanoparticle were acquired on UV-vis-spectrophotometer. TEM image was acquired. UV-vis of colloids measured immediately and two weeks later.

Preparation of ssDNA modified nanoparticle:

Modified nucleotide was dissolved in double distilled water with gold colloids was incubated for 24hrs.The reaction mixture was then added with NaCl solution then incubated for 24hrs at 4°C.Salt concentration was gradually increased and each incubated.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 3/4^th run, gel was viewed under UV transilluminator.

Conjugation drug to functionalized nanoparticles:

Drug was dissolved in buffer and ssDNA modified nanoparticle was added and stirred for 24h at room temperature. The mixture was subsequently freeze dried.

Drug loading efficiency:

The loading efficiency of drug onto the original and Polymer modified nanoparticle with folate were measured. Au nanoparticle (Np’s) modified with ssDNA is mixed with drug with buffer for the given period of time and centrifuged to remove the nanoparticles. The supernatant were measured in a UV-Vis spectrometer for the concentration.

Drug release behaviour:

The drug releasing kinetics of the nanoparticle –ssDNA-drug complex was then evaluated at different pH environment in buffer. The drug releasing rate was calculated as; %drug release=[OD 520 of the supernatant at each time/OD520 of original loaded drugs on Np-ssDNA-drug complex]x100. Each absorbance was subtracted with blank.

Cytotoxicity:

Cancer cell lines were obtained from NCCS pune. To study the in vitro cytotoxicity for Drug loaded nanoparticle, Tryphan blue, Ethidium Bromide assay was performed on cell lines. Each cell line was seeded into 96-well plates. After 24h the medium was changed and various concentration of drug loaded nanoparticle. At intervals the medium was removed and the cells were washed twice with buffer. And then Tryphan blue and dual staining was performed.

CHARACTERIZATION:

Characterization of nanoparticle:

Figure 1 describes the TEM image of nanoparticle. UV-Vis spectra of as prepared colloids characterized as shown in the figure 2.The sharp peak indicates the colloids well-dispersed. From the maximum adsorption at 510nm we can conclude the size of colloid.

Figure 1 Shows the TEM image of an nanoparticle.

Figure 2 Shows the UV-Vis Spectra an analysis of nanoparticle

Figure 3: SEM image of nanoparticle

The SEM image of nanoparticle has been shown above, a) SEM image of nanoparticle, b) magnified image of nanoparticle, c) single image of an nanoparticle.

Fig4: Drug loading efficiency of ssDNA modified nanoparticle with drug

Characterization of Drug loading efficiency:

The loading efficiency of drug onto the ssDNA modified nanoparticle (fig 3 & 4) was measured in 0h and 24h. The ssDNA modified nanoparticle were mixed with drug for the given period of time and centrifuged to remove the nanoparticles^6,7. The supernatant were measured in UV-vis spectrometer for the concentration of drug.

The TEM image of functionalized nanoparticle with drug (fig 5), says that there is an aggregation of nanoparticle with drug. This proves that there is an attachment of nanoparticle withdrug.

Figure 5: TEM image of nanoparticle with drug Drug release behavior:

In this study modified nanoparticle served as the affinity drug carrier and releasing control trigger at the same time. In addition the spectrometer analysis revealed a sustained pH dependent releasing manner of the nano carriers in buffer solution. The loaded drug has a significantly higher release rate in alkaline environment compare to neutral and aciding environment. Thus the modified nanoparticle (fig 6) could serve as an intestine local delivery nano vehicle to pass through stomach and upper GI tract then release therapeutic agents in the lower GI tract, lung and oral.

Figure 6 shows the drug release behavior of ssDNAmodified nanoparticle with drug

In vitro cytotoxicity:

To evaluate the cell cytotoxicity of the free drug and the drug loaded nanoparticles, on cancer cells were used as invitro effect models. The Drug loaded nanoparticleon the viability of three model cell lines, respectively. These results suggest that Drug loaded nanoparticle effectively decreased the in vitro cancer cell viability, which could imply the targeting effects of these nano-carriers in vivo.

CONCLUSION:

Novel Nanoparticle-ssDNA conjugates with drug were prepared for targeted drug delivery. The Drug-loaded NPs showed significant invitro targeting effects for cell lines, which resulted in higher cytotoxicity than with free Drug. In addition, they caused higher cell viability at low concentration. And shows low cell viability at high concentration. Our results imply that the ssDNA functionalized nanoparticles could have high potential to be used for cancer therapy.

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Received on 14.09.2013 Accepted on 01.10.2013

Asian J. Pharm. Tech. 2013; Vol. 3: Issue 4, Pg 161-164