Gold Nanoparticles capped with PEIbisepoxide nanoparticle system for enhanced siRNA delivery into cells for achieving

therapeutics

Name: Karan Shah Poly id: 0414827 Guided by: Prof. Victor Barinov

ABSTRACT: INTRODUCTION: There are number of diseases know till today that occurs due to single gene defect. There are around 4000 human diseases caused by single gene effects which include sickle cell anemia, cystic fibrosis, phenylketonuria and many more. All these diseases can be treated successfully if the gene responsible for causing the disease is made silent and stopped from being expressed. One of the main approaches used is delivery of siRNA and DNA into mammalian cell using various types of vectors. Nanoparticles have shown excellent properties to facilitate in developing such kind of delivery systems. Many kind of nanoparticles are used today for drug delivery approaches. Nanoparticles are also used in delivering RNA/ small interfering RNA (siRNA) into the cell and thus making therapeutic application possible effectively. Various kinds of delivery systems like cationic polymers, lipids, iron oxide nanoparticles, gold nanoparticles, and semiconductor nanocrystals are known [3]. Out of many known different type of nanoparticles, gold nanoparticles are considered as ideal nanoparticle due to its properties like simple synthesis, good biocompatibility, and also it can bind easily.[1] Gold nanoparticles has vast range of application in drug delivery systems and other fields. There are various kind of system developed till date for delivery of siRNA into mammalian cell but they are not so successful due to various issues like biocompatibility, safety and efficacy. Here new delivery system is being developed for enhanced siRNA delivery which consists of gold nanoparticles capped with Polyethyleneimine (PEI). Gold nanoparticles are able to deliver siRNA effectively but are not successful to silence the gene alone. So capping of PEI is done [1]. From this delivery system results are being obtained experimentally to prove its efficacy. This delivery system can also prove to be useful in delivery of small molecule of drugs so that drug which has poor permeability or dissolution property can be delivered effectively by placing it in core of nanoparticles and delivering them to the target. If such an advance system is developed than lot of drugs can be used effectively at low dose with minimum side effects and drug-drug interaction. Such advancement can open gates for various innovations for Drug Delivery Systems. Such an advance system also promises the accurate and easy delivery of RNA to the targeted cell(s). OBJECTIVE: My objective is to review papers, interpret properties of nanoparticle and polymers used in it and combine them to develop a delivery system and relate it to advancement in siRNA delivery systems into the cell.

REVIEW: PAPER 1: In paper 1 gold nanoparticle are used along with polymer Polyethyleneimine (PEI) to produce a delivery system for siRNA into the targeted cells for treatment. The main concept used here is that PEI is capped over the surface of gold nanoparticles so it possesses a positive charge so that it has potential to condense siRNA on it. Gold nanoparticles are used here due to its various benefits like simple synthesis, good biocompatibility and easy conjugation capacity with small drugs as well as large biomolecules. Here the PEI was first conjugated with siRNA and the system was used for delivery of siRNA to the cell. The delivery was successful but it showed cytotoxicity. So gold nanoparticles were introduced and a system was developed such that AuNPs were capped with PEI and then siRNA were attached on it and the system was tested for delivery of siRNA. Here again the delivery was successful and results were confirmed by Green Fluorescent Protein Gene silencing effect. The main benefit of this system compared to PEI/siRNA system was that the cytoxicity was not seen and so the system was proved biocompatible. The GFP protein gene silencing was confirmed by experiment using MDA-MB-435s cells and Lipofectamine 2000/pGEFP-N2 which were subjected to siRNA formulations for doing gene knockdown experiments.

Highest gene silencing with AuNPs/GFP-siRNA

Efficiency for gene silencing confirmed

Figure 1: GFP expression in MDA-MB-435 cells. A] flouroscence images B] quantitative analyses of GFP expression in MDA-MB-435s cells. [1]

PAPER 2: Here, another system comprising of PEI nanoparticles(PN) is used along with 1,4butanediol diglycidyl ether(bisepoxide) as a polymer. The basic concept used here is that the bisepoxide polymer is cross linked with PEI nanoparticles and than DNA/siRNA is attached to it which was found to be successful in delivering the siRNA to the target an also the cytotoxicity was not there. The gene silencing effect was also studied using a well established system of GFP as used before. The main driving force in this system as that due to cross linking there is change in the amine ratio keeping the overall charged preserved and due to that there is reduced cytotoxicity and better transfection efficiency. The main advantage is that instead of using PEI as a layer for capping it is used as nanoparticles as studies has shown that nanoparticles/DNA complexes exhibit increase gene transfer and lower cytotoxicity compared to PEI(25 kDa). Also degree of crosslinking also plays an important role as on increasing the degree of crosslinking, size of nanoparticles was found to be reduced and also the surface charge of nanoparticles was found to be decreased. DNA polymer interaction was also found to be strong but PEI nanoparticles form relatively loose complexes with DNA and so ease the release of DNA in intracellular milieu. Thus this property of PEI nanoparticles can be used for creating advance systems. PAPER 3: Here, Gold nanoparticles and Poly (-amino ester) [PBAEs] are used to form a delivery system for siRNA into cells. Here the basic concept is that the siRNA which is also as small as nanoparticles is linked with siRNA-AuNP conjugates where PBAEs serves as delivery enhancer and gold nanoparticles serves as scaffold to assemble siRNA. Firstly the siRNAAuNPs were synthesized and than library of pBAEs were synthesized using total of 12 primary diamines and they were screened for their ability to deliver siRNA delivery in vitro. Finally to confirm cellular transfection of PBAE-siRNA-AuNPs, gene knockdown in HeLa cell line was done in which luciferase expression was measured as % expression. It was fond that PBAE-siRNA-AuNPs had lowest luciferase expression as compared to siRNAAuNPs and commercially available Lipo2000 thus confirming its potency to deliver siRNA successfully and ability to knock down gene efficiently. The main drawback of this system is tedious work and long process to make it. PROPOSED SYSTEM: This system consists of gold nanoparticles, PEI nanoparticles and bisepoxide polymer and siRNA. Gold nanoparticles are used for its various benefits including ease of synthesis, easy surface modification, biocompatibility and easy binding due to presence of sulfide bonds on it. Instead of using PEI as a polymer layer PEI nanoparticles are prepared and used because PEI nanoparticles shows reduced cytotoxicity and better biocompatibility than normal PEI(25 kDa).[2]. Also bisepoxide polymer is used to cross link with PEI nanoparticles

because if only PEI is used than it will strongly bind to siRNA and than the release is not easy and efficient. But PEI-bisepoxide nanoparticles bind to siRNA loosely and so it can be released easily and delivered accurately. The success ratio for this system is almost 6570%.[2]. This system is made by first synthesizing cross linked complex of PEI nanoparticles and bisepoxide layer and than capping AuNPs with this system. Than the prepared delivery system is conjugated with siRNA via various disulfide bonds of PEI. The main advantage of this system over other system is that accurate delivery with least cytotoxicity. Also the release rate of siRNA can be controlled by changing the degree of cross linking of PN and bisepoxide. By decreasing the degree of crosslinking the size of nanoparticle system increases and so it takes time to enter the specific time and also the binding of siRNA with PEI nanoparticles becomes more tough and so delayed release systems can also be formed with this system. Such advancement can lead to lesser drug administration, decrease drug dosage to acquire therapeutic range and also increased patient compliance. The cellular transfection of this system was evaluated in HeLa cell line where cells were engineered to express firefly luciferase. [3] The result were compared with siRNA-AuNPs, PEI-bisepoxide, PBAEs-siRNA-AuNPs. All the systems were able to suppress the gene for luciferase but the AuNPs-PEI/bisepoxide-siRNA system showed highest luciferase suppression with no cytotoxicity. PEI siRNA siRNA PBAEs

PN AuNP AuNP Figure B Figure C

Figure A

Figure A: system of paper 1(AuNPs capped with PEI) Figure B: system of paper 2(PEI-bisepoxide nanoparticle system) Figure C: system of paper 3(complexation of siRNA-AuNPs with PBAEs) Commercially this system is also a blockbuster system as it uses all the readily available and simple particles required for its production. Also its synthesis is easy and simple. Moreover there are rare drug delivery systems which can deliver both RNA/DNA or any nucleic acid components and drugs effectively at a particular target. Sustained release capability of this system makes it more demanding for many drugs.

CONCLUSION: The objective of creating an advance system for better drug delivery with minimum cytotoxicity is developed by capping gold nanoparticles with complex system of PEIbisepoxide nanoparticle system and measuring its cellular transfection by measuring % luciferase expression in HeLa cell line culture. Also modifying degree of cross linking of PEI-bisepoxide nanoparticle complex, controlled release drug systems can also be made using this system. No side effects and minimum drug-drug interaction are also its additional benefits. Thus this advance system of polymer and nanoparticle can be used for drug delivery and advance research in field of drug delivery systems. REFERENCES: 1. Wen-Jing Song, Jin-Zhi Du, Tian-Meng Sun, Pei-Zhuo Zhang, and Jun Wang(2010) gold nanoparticles capped with PEI for enhance siRNA delivery. small 6, No. 2, 239246 2. Archana Swami, Raj K. Kurupati, Atul Pathak, Y. singh, P. kumar, K.C. Gupta(2007) A unique and highly efficient non-viral DNA/siRNA delivery system based on PEIbisepoxide nanoparticles. Biochemical and biophysical research communications, 362(4):835-41. 3. Lee JS, Gren JJ, Love KT, Sunshine J, Langer R, Anderson DG(2009, jun 9) Gold, poly(betaamino ester) nanoparticles for small interfering RNA delivery. Nano let. (6):2402-6. 4. A. Swami, A. Aggarwal, A. Pathak, S. Patnaik, P. Kumar, Y. Singh and K.C. Gupta, Imidazolyl-PEI modified nanoparticles for enhanced gene delivery, Int. J. Pharm. 335(2007), pp. 180192 5. Marc Lacroix(2009) MDA-MB-435 cells are from melanoma, not from breast cancer. Cancer Chemother Pharmacol 63:567