**3.1 PAMAM dendrimer in gene transfection**

Gene therapy is a promising approach for the treatment of cancer because it enables the production of bioactive agents or the cessation of abnormal functions in the tumor cell. However, the success of gene therapy requires efficient and safe transfer systems because of the degradation of the delivered gene in the systemic circulation.

A variety of molecules including polymers, lipids, and peptides have been studied for their effectiveness as delivery vectors for DNA and RNA molecules. Successful delivery vectors must exhibit a combination of functional attributes. Polymeric carrier molecules should be cationic to complex with nucleic acids, possess a high buffering capacity, exhibit low cytotoxicity, and also contain chemically reactive groups that can be modified for the addition of targeting moieties or other groups.

As a non-viral gene delivery carrier, highly branched, dendritic polymers including poly(amidoamine) (PAMAM) have recently attracted interest as nucleic acid delivery vectors. Previous work has demonstrated that dendrimers can bind to DNA and RNA molecules and mediate modest cellular delivery of these nucleic acids. (PAMAM) dendrimers have attracted great interest due to their high efficacy in vitro gene delivery because of their branched structure. These dendritic polymers bear primary amine groups on their branched surface, which can bind DNA, compact it into polyplexes, and promote the cellular uptake of genes. Therefore, PAMAM dendrimers show high levels of transfection in a wide variety of cultured cells, especially in fractured form of G5 (commercially named SuperFect). Enhanced transfection efficiency has been reported by surface modification of PAMAM with L-arginine. Moreover, the primary amines located on the surface of PAMAM make it possible to conjugate suitable ligands, such as Transferrin, for efficient brain-targeting gene delivery.

Studies that focus on the cell entry mechanisms for several nonviral vectors, including PAMAM dendrimers [16]. The cationic surface charge imparted to the complex through high dendrimer–DNA charge ratios is required for subsequent interaction with the anionic glycoproteins and phospholipids that reside on the cell membrane surface. This interaction initiates the interior movement of the dendrimer–DNA complex into the cell cytosol, either by passive transport caused by membrane perturbations or by endocytosis. Complexes formed without an excess cationic surface charge do not mediate high gene transfection efficiency, which furnishes support for the importance of the initial electrostatic interaction between the complex and cell membrane. Studies following the incorporation of radiolabeled DNA and/or dendrimer components into cells established that the uptake in most cells was primarily via an active endocytosis mechanism. Cells preincubated with

PAMAM Dendrimer as Potential Delivery System for

oligonucleotides in glioma-targeted therapy [22].

the endosome, leading to the release of ASODNs into cytoplasm.

permeation of dendrimers.

Combined Chemotherapeutic and MicroRNA-21 Gene Therapy 505

dendrimers (i.e. G=3 and 4) which had been modified with PEG monomethyl ether chains (i.e. 550 and 2000 Da respectively) attached to their surfaces. A similar construct involving PEG chains and PAMAM dendrimers was used to deliver the anticancer drug 5 fluorouracil. Encapsulation of 5-fluorouracil into G=4 increase in the cytotoxicity and

Dendrimers have ideal properties which are useful in targeted drug-delivery system. One of the most effective cell-specific targeting agents delivered by dendrimers is folic acid PAMAM dendrimers modified with carboxymethyl PEG5000 surface chains revealed reasonable drug loading, a reduced release rate and reduced haemolytic toxicity compared with the non-PEGylated dendrimer. A third-generation dendritic unimolecular micelle with indomethacin entrapped as model drug gives slow and sustained in vitro release, as compared to cellulose membrane control [20]. Controlled release of the Flurbiprofen could be achieved by formation of complex with amine terminated generation 4 (G4) PAMAM Dendrimers [21]. The results found that PEG-dendrimers conjugated with encapsulated

drug and sustained release of methotrexate as compare to unencapsulated drug.

**4. Multifunctional dendrimer nanodevices: In vitro and in vivo testing 4.1 Target gene therapy to rat C6 glioma cells rhrough folate receptor-PAMAM** 

Despite the progress in the PAMAM mediated gene delivery, few studies have investigated the suitability of PAMAM dendrimers for ASODN delivery in vivo, especially for brain gliomas. The purpose of the present study is to evaluate whether in vivo gene delivery by folate-PAMAM (G5) conjugates can inhibit the development of gliomas. We selected the EGFR gene as an antisense target and the rat C6 intracranial glioma model for the in vivo study. Synthetic foliated (FA-)PAMAM was complexed with EGFR ASODN, and then the gene transfection efficacy, dynamic uptake, and biological effects of the FA-PAMAM delivery system on C6 rat glioma cells were investigated both in vitro and in vivo. Our results showed that the FA-PAMAM dendrimer conjugates transported EGFR-ASODNs into glioma cells in vitro, and yielded a favorable therapeutic effect in vivo on administration by local perfusion. Therefore, FA-PAMAM may represent a potential delivery system for short

We chose G5 PAMAM as the gene vector in the present study because its many surface amine groups enable efficient complex formation with ASODNs through charge-based interactions. Western blot analysis demonstrated the binding of G5 PAMAM to ASODNs, with an optimum ASODN/PAMAM ratio of 16:1. TEM analysis revealed that the complexes were >70 nm in size, and this small size likely enabled the efficient transfer of ASODNs to cells that we observed by flow cytometry. We used ASODNs directly labeled with fluorescent probes, such that flow cytometry directly reflected the uptake of the ASODN by the tumor. ASODN uptake mediated by PAMAM increased twofold in comparison with oligofectamine. The high uptake of ASODNs resulted in significant down-regulation of EGFR, suggesting that PAMAM mediated high efficiency transfection of C6 tumor cells with ASODNs. This high transfection efficiency can be attributed to not only the small size of the complexes, but also to the 'proton sponge' effect of PAMAM,31 in which the acidification of tertiary amino groups on PAMAM in the endosome increases the osmotic pressure within

However, while nonderivatized PAMAM achieves high efficiency transfection, its low targeting efficiency needs to be improved. One strategy to achieve this is the

inhibitors of endocytosis (i.e. cytochalasin B and deoxyglucose) or cellular metabolism (i.e. sodium azide) reduced the uptake that corresponded to lower transgene expression, regardless of cell type.

These dendrimers as nanocarriers possess the following advantages: (1) neutral surface of the dendrimer for low cytotoxicity; (2) existence of cationic charges inside the dendrimer (not on the outer surface) resulting in highly organized compact nanoparticles, which can potentially protect nucleic acids from degradation. Noteworthly, surface modified QPAMAM-NHAc dendrimer demonstrated enhanced cellular uptake of siRNA when compared with the internally cationic QPAMAM-OH dendrimer (degree of quaternization 97%).

George's study shows PEG-G5 and PEG-G6 dendrimers, with PEG conjugation molar ratio at 8% (PEG to surface amine per PAMAM), can facilitate dramatic intramuscular gene delivery in neonatal mice [17]. Park's group concluded that di-arginine conjugation to PAMAM dendrimers can improve polyplex stability, ntra-nuclear localization, and transfection efficiency but also induce charge density- and generation-dependent cytotoxicity. Therefore, a novel strategy for highly densed arginine conjugation maintaining low cytotoxicity will be needed for the development of efficient gene delivery carriers [18].
