**2.3 miR-21 regulated GBM cell growth in vitro and in vivo**

To evaluate the significance of miR-21 overexpression in glioma cells, we used a loss-offunction antisense approach. An As-miR-21 oligonucleotide (ODN) was used to knock down miR-21 expression in U251 and LN229 cells. RT-realtime PCR results determined that the relative expression level of miR-21 in As-miR-21 ODN-treated U251 cell was 6.25% (P<0.01) and 12.5% for LN229 cells (P<0.01) compared with their control cells, respectively. In addition, LNA-based in situ hybridization showed that transfection of a scrambled ODN had no effect on miR-21 expression. In contrast, the cy3 red fluorescence signal in As-miR-21-transfected U251 cells was lower (Figure 1B). These data suggested that As-miR-21 can specifically inhibit the endogenous miR-21 expression in U251 and LN229 cells.

The GBM cell growth inihibitory effect (MTT assay) of decreased miR-21 reached maximum three days post transfection. G1 phase blockage was observed to indicate cell cycle distribution changed significantly after miR-21 inhibitory. Additionally, the Annexin V positive early phase apoptotic cells were significantly increased in cells transfected with ASmiR-21 as compared to that in parental cells and cells treated with scrambled ODN.

The in vitro experiments suggest that miR-21 is a potential target for therapy in GBM. To further confirm this, we performed a proof-of-principle experiment using a U251 glioma xenograft model and a lipofectamine-mediated gene therapy approach. The xenograft tumors volume suppression indicated that miR-21 contributed a lot to U251 GBM cell proliferation in vivo. Pathological examination found micro-vessel density indicated that was and evaluated miR-21 expression by in situ hybridization and cellular apoptosis by TUNEL assay.

Despite the apparently predominance of microRNA in cancer therapy, several problems have to be overcome for successful clinical application. They show a poor stability towards nuclease activity, low intracellular penetration and low bioavailability. Although chemical modifications were brought to the basic microRNA, their sensitivity to degradation and poor intracellular penetration is still hampering their widespread clinical applications. In fact, the major bottleneck in the development of miRNA strategy is the delivery of these

PAMAM Dendrimer as Potential Delivery System for

**3. Polyamidoamine (PAMAM) dendrimers** 

**3.1 PAMAM dendrimer in gene transfection** 

addition of targeting moieties or other groups.

for efficient brain-targeting gene delivery.

the degradation of the delivered gene in the systemic circulation.

important substances.

Combined Chemotherapeutic and MicroRNA-21 Gene Therapy 503

Recently, a great deal of attention has been given to polyamidoamine (PAMAM) dendrimers; these are one of the most appropriate candidates for suitable carrier systems. PAMAM dendrimers represent an exciting new class of macromolecular architecture called dense star polymers. Unlike classical polymers, dendrimers have a high degree of molecular uniformity, a narrow molecular weight distribution, specific size and shape character [15]. The terminal amine groups of PAMAM dendrimers can be modified with different functionalities and can be linked with various biomolecules. These unique structural features of PAMAM dendrimers make them ideal nanoplatforms to conjugate biologically

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

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

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,

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

macromolecules to the target cells, tissues or organs. Therefore the development of more efficient delivery systems is regarded as one of the most promising strategies to solve these pharmaceutical hurdles. Specifically, delivery vectors must be designed to effectively complex with nucleic acid molecules and aid in overcoming intracellular barriers such as endosomal escape and cytoplasmic vector dissociation. For that reason, improvements on effective delivery have progressed rapidly. Among the different approaches under study, dendrimers are attracting a great interest for their well defined structure and great versatility in their chemistry that offer a unique platform for the rational design of efficient antisense delivery systems.

Fig. 1. The effect of miR-21 knockdown on U251 and LN229 GBM cell proliferation. (A) miR-21 was overexpressed in six glioma cells. (B) In situ examination of miR-21 expression in U251 cells. Arrows highlight miR-21 in situ expression in U251 cells. Bar¼20 mm. (C、D) MTT cell proliferation assay. miR-21 knockdown in U251 and LN229 GBM inhibits cell proliferation in vitro and in vivo. (E) Cell-cycle profiles after PI staining. miR-21 knockdown induced G1 arrest in both U251 and LN229 GBM cells. (F). As-miR-21 and scramble ODN-transfected U251 and LN229 GBM cells were analyzed using FCM to determine cell-cycle status.
