**5. Conclusion**

510 Non-Viral Gene Therapy

Fig. 4. Effect of the miR-21 inhibitor on the chemo-sensitivity of U251 and LN229 cells to taxol treatment. The growth of U251 and LN229 cells were inhibited by the miR-21 inhibitor, taxol only, and the indicated combinations. The cells were treated with the miR-21 inhibitor

determine cell viability. Each value represents the mean ±SD from triplicate determinations. An aqueous solution of taxol (circles) and miR-21 inhibitor-loaded PAMAM (triangle) was incubated with human glioblastoma U251 and LN229 cells for six days. Druginduced decrease in cell numbers was measured using the MTT assay. The miR-21 inhibitor enhanced taxol induced apoptosis. Flow cytometry analyses of propidium iodide-stained cells were performed in triplicate (B). miR-21 inhibitor and taxol induce G1 and S phase arrest on cell cycle distribution. U251 and LN229 cells were treated with the miR-21 inhibitor and taxol alone or in combination, and cell cycle distributions were detected by Flow cytometry 48 h later (C). Evaluation of the expression of PTEN, EGFR, STAT3, and p-STAT3 in human glioblastoma LN229 and U251 cell lines. Western blot of protein extracts from cells treated with the miR-21 inhibitor or taxol, alone or combination (D). The expression of b-actin was examined to ensure uniform protein loading in all lanes.

complexed to PAMAM for 6 h at 37°C. The medium was then replaced with media containing various concentrations of taxol. After 72 h of incubation, an MTT assay was performed. Absorbance at 570 nm was normalized to the control (untreated cells) to

MiR-21 was one of the most frequently overexpressed miRNA in human glioblastoma (GBM) cell lines which can serve as a therapeutic target for glioblastoma. We validated that downregulation of miR-21 inhibited the growth of GBM cell lines and induced apoptosis. These effects were only partially dependent on PTEN, highlighting the existence of multiple, and possibly yet unknown, targets of miR-21. Inhibition of miR-21 also suppressed EGFR and Akt activity. These observations were confirmed in in vivo xenograft experiments that showed the potential clinical relevance of miR-21-targeting agents. Targeting miR-21 by antisense or small-molecule compounds may represent new targeted therapeutic strategies for human cancers, including gliomas.

PAMAM dendrimer has been reported to be good gene delivery candidate. Although the biological effects obtained from in vitro analysis of PAMAM and FA-PAMAM are approximate, our in vivo study implies that FAPAMAM is functionally effective for gene delivery into three-dimensional tissues. This may be due to folate-mediated targeting of ASODNs to folate receptor-expressing cells in solid tumors. Stereotactic administration, which enables FA-PAMAM-ASODNs to be injected directly into a tumor, may also produce better results than intravenous injection. Site-specific delivery remains the best choice to overcome gene delivery side effects and to increase its efficacy.

Next, we exhibit the anticancer potential of a combination of 5-FU treatment and antisense miR-21 technology using PAMAM dendrimers. PAMAM dendrimers, an available cocarrier of chemotherapeutant and as-miR-21, could effectively deliver 5-FU and as-miR-21 simultaneously, forming complexes smaller than 100 nm in diameter. The small size of the complexes facilitated their effective uptake by tumor cells, so the chemotherapeutant and asmiR-21 could be synchronously introduced to glioma cell for combined actions. The codelivery of as-miR-21 significantly improved the cytotoxicity of 5-FU and dramatically increased the level of apoptosis of U251 cells; it also decreased the migration abilities of the tumor cells. Our results provide invaluable information regarding the future application of

PAMAM Dendrimer as Potential Delivery System for

9232

0006-291X

647–658, ISSN: 0016-5085

ISSN: 1461-5347

1367-5931

May), pp: 539-50, ISSN: 1015-6305

(2009 Jun) pp: 132-9, ISSN: 0168-3659

No.11, (2008 Nov) pp:88-98, ISSN:

*Hematol Educ Program*. pp: 708-19 , ISSN: 1520-4391

Combined Chemotherapeutic and MicroRNA-21 Gene Therapy 513

Zhu S, et al (2007). MicroRNA-21 targets the tumor suppressor gene tropomyosin 1 (TPM1). *J Biol Chem*, Vol. 282, No.19, (2007 May), pp: 14328–14336, ISSN: 0021-9258 Asangani IA, et al (2008). MicroRNA-21 (miR-21) post-transcriptionally downregulates

Moore LM, Zhang W. Targeting miR-21 in glioma: a small RNA with big potential. Expert Opin Ther Targets. Vol. 14, No.11, (2010 Nov r), pp: 1247-57, ISSN: 1472-8222 Ciafre` SA, et al. Extensive modulation of a set of microRNAs in primary glioblastoma.

Meng F, et al (2007). MicroRNA-21 regulates expression of the PTEN tumor suppressor gene

Malzkorn B, et al (2010). Identification and functional characterization of microRNAs

Baker JR Jr (2009). Dendrimer-based nanoparticles for cancer therapy. *Hematology Am Soc* 

Eichman JD, et al (2000). The use of PAMAM dendrimers in the efficient transfer of genetic

Qi R, et al (2009). PEG-conjugated PAMAM dendrimers mediate efficient intramuscular gene expression. *AAPS J.* Vol. 11 , No. 3, (2009 Sep) pp: 395-405, ISSN: 1550-7416 Kim TI, et al (2009). Comparison between arginine conjugated PAMAM dendrimers with

Tolia,G.T et al (2008). The role of dendrimers in drug delivery, Pharmaceut. Tech., Vol. 32,

Patri, A.K., Majoros and. Baker, J.R., (2002). Dendritic polymer macromolecular carriers for

Asthana ,A.,et al, 2005 . Poly (amidoamine) (pamam) dendritic nanostructures for controlled

Ren Y, et al (2010). Co-delivery of as-miR-21 and 5-FU by poly(amidoamine) dendrimer

Karmakar S, et al (2007). Combination of all- trans retinoic acid and taxol regressed

*PharmSciTech*. Vol. 6 , No. 3, (2005 Oct) pp: E536-42, ISSN: 1530-9932 Kang C, et al (2010). Evaluation of folate-PAMAM for the delivery of antisense

(2010 May), Vol. 93 , No. 2, pp: 585-94, ISSN: 1549-3296

3, (2010 Mar) pp: 303-14., ISSN: 0920-5063

pp: 2077-87, ISSN: 1360-8185.

tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. *Oncogene*, Vol. 27, No. 15, (2008 Apr), pp: 2128–2136, ISSN: 0950-

*Biochem Biophys Res Commun* Vol. 334 , No.4 , (2005 Sep), pp: 1351–1358 , ISSN:

in human hepatocellular cancer. *Gastroenterology*, Vol. 133, No. 2, (2007 Aug), pp:

involved in the malignant progression of gliomas. *Brain Pathol.* Vol.20, No.3, (2010

material into cells. *Pharm Sci Technolo Today*, Vol. 3, No. 7, (2000 Jul), pp: 232-245,

structural diversity for gene delivery systems. *J Control Release*. Vol. 136, No. 2,

drug delivery, *Curr. Opin. Chem. Biol.* Vol. 6, No. 4, (2002 Aug) pp: 466-71, ISSN:

site specific delivery of acidic anti-inflammatory active ingredient, *AAPS* 

oligonucleotides to rat C6 glioma cells in vitro and in vivo. *J Biomed Mater Res A*.

attenuates human glioma cell growth in vitro. *J Biomater Sci Polym Ed*. Vol. 21 , No.

glioblastoma T98G xenografts in nude mice. *Apoptosis*, Vol.12, No.11, (2007 Nov)

drug–polymer complexes combined with gene therapy for cancer treatments. Taken together, our findings suggest that the combination of 5-FU treatment and as-miR-21 might be a potential clinical strategy for cancer chemotherapy.

Furthermore, the miR-21 inhibitor could enhance the chemo-sensitivity of human glioblastoma cells to taxol via PAMAM dendrimer. A combination of miR-21 inhibitor and taxol could be an effective therapeutic strategy for controlling. The above data suggested that in both the PTEN mutant U251 cell line and the PTEN wild-type LN229 cells, miR-21 blockage could increase the chemosensitivity to taxol. It is worth noting that the miR-21 inhibitor additively interacted with taxol on U251cells and synergistically on LN229 cells. Thus, the miR-21 inhibitor might interrupt the activity of EGFR pathways, independently of PTEN status. The miR-21 inhibitor enhanced the chemo-sensitivity of human glioblastoma cells to taxol and combination of the miR-21 inhibitor and taxol could be an effective therapeutic strategy for suppressing the growth of GBM.

the growth of GBM by inhibiting STAT3 expression and phosphorylation.
