**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 oligonucleotides in glioma-targeted therapy [22].

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

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

PAMAM Dendrimer as Potential Delivery System for

Combined Chemotherapeutic and MicroRNA-21 Gene Therapy 507

Fig. 2. Contrast-enhanced coronal MR images of representative animals in the ASODN, Oligofectamine/ASODN, PAMAM/ASODN, FA-PAMAM/ASODN group at 1, 2, and

PAMAM/ASODN reached the 3-week time point, preventing a valid statistical comparison at that interval. The tumor in this animal is smaller in diameter and less contrast-enhanced

With positively charged primary amino groups on the surface, the PAMAM dendrimer can feasibly interact with biomolecules to form complexes through charge-based interactions, and protect them from rapid degradation by cellular endo- and exonucleases. Thus, the PAMAM dendrimer may be suitable for gene transfer or oligonucleotide delivery. Besides, because PAMAM has well-defined internal cavities and an open architecture, guest

3weeks after tumor xenograft. Four animals in PAMAM/ASODN and FA-

than the one in the animal without gene therapy.

derivatization of PAMAM with ligands. Various ligands such as folic acid, transferring, and lactoferrin have been conjugated to PAMAM, thus enabling efficient gene targeting to tumors or brain. We chose folic acid as the functional ligand with which to modify PAMAM because of its low immunogenicity, unlimited availability, functional stability, defined conjugation chemistry, and a favorable nondestructive cellular internalization pathway.23 More importantly, the receptor for folic acid is a cell-proliferation protein that is overexpressed in many types of cancer cells.32–34 The expression levels of folate receptor in tumors have been reported to be 100–300 times higher than those observed in normal tissue.35 Although some ambiguity surrounds the expression level of the folate receptor in brain tumors,36 our results demonstrate that conjugation with folic acid enhanced the uptake of ASOND/PAMAM complexes by tumor cells and resulted in greater inhibition of EGFR expression in comparison with the native dendrimer. The in vivo study also demonstrates the superiority of FAPAMAM over either PAMAM or oligofectamine as a vector for mediating ASODN gene therapy. Dynamic contrast MRI scanning indicated significant suppression of tumor growth 2 weeks after C6 cell implantation (Fig. 2), which prolonged the survival time of rats in the FA-PAMAMmediated therapeutic groups.

In the first place, we evaluated the efficiency of folate-PAMAM dendrimers conjugates (FA-PAMAM) for the in situ delivery of therapeutic antisense oligonucleotides (ASODN) that could inhibit the growth of C6 glioma cells. Folic acid was coupled to the surface amino groups of G5-PAMAM dendrimer (G5D) through a 1-[3-(dimethylamino)propyl]-3 ethylcarbodiimide bond, and ASODNs corresponding to rat epidermal growth factor receptor (EGFR) were then complexed with FA-PAMAM. At an ASODN to PAMAM ratio of 16:1, agarose electrophoresis indicated that antisense oligonucleotides were completely complexed with PAMAM or FA-PAMAM. The ASODN transfection rates mediated by FA-PAMAM and PAMAM were superior to oligofectamine, resulting in greater suppression of EGFR expression and glioma cell growth. Stereotactic injection of EGFR ASODN:FA-PAMAM complexes into established rat C6 intracranial gliomas resulted in greater suppression of tumor growth and longer survival time of tumor-bearing rats compared with PAMAM and oligofectamine-mediated EGFR-ASODN therapy. The current study demonstrates the suitability of folate-PAMAM dendrimer conjugates for efficient EGFR ASODN delivery into glioma cells, wherein they release the ASODN from the FA-PAMAM to knock down EGFR expression in C6 glioma cells, both in vitro and in vivo. FA-PAMAM may thus represent a novel delivery system for short oligonucleotides in glioma-targeted therapy.

#### **4.2 Co-delivery of as-mir-21 and 5-fu by poly(amidoamine) dendrimer attenuates human glioma cell growth in vitro**

The efficacy of conventional chemotherapy is limited owing to the low therapeutic index of many anticancer drugs, as well as intrinsic or acquired drug resistance. To circumvent these difficulties, novel therapeutic strategies have been developed, and one attractive strategy is the combination of gene therapy with chemotherapy.

MicroRNAs have been demonstrated to be deregulated in different types of cancer. miR-21 is a key player in the majority of cancers. Down-regulation of miR-21 in glioblastoma cells leads to repression of cell growth, increased cellular apoptosis and cell-cycle arrest, which can theoretically enhance the chemotherapeutic effect in cancer therapy.

derivatization of PAMAM with ligands. Various ligands such as folic acid, transferring, and lactoferrin have been conjugated to PAMAM, thus enabling efficient gene targeting to tumors or brain. We chose folic acid as the functional ligand with which to modify PAMAM because of its low immunogenicity, unlimited availability, functional stability, defined conjugation chemistry, and a favorable nondestructive cellular internalization pathway.23 More importantly, the receptor for folic acid is a cell-proliferation protein that is overexpressed in many types of cancer cells.32–34 The expression levels of folate receptor in tumors have been reported to be 100–300 times higher than those observed in normal tissue.35 Although some ambiguity surrounds the expression level of the folate receptor in brain tumors,36 our results demonstrate that conjugation with folic acid enhanced the uptake of ASOND/PAMAM complexes by tumor cells and resulted in greater inhibition of EGFR expression in comparison with the native dendrimer. The in vivo study also demonstrates the superiority of FAPAMAM over either PAMAM or oligofectamine as a vector for mediating ASODN gene therapy. Dynamic contrast MRI scanning indicated significant suppression of tumor growth 2 weeks after C6 cell implantation (Fig. 2), which prolonged the survival time of rats in the FA-PAMAM-

In the first place, we evaluated the efficiency of folate-PAMAM dendrimers conjugates (FA-PAMAM) for the in situ delivery of therapeutic antisense oligonucleotides (ASODN) that could inhibit the growth of C6 glioma cells. Folic acid was coupled to the surface amino groups of G5-PAMAM dendrimer (G5D) through a 1-[3-(dimethylamino)propyl]-3 ethylcarbodiimide bond, and ASODNs corresponding to rat epidermal growth factor receptor (EGFR) were then complexed with FA-PAMAM. At an ASODN to PAMAM ratio of 16:1, agarose electrophoresis indicated that antisense oligonucleotides were completely complexed with PAMAM or FA-PAMAM. The ASODN transfection rates mediated by FA-PAMAM and PAMAM were superior to oligofectamine, resulting in greater suppression of EGFR expression and glioma cell growth. Stereotactic injection of EGFR ASODN:FA-PAMAM complexes into established rat C6 intracranial gliomas resulted in greater suppression of tumor growth and longer survival time of tumor-bearing rats compared with PAMAM and oligofectamine-mediated EGFR-ASODN therapy. The current study demonstrates the suitability of folate-PAMAM dendrimer conjugates for efficient EGFR ASODN delivery into glioma cells, wherein they release the ASODN from the FA-PAMAM to knock down EGFR expression in C6 glioma cells, both in vitro and in vivo. FA-PAMAM may thus represent a novel delivery system for short oligonucleotides in glioma-targeted

**4.2 Co-delivery of as-mir-21 and 5-fu by poly(amidoamine) dendrimer attenuates** 

The efficacy of conventional chemotherapy is limited owing to the low therapeutic index of many anticancer drugs, as well as intrinsic or acquired drug resistance. To circumvent these difficulties, novel therapeutic strategies have been developed, and one attractive strategy is

MicroRNAs have been demonstrated to be deregulated in different types of cancer. miR-21 is a key player in the majority of cancers. Down-regulation of miR-21 in glioblastoma cells leads to repression of cell growth, increased cellular apoptosis and cell-cycle arrest, which

mediated therapeutic groups.

**human glioma cell growth in vitro** 

the combination of gene therapy with chemotherapy.

can theoretically enhance the chemotherapeutic effect in cancer therapy.

therapy.

Fig. 2. Contrast-enhanced coronal MR images of representative animals in the ASODN, Oligofectamine/ASODN, PAMAM/ASODN, FA-PAMAM/ASODN group at 1, 2, and 3weeks after tumor xenograft. Four animals in PAMAM/ASODN and FA-PAMAM/ASODN reached the 3-week time point, preventing a valid statistical comparison at that interval. The tumor in this animal is smaller in diameter and less contrast-enhanced than the one in the animal without gene therapy.

With positively charged primary amino groups on the surface, the PAMAM dendrimer can feasibly interact with biomolecules to form complexes through charge-based interactions, and protect them from rapid degradation by cellular endo- and exonucleases. Thus, the PAMAM dendrimer may be suitable for gene transfer or oligonucleotide delivery. Besides, because PAMAM has well-defined internal cavities and an open architecture, guest

PAMAM Dendrimer as Potential Delivery System for

Combined Chemotherapeutic and MicroRNA-21 Gene Therapy 509

Fig. 3. Characterization of 5-FU/PAMAM/As-miR-21 complex. TEM image of 5-FU– PAMAM–as-miR-21 complex, N/P = 16. Magnification 58 000×, scale bar = 100 nm (A). Release profiles of 5-FU from PAMAMand PAMAM–as-miR-21 complexes compared with free 5-FU (B). Cell uptake detected by flow cyctometry and fluorescent microscopy image of

Substantial data indicate that the oncogene microRNA 21 (miR-21) is significantly elevated in glioblastoma multiforme (GBM) and regulates multiple genes associated with cancer cell proliferation, apoptosis, and invasiveness. Thus, miR-21 can theoretically become a target to enhance the chemotherapeutic effect in cancer therapy. So far, the effect of downregulating miR-21 to enhance the chemotherapeutic effect to taxol has not been studied in human GBM. In this study, we combine taxol chemotherapy and miR-21 inhibitor treatment via polyamidoamine (PAMAM) dendrimers vector to evaluate the effects of combination therapy on suppression of glioma cells. The result indicated that the miR-21 inhibitor can decrease the proliferation of both U251 and LN229 cells and increase the cells' sensitivity to taxol treatment. The taxol concentration causing 50% growth inhibition (IC50) of U251 cells is 400 nmol/mL; whereas, in combination with the miR-21 inhibitor (20 μmol/L) the IC50

U251 cells after transfection with different complexes (C).

molecules can become directly encapsulated into the macromolecule interior through hydrophobic interactions. In this study, the poly(amidoamine) (PAMAM) dendrimer was employed as a carrier to co-deliver antisense-miR-21 oligonucleotide (as-miR-21) and 5 fluorouracil (5-FU) to achieve delivery of as-miR-21 to human glioblastoma cells and enhance the cytotoxicity of 5-FU antisense therapy.

Taking advantage of hydrogen-bond interaction, we encapsulated 5-FU in the PAMAM nanoparticles simply by a membrane dialysis method. the encapsulation efficiency and loading efficiency of the drug were determined by UV spectroscopy to be 66.21 and 31.77%, respectively. Through their charge-based interactions, 5-FU-PAMAM could conjugate with as-miR-21. The co-delivery of as-miR-21 not only significantly improved the cytotoxicity and chemosensitivity of 5-FU and dramatically increased the apoptotic percentage of the U251 cells but also brought down the migration ability of the tumor cells. The inhibitory effect toward brain tumors was evaluated by MTT assay, and measurements of cell apoptosis and invasion using the human brain glioma cell line U251. PAMAM could be simultaneously loaded with 5-FU and as-miR-21, forming a complex smaller than 100 nm in diameter. Both the chemotherapeutant and as-miR-21 could be efficiently introduced into tumor cells. The co-delivery of as-miR-21 significantly improved the cytotoxicity of 5-FU and dramatically increased the apoptosis of U251 cells, while the migration ability of the tumor cells was decreased. These results suggest that our co-delivery system may have important clinical applications in the treatment of miR-21-overexpressing glioblastoma.

We report the anticancer potential of a combination of 5-FU treatment and antisense miR-21 technology using PAMAM dendrimers. PAMAM dendrimers, an available co-carrier 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 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 [23].

#### **4.3 MicroRNA-21 inhibitor sensitizes human glioblastoma cells to taxol using PAMAM dendrimer**

Chemotherapeutic drugs are fundamental in cancer management and are responsible for most cases of adjuvant treatment in patients with GBMs after surgical procedures. Recently, much attention has been focused on the use taxol on glioma, both in experimental studies and in clinical trails [24]. However, the median overall survival did not increase in patients treated by concurrent chemoradiotherapy.

The successful of anti-cancer treatment are often limited by the development of drug resistance. Consequently, further studies that could enhance the therapeutic effect of taxol should be encouraged.Recent work has highlighted the involvement of non-coding RNAs, microRNAs(miRNAs) in cancer development, and their possible involvement in the evolution of drug resistance has been proposed.

molecules can become directly encapsulated into the macromolecule interior through hydrophobic interactions. In this study, the poly(amidoamine) (PAMAM) dendrimer was employed as a carrier to co-deliver antisense-miR-21 oligonucleotide (as-miR-21) and 5 fluorouracil (5-FU) to achieve delivery of as-miR-21 to human glioblastoma cells and

Taking advantage of hydrogen-bond interaction, we encapsulated 5-FU in the PAMAM nanoparticles simply by a membrane dialysis method. the encapsulation efficiency and loading efficiency of the drug were determined by UV spectroscopy to be 66.21 and 31.77%, respectively. Through their charge-based interactions, 5-FU-PAMAM could conjugate with as-miR-21. The co-delivery of as-miR-21 not only significantly improved the cytotoxicity and chemosensitivity of 5-FU and dramatically increased the apoptotic percentage of the U251 cells but also brought down the migration ability of the tumor cells. The inhibitory effect toward brain tumors was evaluated by MTT assay, and measurements of cell apoptosis and invasion using the human brain glioma cell line U251. PAMAM could be simultaneously loaded with 5-FU and as-miR-21, forming a complex smaller than 100 nm in diameter. Both the chemotherapeutant and as-miR-21 could be efficiently introduced into tumor cells. The co-delivery of as-miR-21 significantly improved the cytotoxicity of 5-FU and dramatically increased the apoptosis of U251 cells, while the migration ability of the tumor cells was decreased. These results suggest that our co-delivery system may have important clinical applications in the treatment of miR-

We report the anticancer potential of a combination of 5-FU treatment and antisense miR-21 technology using PAMAM dendrimers. PAMAM dendrimers, an available co-carrier 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 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

enhance the cytotoxicity of 5-FU antisense therapy.

21-overexpressing glioblastoma.

**PAMAM dendrimer** 

be a potential clinical strategy for cancer chemotherapy [23].

treated by concurrent chemoradiotherapy.

tion of drug resistance has been proposed.

**4.3 MicroRNA-21 inhibitor sensitizes human glioblastoma cells to taxol using** 

Chemotherapeutic drugs are fundamental in cancer management and are responsible for most cases of adjuvant treatment in patients with GBMs after surgical procedures. Recently, much attention has been focused on the use taxol on glioma, both in experimental studies and in clinical trails [24]. However, the median overall survival did not increase in patients

The successful of anti-cancer treatment are often limited by the development of drug resistance. Consequently, further studies that could enhance the therapeutic effect of taxol should be encouraged.Recent work has highlighted the involvement of non-coding RNAs, microRNAs(miRNAs) in cancer development, and their possible involvement in the evolu-

Fig. 3. Characterization of 5-FU/PAMAM/As-miR-21 complex. TEM image of 5-FU– PAMAM–as-miR-21 complex, N/P = 16. Magnification 58 000×, scale bar = 100 nm (A). Release profiles of 5-FU from PAMAMand PAMAM–as-miR-21 complexes compared with free 5-FU (B). Cell uptake detected by flow cyctometry and fluorescent microscopy image of U251 cells after transfection with different complexes (C).

Substantial data indicate that the oncogene microRNA 21 (miR-21) is significantly elevated in glioblastoma multiforme (GBM) and regulates multiple genes associated with cancer cell proliferation, apoptosis, and invasiveness. Thus, miR-21 can theoretically become a target to enhance the chemotherapeutic effect in cancer therapy. So far, the effect of downregulating miR-21 to enhance the chemotherapeutic effect to taxol has not been studied in human GBM. In this study, we combine taxol chemotherapy and miR-21 inhibitor treatment via polyamidoamine (PAMAM) dendrimers vector to evaluate the effects of combination therapy on suppression of glioma cells. The result indicated that the miR-21 inhibitor can decrease the proliferation of both U251 and LN229 cells and increase the cells' sensitivity to taxol treatment. The taxol concentration causing 50% growth inhibition (IC50) of U251 cells is 400 nmol/mL; whereas, in combination with the miR-21 inhibitor (20 μmol/L) the IC50

PAMAM Dendrimer as Potential Delivery System for

modulating EGFR/STAT3 signaling [25].

for human cancers, including gliomas.

overcome gene delivery side effects and to increase its efficacy.

LN229 cells.

**5. Conclusion** 

Combined Chemotherapeutic and MicroRNA-21 Gene Therapy 511

was 60 nmol/mL. Taxol can also increase the efficacy of the miR-21 inhibitor. For example, combination treatment reduced cell viability to 20% compared with 86% viability for miR-21 inhibitor gene therapy alone. In LN229 cells, combination treatment with 20 μmol/L of the miR-21 inhibitor reduced the IC50 of taxol from 820 to 160 nmol/L . It is worth noting that the miR-21 inhibitor additively interacted with taxol on U251cells and synergistically on

Taxol treatment also increased the percentage of apoptotic cancer cells in miR-21 inhibitor transfected cells compared with control cells. Furthermore, treatment of the miR-21 inhibitor-transfected cells with the anti-cancer drugs taxol resulted in significantly reduced cell viability and invasiveness compared with control cells. These results indicated that the miR-21 plays an important role in the resistance of brain cancer cells to chemotherapeutic drugs. Therefore, miR-21 inhibitor gene therapy combined with taxol chemotherapy might

Thus, the miR-21 inhibitor might interrupt the activity of EGFR pathways, independently of PTEN status. Meanwhile, the expression of STAT3 and p-STAT3 decreased to relatively low levels after miR-21 inhibitor and taxol treatment. The data strongly suggested that a regulatory loop between miR-21 and STAT3 might provide an insight into the mechanism of

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

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

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

represent a promising novel therapeutic approach for the treatment of glioblastoma.

was 60 nmol/mL. Taxol can also increase the efficacy of the miR-21 inhibitor. For example, combination treatment reduced cell viability to 20% compared with 86% viability for miR-21 inhibitor gene therapy alone. In LN229 cells, combination treatment with 20 μmol/L of the miR-21 inhibitor reduced the IC50 of taxol from 820 to 160 nmol/L . It is worth noting that the miR-21 inhibitor additively interacted with taxol on U251cells and synergistically on LN229 cells.

Taxol treatment also increased the percentage of apoptotic cancer cells in miR-21 inhibitor transfected cells compared with control cells. Furthermore, treatment of the miR-21 inhibitor-transfected cells with the anti-cancer drugs taxol resulted in significantly reduced cell viability and invasiveness compared with control cells. These results indicated that the miR-21 plays an important role in the resistance of brain cancer cells to chemotherapeutic drugs. Therefore, miR-21 inhibitor gene therapy combined with taxol chemotherapy might represent a promising novel therapeutic approach for the treatment of glioblastoma.

Thus, the miR-21 inhibitor might interrupt the activity of EGFR pathways, independently of PTEN status. Meanwhile, the expression of STAT3 and p-STAT3 decreased to relatively low levels after miR-21 inhibitor and taxol treatment. The data strongly suggested that a regulatory loop between miR-21 and STAT3 might provide an insight into the mechanism of modulating EGFR/STAT3 signaling [25].
