**2. Nanobased cancer treatment strategies**

Most neoplasm's are derived from multiple mutations and rarely can be controlled through the targeting of a single mutation. The efficacy of gene therapy in tumor treatment will undoubtedly rely upon the simultaneous targeting of multiple cellular processes or the ability to invoke various antitumor responses. Current clinical trials in cancer exploit a variety of different treatment approaches. Tumor suppressor modalities compensate for a genetic mutation via gene transfer or replacement of an altered tumor suppressor gene (e.g. *p53, BRCA1*). Molecular chemotherapy, involves the transfer of a suicide gene (e.g. Herpes Simplex Virus-thymidine kinase [*HSV-tk*], CD::upp) targeted to specific tissues by extracellular tumor/tissue targeting strategies, and/or via tissue-specific expression. The delivered gene then makes the cell susceptible to a prodrug (e.g. gancyclovir, 5-FC) making tumor-specific expression critical. Tumor immunotherapy involves the gene transfer of cytokines (e.g. IL-2, IL-12) that impart antitumor immunomodulatory properties. Oncofactor inhibition strategies such as growth factor inhibition and oncogene inhibition (e.g. erb-B2 silencing) aim to prevent tumor progression by inhibiting key growth factors. Antiangiogenesis therapy seeks to destroy the vasculature supplying the tumor in the hopes of starving it of essential nutrients to diminish or prevent its progression. Multi Drug Resistance associated genes strategies, involve knocking down gene associated with or conferring MDR, such as *PRP-4* and *survivin* to improve chemosensitivity. We will examine in detail each of these strategies.

#### **2.1 Tumor suppressor gene therapy**

As a critical player in cancer onset, *p53* has come to the forefront of oncological research and is now recognized to be the single most frequently inactivated gene in human cancers (Ning et al. 2011;Olivier et al. 2002;Olivier et al. 2010). The *p53* gene enforces a variety of anticancer functions by encouraging cells to arrest or die in the face of DNA damage, hypoxia, oxidative stress, excessive mitogenic stimuli or denuded telomers. In addition, the protein influences several biological functions such as involvement in cell cycle regulation, programmed cell death, senescence, differentiation and development, transcription, DNA replication, DNA repair and maintenance of genomic stability. Thus, it's not surprising that *p53* is regarded as the "*Guardian of the Genome*" in preventing human neoplasia (Lane 1992). P*53* protein has emerged as a key tumor suppressor protein in cellular stress response pathways. The *p53* gene, mapped to chromosome 17p13.1, consists of 11 exons spanning over 20 kb of DNA encoding a 393 amino acid and 53 kDa nuclear protein. Its structure consists of an acidic N-terminus with a transactivation domain, a hydrophobic central DNAbinding core and a basic C-terminus with regulatory and oligomerization domains (Hainaut and Vahakangas 1997). Although greater than 90% of the isolated mutations in this gene have been localized to the DNA binding site of *p53*, coded by exons 5 to 8; some mutations have also been reported outside the evolutionary conserved regions (Hainaut and Hollstein 2000). The *p53* gene product is a sequence-specific nuclear transcription factor that binds to defined consensus sites within DNA as a tetramer and affects the transcription of its target genes. These target genes are involved in critical cell processes such as:


516 Non-Viral Gene Therapy

specificity and high drug toxicities respectively. The success of nanoparticle delivery systems will ultimately depend on the ability to efficiently deliver the gene of interest and express a therapeutic gene(s) in tumor cells in a targeted manner in order to mitigate toxicity. This chapter examines current existing nanoparticle-based gene therapy approaches

Most neoplasm's are derived from multiple mutations and rarely can be controlled through the targeting of a single mutation. The efficacy of gene therapy in tumor treatment will undoubtedly rely upon the simultaneous targeting of multiple cellular processes or the ability to invoke various antitumor responses. Current clinical trials in cancer exploit a variety of different treatment approaches. Tumor suppressor modalities compensate for a genetic mutation via gene transfer or replacement of an altered tumor suppressor gene (e.g. *p53, BRCA1*). Molecular chemotherapy, involves the transfer of a suicide gene (e.g. Herpes Simplex Virus-thymidine kinase [*HSV-tk*], CD::upp) targeted to specific tissues by extracellular tumor/tissue targeting strategies, and/or via tissue-specific expression. The delivered gene then makes the cell susceptible to a prodrug (e.g. gancyclovir, 5-FC) making tumor-specific expression critical. Tumor immunotherapy involves the gene transfer of cytokines (e.g. IL-2, IL-12) that impart antitumor immunomodulatory properties. Oncofactor inhibition strategies such as growth factor inhibition and oncogene inhibition (e.g. erb-B2 silencing) aim to prevent tumor progression by inhibiting key growth factors. Antiangiogenesis therapy seeks to destroy the vasculature supplying the tumor in the hopes of starving it of essential nutrients to diminish or prevent its progression. Multi Drug Resistance associated genes strategies, involve knocking down gene associated with or conferring MDR, such as *PRP-4* and *survivin* to improve chemosensitivity. We will examine

As a critical player in cancer onset, *p53* has come to the forefront of oncological research and is now recognized to be the single most frequently inactivated gene in human cancers (Ning et al. 2011;Olivier et al. 2002;Olivier et al. 2010). The *p53* gene enforces a variety of anticancer functions by encouraging cells to arrest or die in the face of DNA damage, hypoxia, oxidative stress, excessive mitogenic stimuli or denuded telomers. In addition, the protein influences several biological functions such as involvement in cell cycle regulation, programmed cell death, senescence, differentiation and development, transcription, DNA replication, DNA repair and maintenance of genomic stability. Thus, it's not surprising that *p53* is regarded as the "*Guardian of the Genome*" in preventing human neoplasia (Lane 1992). P*53* protein has emerged as a key tumor suppressor protein in cellular stress response pathways. The *p53* gene, mapped to chromosome 17p13.1, consists of 11 exons spanning over 20 kb of DNA encoding a 393 amino acid and 53 kDa nuclear protein. Its structure consists of an acidic N-terminus with a transactivation domain, a hydrophobic central DNAbinding core and a basic C-terminus with regulatory and oligomerization domains (Hainaut and Vahakangas 1997). Although greater than 90% of the isolated mutations in this gene have been localized to the DNA binding site of *p53*, coded by exons 5 to 8; some mutations have also been reported outside the evolutionary conserved regions (Hainaut and Hollstein

to cancer treatment, and assesses their therapeutic utility.

**2. Nanobased cancer treatment strategies** 

in detail each of these strategies.

**2.1 Tumor suppressor gene therapy** 

3. Apoptosis: Bax, Bcl-xL, Fas, FasL, DR5/Killer, Apaf-1, Puma and Noxa.

The loss of *p53* function is of relevance to a broad array of cancer types, including 15–50% of breast cancer cases, 25–70% of metastatic prostate cancers, 25–75% of lung cancers, 33–100% of head and neck cancers and 60-80% advanced ovarian cancers (Ruley, 1996). Furthermore, the null mutation of the gene imparts strongly unfavorable prognosis when associated within human ovarian, lung, colon and breast cancer cases. *P53* also represses genes involved in tumor angiogenesis, and recent evidence suggests that tumor cells possessing a wild-type *p53* allele are more sensitive to chemotherapeutic agents and radiation than *p53* null mutants (Lowe et al. 1994). The pleiotropic abnormalities imparted by deficient *p53* in a significant fraction of human cancers make it one of the primary candidates for cancer gene therapy, whereby the effective expression or replacement of *p53* may re-establish cell growth control, restore appropriate responses to DNA-damaging agents (e.g. chemotherapy and radiotherapy) and preclude tumor angiogenesis. In human neoplasia's that express the wild-type protein, aberrations of p53 regulators, such as MDM2, account for p53 inhibition. For this reason, improved understanding of the p53 pathway should lead to better diagnosis and treatment of cancer in the future.

The *p53* gene therapeutic, Gendicine, is currently approved in China and its US counterpart, Advexin, has shown activity in number of clinical trials. In more conventional approaches a range of small drug like molecules targeting the *p53* mediated system have been developed and several are now in clinical trials. Of critical importance has been the development of small-molecule inhibitors of the p53–Mdm2 protein interaction such as the Nutlins (Vassilev 2004), which have shown activity against human xenografts in preclinical models. Therefore, designing small-molecule inhibitors of the *p53*-MDM2 protein-protein interaction is a promising strategy for the treatment of cancers retaining wild-type *p53* (Lauria et al. 2010). Advanced structural approaches have provided compelling support for the idea that some *p53* mutants can be targets for small molecules that would cause them to regain wildtype function (Joerger et al. 2006). Many adenoviral vectors with cancer specific conditional replication properties have been preclinically evaluated to date. Most promising among these are: i) the Ad dl1520 that possesses a deletion of the 55-kd E1B gene limiting growth to only *p53*-deficient tumor cells. This strategy has shown preclinical efficacy against *p53* deficient nude mouse-human ovarian carcinomatosis xenografts (Vasey et al. 2002); and ii) the integrin-targeted Ad5-D24RGD and serotype 3 receptor-targeted Ad5/3-D24 that possesses a 24 bp deletion in the retinoblastoma binding site of E1A, conferring selective replication in cancer cells that are deficient in the Rb/p16 pathway.

#### **2.2 Molecular chemotherapy**

Suicide gene therapy/molecular chemotherapy is a new experimental form of cancer chemotherapy that is currently being evaluated in human trials (Bhaumik 2011; Onion et al. 2009;Xu et al. 2009). This approach involves intra-tumoral delivery of genes encoding enzymes that convert nontoxic prodrugs into toxic anti-metabolites. Two suicide genes that are being evaluated in the clinic are the *Escherichia coli* CD (*codA* cytosine deaminase) and

Nanomedicine Based Approaches to Cancer Diagonsis and Therapy 519

considerable promise for future immunotherapy protocols (Weiss et al. 2007). Recent studies using animal models have shown that genetically modified tumor cells expressing cytokines such as IL-2, IL-4, IL-6, IFN-γ or GM-CSF were capable of inducing an immune response against preexisting tumors (Connor et al. 1993). In preclinical models and clinical trials, cytokines have been delivered to melanoma lesions by intratumoral injection of naked DNA, recombinant viral vectors and transduced tumor cells and fibroblasts (Sun et al. 1998). Initial clinical trials evaluating the systemic delivery of IL-2 and TNF-α were complicated by significant systemic toxicity (Rosenberg 1999), thereby generating interest in gene transfer strategies that limit cytokine production to the tumor milieu. Preclinical studies employing TAAs to generate tumor vaccines have been promising, and seem to suggest that immunization with recombinant, virus-encoding TAAs can confer suppression of growth in pre-established tumors and the production of specific cytotoxic T lymphocyte responses.

The cytokine IFN-γ has been shown to up-regulate MHC expression, antigen processing, antigen processing and presentation expression in many cell populations, including dendritic cells, B cells, macrophages, and endothelial cells, producing more potent antigen presenting cells (APCs), as IFN-γ can induce multiple gene expressions that are related to MHC processing and presentation. Dendritic cells (DCs) play a vital role in the initiation of immune response as professional antigen presenting cells to T cells promoting CD8+ T-cellmediated cytotoxic responses. Reported research in animal studies indicates that vaccine immunity may represent a promising alternative therapy for cancer patients. However, broad clinical utility has yet to be achieved, owing to the low transfection efficiency of DCs (Chen et al. 2010b). Different formulations of liposomes have been designed to improve the uptake by DCs through different receptor-mediated routes. These formulations include liposomes prepared with mannosylated phosphatidylethanolamine (Man-PE), trimethyl ammonium propane, and phosphatidylserine targeted to mannose receptor (MR), negatively charged surface proteins and phosphatidylserine receptor (PSR) of DCs, respectively. Several other immunomodulatory clinical trials are under way with cytokines currently comprising 18.3% of immunotherapy clinical trials (http://www.wiley.com/ legacy/wileychi/genmed/clinical/). Table 1 provides a list of various types of genes currently involved in various gene therapy clinical trials. A number of other monoclonal antibodies; namely EC90, Removab, and CNTO-95 which target FOLR1, EpCAM and αintegrin (CD51), respectively are in Phase I/II studies, and indicate that further clinical investigation in this area is warranted (Bell-McGuinn et al. 2011;Kowalski et al. 2010).

The epidermal growth factor receptor (EGFR) belongs to the proto-oncogene family, which consists of four structurally-related transmembrane receptors (i.e., EGFR, ErbB2, ErbB3, and ErbB4) and is a key therapeutic target in the treatment of many types of cancer. The most extensively studied growth factors are ErbB1 (Javle et al. 2010) and ErbB2 (Nihira 2003). These ErbB/EGF receptors are tyrosine kinases and play important physiologic roles in cell proliferation, survival, adhesion, motility, invasion, and angiogenesis. High expression of EGF receptor protein is observed in several types of cancer including breast, bladder, colon, lung and gastric cancers, making them potential targets for targeted therapies, which represent some of the most successful therapeutic approaches to date (Iqbal et al. 2011;Lai et

However, conversion to clinical trials has failed to show significant efficacy.

**2.4 Oncofactor inhibition strategies 2.4.1 Growth factor inhibition** 

al. 2009;Yarom and Jonker 2011).

HSV-1 *tk* (thymidine kinase) genes, which confer sensitivity to 5-FC (5-flourocytosine) and GCV , respectively. The rationale behind the suicide gene therapy approach is that after "targeted" transfer of these genes to the tumor, only tumor and neighboring cells will be rendered sensitive to their cytotoxic action. A variety of tumor models have demonstrated that both the CD/5-FC (by inhibiting thymidylate synthase) and HSV-1 *tk*/GCV prodrug systems (by inhibiting DNA chain elongation) can enhance the efficacy of radiation therapy, resulting in significantly better tumor control and/or cure (Rogulski et al., 1997). Suicide gene therapy is a particularly attractive approach to the treatment of cancer as it is essentially a tumor-targeted chemotherapy. As such, the systemic toxicity commonly associated with, and a major limitation of, conventional chemotherapy is avoided. Using a pair of adenoviral vectors that express a *CD*/HSV-1 *tk* fusion gene without or with the wt human *p53* gene, it has been found that co-expression of *p53* did not enhance the cytotoxicity of *CD*/5-FC or HSV-1 *tk*/GCV suicide gene therapies using the SK-OV-3 and Hep3B tumor models *in vitro* or *in vivo* (Xie et al., 1999). This notion is consistent with the fact that *CD*/5- FC and HSV-1 *tk*/GCV suicide gene therapies have demonstrated effectiveness against a variety of tumors that lack functional *p53* (e.g., SK-OV-3, Hep3B, 9L, WiDr, U251, DU145, PC-3, C33A, and many others). Azatian et al. (Azatian et al. 2009) investigated the effectiveness of HSV-*tk* activation as gene therapy for gastroesophageal junction and gastric adenocarcinomas using a stress-inducible Grp78 promoter. The gastric adenocarcinoma cell line MKN-74/*tk* cells were completely killed when cultured with 1 µg/ml GCV for 10 days. Cell viability was also significantly lower under glucose starvation conditions when HSV*-tk* expression was regulated by the Grp78 promoter . Furthermore, non-viral approaches are being investigated for use in combination with suicide gene therapy for treatment of various carcinomas. Using transferrin lipoplexes, prepared from cationic liposomes and cholesterol, significant tumor reduction was achieved upon intratumoral delivery of HSV-*tk* or *CD* genes, followed by intraperitoneal injection of GCV or 5-FC, respectively (Neves et al. 2009). Enhanced apoptosis, the recruitment of NK cells, CD4 and CD8 T-lymphocytes and an increase in the levels of several cytokines/chemokines were observed within the tumors. These observations suggest that suicide gene therapy with lipoplexes modifies the tumor microenvironment, and leads to the recruitment of immune effector cells that can act as adjuvants in reducing the tumor size.

#### **2.3 Immunomodulatory strategies**

Tumor cells alter antigen presentation on their surface compromising recognition by the immune system and immunosurveillance evasion. Tumor immunotherapy strategies aim to boost the anti-tumor immune response by stimulating the cell-mediated arm of the immune system (Wojtowicz-Praga 1997). Gene therapy approaches to enhance immune responses include delivery of cytokines to the tumor, administration of tumor vaccines based on tumor-associated antigens (TAAs), upregulation of Major Histocompatibitlity Complexes (MHC)(Nabel et al. 1993) or co-stimulatory molecules, and inhibition of immunosuppressive molecules. Many cytokines activate the immune system, including interleukins (IL) 2, 4, 7, 12 and 18, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), and granulocyte– macrophage colony-stimulating factor (GM-CSF), which are among the most potent inducers of anti-tumor activity in a variety of preclinical studies. More recently, some exciting new cytokines have been characterized, such as IL-21, IL-23, IL-27, and their immunomodulatory and antitumor effects *in vitro* and *in vivo* suggest that they may have

HSV-1 *tk* (thymidine kinase) genes, which confer sensitivity to 5-FC (5-flourocytosine) and GCV , respectively. The rationale behind the suicide gene therapy approach is that after "targeted" transfer of these genes to the tumor, only tumor and neighboring cells will be rendered sensitive to their cytotoxic action. A variety of tumor models have demonstrated that both the CD/5-FC (by inhibiting thymidylate synthase) and HSV-1 *tk*/GCV prodrug systems (by inhibiting DNA chain elongation) can enhance the efficacy of radiation therapy, resulting in significantly better tumor control and/or cure (Rogulski et al., 1997). Suicide gene therapy is a particularly attractive approach to the treatment of cancer as it is essentially a tumor-targeted chemotherapy. As such, the systemic toxicity commonly associated with, and a major limitation of, conventional chemotherapy is avoided. Using a pair of adenoviral vectors that express a *CD*/HSV-1 *tk* fusion gene without or with the wt human *p53* gene, it has been found that co-expression of *p53* did not enhance the cytotoxicity of *CD*/5-FC or HSV-1 *tk*/GCV suicide gene therapies using the SK-OV-3 and Hep3B tumor models *in vitro* or *in vivo* (Xie et al., 1999). This notion is consistent with the fact that *CD*/5- FC and HSV-1 *tk*/GCV suicide gene therapies have demonstrated effectiveness against a variety of tumors that lack functional *p53* (e.g., SK-OV-3, Hep3B, 9L, WiDr, U251, DU145, PC-3, C33A, and many others). Azatian et al. (Azatian et al. 2009) investigated the effectiveness of HSV-*tk* activation as gene therapy for gastroesophageal junction and gastric adenocarcinomas using a stress-inducible Grp78 promoter. The gastric adenocarcinoma cell line MKN-74/*tk* cells were completely killed when cultured with 1 µg/ml GCV for 10 days. Cell viability was also significantly lower under glucose starvation conditions when HSV*-tk* expression was regulated by the Grp78 promoter . Furthermore, non-viral approaches are being investigated for use in combination with suicide gene therapy for treatment of various carcinomas. Using transferrin lipoplexes, prepared from cationic liposomes and cholesterol, significant tumor reduction was achieved upon intratumoral delivery of HSV-*tk* or *CD* genes, followed by intraperitoneal injection of GCV or 5-FC, respectively (Neves et al. 2009). Enhanced apoptosis, the recruitment of NK cells, CD4 and CD8 T-lymphocytes and an increase in the levels of several cytokines/chemokines were observed within the tumors. These observations suggest that suicide gene therapy with lipoplexes modifies the tumor microenvironment, and leads to the recruitment of immune effector cells that can act as

Tumor cells alter antigen presentation on their surface compromising recognition by the immune system and immunosurveillance evasion. Tumor immunotherapy strategies aim to boost the anti-tumor immune response by stimulating the cell-mediated arm of the immune system (Wojtowicz-Praga 1997). Gene therapy approaches to enhance immune responses include delivery of cytokines to the tumor, administration of tumor vaccines based on tumor-associated antigens (TAAs), upregulation of Major Histocompatibitlity Complexes (MHC)(Nabel et al. 1993) or co-stimulatory molecules, and inhibition of immunosuppressive molecules. Many cytokines activate the immune system, including interleukins (IL) 2, 4, 7, 12 and 18, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), and granulocyte– macrophage colony-stimulating factor (GM-CSF), which are among the most potent inducers of anti-tumor activity in a variety of preclinical studies. More recently, some exciting new cytokines have been characterized, such as IL-21, IL-23, IL-27, and their immunomodulatory and antitumor effects *in vitro* and *in vivo* suggest that they may have

adjuvants in reducing the tumor size.

**2.3 Immunomodulatory strategies** 

considerable promise for future immunotherapy protocols (Weiss et al. 2007). Recent studies using animal models have shown that genetically modified tumor cells expressing cytokines such as IL-2, IL-4, IL-6, IFN-γ or GM-CSF were capable of inducing an immune response against preexisting tumors (Connor et al. 1993). In preclinical models and clinical trials, cytokines have been delivered to melanoma lesions by intratumoral injection of naked DNA, recombinant viral vectors and transduced tumor cells and fibroblasts (Sun et al. 1998). Initial clinical trials evaluating the systemic delivery of IL-2 and TNF-α were complicated by significant systemic toxicity (Rosenberg 1999), thereby generating interest in gene transfer strategies that limit cytokine production to the tumor milieu. Preclinical studies employing TAAs to generate tumor vaccines have been promising, and seem to suggest that immunization with recombinant, virus-encoding TAAs can confer suppression of growth in pre-established tumors and the production of specific cytotoxic T lymphocyte responses. However, conversion to clinical trials has failed to show significant efficacy.

The cytokine IFN-γ has been shown to up-regulate MHC expression, antigen processing, antigen processing and presentation expression in many cell populations, including dendritic cells, B cells, macrophages, and endothelial cells, producing more potent antigen presenting cells (APCs), as IFN-γ can induce multiple gene expressions that are related to MHC processing and presentation. Dendritic cells (DCs) play a vital role in the initiation of immune response as professional antigen presenting cells to T cells promoting CD8+ T-cellmediated cytotoxic responses. Reported research in animal studies indicates that vaccine immunity may represent a promising alternative therapy for cancer patients. However, broad clinical utility has yet to be achieved, owing to the low transfection efficiency of DCs (Chen et al. 2010b). Different formulations of liposomes have been designed to improve the uptake by DCs through different receptor-mediated routes. These formulations include liposomes prepared with mannosylated phosphatidylethanolamine (Man-PE), trimethyl ammonium propane, and phosphatidylserine targeted to mannose receptor (MR), negatively charged surface proteins and phosphatidylserine receptor (PSR) of DCs, respectively. Several other immunomodulatory clinical trials are under way with cytokines currently comprising 18.3% of immunotherapy clinical trials (http://www.wiley.com/ legacy/wileychi/genmed/clinical/). Table 1 provides a list of various types of genes currently involved in various gene therapy clinical trials. A number of other monoclonal antibodies; namely EC90, Removab, and CNTO-95 which target FOLR1, EpCAM and αintegrin (CD51), respectively are in Phase I/II studies, and indicate that further clinical investigation in this area is warranted (Bell-McGuinn et al. 2011;Kowalski et al. 2010).

#### **2.4 Oncofactor inhibition strategies**

#### **2.4.1 Growth factor inhibition**

The epidermal growth factor receptor (EGFR) belongs to the proto-oncogene family, which consists of four structurally-related transmembrane receptors (i.e., EGFR, ErbB2, ErbB3, and ErbB4) and is a key therapeutic target in the treatment of many types of cancer. The most extensively studied growth factors are ErbB1 (Javle et al. 2010) and ErbB2 (Nihira 2003). These ErbB/EGF receptors are tyrosine kinases and play important physiologic roles in cell proliferation, survival, adhesion, motility, invasion, and angiogenesis. High expression of EGF receptor protein is observed in several types of cancer including breast, bladder, colon, lung and gastric cancers, making them potential targets for targeted therapies, which represent some of the most successful therapeutic approaches to date (Iqbal et al. 2011;Lai et al. 2009;Yarom and Jonker 2011).

Nanomedicine Based Approaches to Cancer Diagonsis and Therapy 521

study S0413, a phase II trial of lapatinib was conducted as first-line therapy in patients with advanced or metastatic gastric cancer**.** Median (95% CI) time to treatment failure was 1.9 (1.6-3.1) months and overall survival (OS) was 4.8 (3.2-7.4) months. Lapatinib is well tolerated, with modest single-agent activity in advanced/metastatic gastric cancer patients. Potential molecular correlatives such as HER2, interleukin (IL)-8 and genomic polymorphisms IL-8, and vascular endothelial growth factor correlated with OS (Iqbal et al. 2011). Similarly, in other Phase II studies with single agent Gefitinib or Erlotinib only modest activity was observed. Gefitinib cytotoxic combinations are also currently under examination (Wagner et al. 2007). Further, very recently it was observed that direct covalent coupling of antibodies to glutaraldehyde activated nanoparticles is an appropriate method to achieve cell-type specific drug carrier systems based on polymeric nanoparticles that have potential to be applied for targeted chemotherapy in EGFR

Trastuzumab (trade name Herceptin) is the first anticancer drug generated by Genentech, Inc. whose use as a treatment for breast cancer patients is decided based on the status of the HER2 gene amplification/HER2 protein over-expression. The development and standardization of an HER2 test was a key strategy in clinical development of this drug, since appropriate selection of patients with HER2 over-expression was critical for success. This also highlights the important and evolving era of personalized and targeted therapies. In the clinic, the success of imatinib (Gleevec®, STI571) and trastuzumab, both firsts of their kind, spurred further development of new, second-generation drugs that target kinases in cancer. Further, these targeted drugs combined with chemotherapeutic drugs are found effective and well tolerated in nanoparticle based drug delivery (e.g. albumin-bound paclitaxel combined with carboplatin and trastuzumab or as trastuzumab-dextran iron

Phosphoinositide 3-kinase (PI3K) is a major signaling component downstream of growth factor receptor tyrosine kinases (RTKs). PI3K catalyzes the production of the lipid second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3) at the cell membrane. PIP3 in turn contributes to the recruitment and activation of a wide range of downstream targets, including the serine-threonine protein kinase Akt (also known as protein kinase B). The PI3K-Akt-mTOR signaling pathway regulates many normal cellular processes including cell proliferation, survival, growth, and motility—processes that are critical for tumorigenesis. Components of this pathway are frequently abnormal in a variety of tumors, making them an attractive target for anti-cancer therapy. In contrast to *p53* and other tumor-suppressor pathways, the PI3K pathway is activated in cancer, making this an optimal target for therapy as it is easier to inhibit activation events than to replace lost tumor-suppressor function. Studies have demonstrated that phosphorylative activation via the mTOR pathway results in increased G1 cell cycle progression, cell survival, and tumor cell proliferation (Campbell et al. 2004;Vega et al. 2006). The mTOR pathway has been shown in preclinical studies to play a role in the carcinogenesis of breast, ovary and prostate as well as gastrointestinal malignancies. Novel analogs of rapamycin (temsirolimus, everolimus, and deforolimus), which have improved pharmaceutical properties, have been designed for oncology indications. Clinical trials of these analogs have already validated the importance of mTOR inhibition as a novel treatment strategy

oxide nanoparticles) for the treatment of cancer (Conlin et al. 2010).

**2.4.2 Signal transduction (P13K/AKT/mTOR pathway) inhibition** 

for several malignancies (Gibbons et al. 2009).

positive cancer (Aggarwal et al. 2011).


Table 1. List of various types of genes presently involved in various gene therapy clinical trials (Source: http://www.wiley.com/legacy/wileychi/genmed/clinical/).

Gefitinib and Erlotinib are small molecules that exert their function by inhibiting the intracellular tyrosine kinase domain of EGFR. Recently, the southwest oncology group

Adhesion molecule 10 0.6 Antigen 334 20.7 Antisense 13 0.8 Cell cycle 8 0.5 Cell protection/Drug resistance 19 1.1 Cytokine 317 18.5 Deficiency 136 7.9 Growth factor 128 7.5 Hormone 8 0.5 Marker 54 3.2 Oncogene regulator 11 0.6 Oncolytic virus 37 2.2

Receptor 113 6.6 Replication inhibitor 74 4.3 Ribozyme 6 0.4 siRNA 11 0.6 Suicide 144 8.4 Transcription factor 28 1.6 Tumor suppressor 150 8.8 Viral vaccine 6 0.4 Others 26 1.5 Unknown 49 2.9

Total 1714

trials (Source: http://www.wiley.com/legacy/wileychi/genmed/clinical/).

Table 1. List of various types of genes presently involved in various gene therapy clinical

Gefitinib and Erlotinib are small molecules that exert their function by inhibiting the intracellular tyrosine kinase domain of EGFR. Recently, the southwest oncology group

**Gene Therapy Clinical Trials Number %** 

12 0.7

**Gene type** 

Porins, ion channels,

transporters

study S0413, a phase II trial of lapatinib was conducted as first-line therapy in patients with advanced or metastatic gastric cancer**.** Median (95% CI) time to treatment failure was 1.9 (1.6-3.1) months and overall survival (OS) was 4.8 (3.2-7.4) months. Lapatinib is well tolerated, with modest single-agent activity in advanced/metastatic gastric cancer patients. Potential molecular correlatives such as HER2, interleukin (IL)-8 and genomic polymorphisms IL-8, and vascular endothelial growth factor correlated with OS (Iqbal et al. 2011). Similarly, in other Phase II studies with single agent Gefitinib or Erlotinib only modest activity was observed. Gefitinib cytotoxic combinations are also currently under examination (Wagner et al. 2007). Further, very recently it was observed that direct covalent coupling of antibodies to glutaraldehyde activated nanoparticles is an appropriate method to achieve cell-type specific drug carrier systems based on polymeric nanoparticles that have potential to be applied for targeted chemotherapy in EGFR positive cancer (Aggarwal et al. 2011).

Trastuzumab (trade name Herceptin) is the first anticancer drug generated by Genentech, Inc. whose use as a treatment for breast cancer patients is decided based on the status of the HER2 gene amplification/HER2 protein over-expression. The development and standardization of an HER2 test was a key strategy in clinical development of this drug, since appropriate selection of patients with HER2 over-expression was critical for success. This also highlights the important and evolving era of personalized and targeted therapies. In the clinic, the success of imatinib (Gleevec®, STI571) and trastuzumab, both firsts of their kind, spurred further development of new, second-generation drugs that target kinases in cancer. Further, these targeted drugs combined with chemotherapeutic drugs are found effective and well tolerated in nanoparticle based drug delivery (e.g. albumin-bound paclitaxel combined with carboplatin and trastuzumab or as trastuzumab-dextran iron oxide nanoparticles) for the treatment of cancer (Conlin et al. 2010).

#### **2.4.2 Signal transduction (P13K/AKT/mTOR pathway) inhibition**

Phosphoinositide 3-kinase (PI3K) is a major signaling component downstream of growth factor receptor tyrosine kinases (RTKs). PI3K catalyzes the production of the lipid second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3) at the cell membrane. PIP3 in turn contributes to the recruitment and activation of a wide range of downstream targets, including the serine-threonine protein kinase Akt (also known as protein kinase B). The PI3K-Akt-mTOR signaling pathway regulates many normal cellular processes including cell proliferation, survival, growth, and motility—processes that are critical for tumorigenesis. Components of this pathway are frequently abnormal in a variety of tumors, making them an attractive target for anti-cancer therapy. In contrast to *p53* and other tumor-suppressor pathways, the PI3K pathway is activated in cancer, making this an optimal target for therapy as it is easier to inhibit activation events than to replace lost tumor-suppressor function. Studies have demonstrated that phosphorylative activation via the mTOR pathway results in increased G1 cell cycle progression, cell survival, and tumor cell proliferation (Campbell et al. 2004;Vega et al. 2006). The mTOR pathway has been shown in preclinical studies to play a role in the carcinogenesis of breast, ovary and prostate as well as gastrointestinal malignancies. Novel analogs of rapamycin (temsirolimus, everolimus, and deforolimus), which have improved pharmaceutical properties, have been designed for oncology indications. Clinical trials of these analogs have already validated the importance of mTOR inhibition as a novel treatment strategy for several malignancies (Gibbons et al. 2009).

Nanomedicine Based Approaches to Cancer Diagonsis and Therapy 523

cycloaddition known as a "click" reaction. Nanoparticles were found to be spherical, dense, monodisperse and stable. No cytotoxicity was observed after four days of incubation demonstrating the biocompatibility of nanoparticles. Fluorescence highlighted the specific interaction of these functionalized nanoparticles for VEGF receptors, suggesting that the targeting peptide bioactivity was retained (Deshayes et al. 2011). Therapies based on mAb targeting and blocking of vascular endothelial growth factor, such as that conferred by the monoclonal bevacizumAb, have clearly demonstrated remarkable antitumor efficacy, although the mechanism of action here is not well understood. Combined treatment of bevacizumAb with Sorafenib (a small molecular inhibitor of several tyrosine protein kinases such as VEGFR and PDGFR) resulted in partial response or disease stabilization for ≥ 4 months (median, 6 months; range, 4 to 22+ months) in 22 (59%) of 37 assessable patients

Matrix metalloproteases (MMPs) are a family of more than 20 zinc and calcium dependent enzymes that degrade all major basement membrane and extracellular matrix components. These enzymes also promote tumor invasion and metastasis, regulating host defense mechanisms and normal cell function. They are well adapted to serve as signaling conduits in the tumor-stromal microenvironment given their crucial roles in the complex processes of tissue invasion, blood vessel homeostasis, and metastasis. Specifically, MMP-1, MMP-2 and MMP-9 mRNA expression in multivariate analyses has been correlated with a wellcharacterized clinical significance in solid tumors but have no such role in effusions, suggesting the clinical role of MMP is limited to solid lesions. MMP inhibitors (MMPIs) are expected to be useful for the treatment of diseases such as cancer, osteoarthritis, and rheumatoid arthritis. A vast number of MMPIs have been developed in recent years. With the failure of these inhibitors in clinical trials, more efforts have been directed to the design

Cancer cells counter the influx of a chemotherapeutic by draining the drug from cells, deactivating the protein that transports the drug across cell walls, restoring DNA breaks, or developing some other mechanism to deactivate the drug. Multidrug resistance continues to be a major problem in the management of cancer and knockdown of drug resistance associated genes such as MDR1 (ABCB1), survivin, and pre-mRNA processing factor-4 (PRP-4) has been attempted in cancer cell lines. PRP-4 belongs to the serine/threonine protein kinase family, plays a role in pre-mRNA splicing and cell mitosis whereas survivin, a member of the inhibitor of apoptosis family of proteins, is implicated in both apoptosis inhibition and cell cycle control. Recently, Chen et al., (2010) developed two nanoparticle formulations, cationic liposome-polycation-DNA (Whitmore et al. 1999) and anionic liposome-polycation-DNA (LPD-II), for systemic co-delivery of doxorubicin (Dox) and a therapeutic small interfering RNA (siRNA) to multiple drug resistance (Hesdorffer et al. 1998) tumors. In this study, they have provided four strategies to overcome drug resistance. First, the investigators formed the LPD nanoparticles with a guanidinium-containing cationic lipid, i.e. N,N-distearyl-N-methyl-N-2-(N'-arginyl) aminoethyl ammonium chloride, which can induce reactive oxygen species, down-regulate MDR transporter expression, and increase Dox uptake. Second, to block angiogenesis and increase drug penetration, the authors have further formulated LPD nanoparticles to co-deliver vascular endothelial growth factor siRNA and Dox. An enhanced Dox uptake and a therapeutic effect were observed when combined with vascular endothelial growth factor siRNA in the

of specific inhibitors with different Zn-binding groups (Tu et al. 2008).

**2.6 Multidrug resistance inhibition/gene transfer strategies** 

with advanced solid tumors .

#### **2.4.3 Oncogene inhibition**

Gene therapy has also been employed in a reversed approach, namely by inhibition of oncogenes. The use of vectors to express anti-sense (ODN), ribozymes or small interfering RNAs (siRNA) to silence a variety of oncogenes has enabled genetic loss-of-function studies in tissue culture systems. Gene expression can be disrupted at the transcriptional (triplex DNA) or translational (antisense DNA or short inference RNA) level (Braasch et al. 2002). In the triplex DNA-based antigene approach, transcription is disrupted by the binding of a triplex-forming oligonucleotide at the promoter region of a target gene. In the antisense strategy, the oligonucleotide (ON) molecule corresponding to a target gene is delivered inside a cell where it binds to its complementary, targeted messenger RNA (mRNA), to produce a partially double-stranded ON/mRNA complex. Extensive work in this area has already led to one antisense ON product approved for local therapy of cytomegalovirus retinitis (Vitravene) and nearly twenty others in late-stage clinical trials (Cheung et al. 2010).

Silencing of specific mRNA using double stranded RNA oligonucleotides represents one of the newest technologies for suppressing a specific gene product. Small interfering RNA (siRNA) are 21 nucleotides long, double stranded RNA fragments that are identical in sequence to the target mRNA. Silencing of specific gene targets, such as cancer-associated mutations in oncogenes and their amplification in tumors therefore represents promising therapeutic approach in treating cancer, and strategies focusing on classic oncogenes (such as *bcl-2*, *ras* and *HER-2/neu)* have been extensively explored in esophageal, breast, ovarian and other carcinomas. Other candidate targets may include genes associated with cell proliferation, metastasis, angiogenesis, and drug resistance. Very recently, Liang et al., (2011) showed that DNA vector-based Stat3-specific RNA interference (si-Stat3) blocks Stat3 signalling which was delivered by hydroxyapatite nanoparticles and suppresses mouse prostate tumor growth *in vivo*. In this study, the Stat3 downstream genes Bcl-2, VEGF and cyclin D1 were also strongly downregulated in the tumor tissues that also displayed significant increases in Bax expression and Caspase3 activity (Liang et al. 2011). While the application of antisense technology to the treatment of human cancer is conceptually straightforward, selection of appropriate gene targets is an important parameter in the potential success of siRNA cancer therapies (Ashihara 2010), and in practice there are many complicated, mechanistically based questions that must be considered. Importantly, folding of target RNAs or their association with specific proteins in the cell often prevents the ON molecules from binding to their targets. In addition, silencing of such genes must not affect the functions of normal cells.

#### **2.5 Anti-angiogenic therapy**

The ability of solid tumors to grow locally, and subsequently disseminate to distant organs, is dependent upon the formation of new blood vessels (Azad et al. 2008) and involves angiogenic factors such as vascular endothelial growth factor (VEGF). VEGF plays an important role in the proliferation of cancer cells, angiogenesis and vascular permeability in peritoneal dissemination. BevacizumAb, the only United States Food and Drug Administration (FDA)-approved anti-VEGF agent, is a monoclonal antibody that inhibits the binding of VEGF to VEGF receptors. CBO-P11, a cyclo-peptide, has proven to specifically bind to receptors of VEGF and may be used as targeting ligand for tumor angiogenesis. Deshayes et al., (2011) investigated the conjugation of CBO-P11 on the surface of poly(vinylidene fluoride) nanoparticles using the copper(I)-catalyzed Huisgen 1,3-dipolar

Gene therapy has also been employed in a reversed approach, namely by inhibition of oncogenes. The use of vectors to express anti-sense (ODN), ribozymes or small interfering RNAs (siRNA) to silence a variety of oncogenes has enabled genetic loss-of-function studies in tissue culture systems. Gene expression can be disrupted at the transcriptional (triplex DNA) or translational (antisense DNA or short inference RNA) level (Braasch et al. 2002). In the triplex DNA-based antigene approach, transcription is disrupted by the binding of a triplex-forming oligonucleotide at the promoter region of a target gene. In the antisense strategy, the oligonucleotide (ON) molecule corresponding to a target gene is delivered inside a cell where it binds to its complementary, targeted messenger RNA (mRNA), to produce a partially double-stranded ON/mRNA complex. Extensive work in this area has already led to one antisense ON product approved for local therapy of cytomegalovirus retinitis (Vitravene) and nearly twenty others in late-stage clinical trials

Silencing of specific mRNA using double stranded RNA oligonucleotides represents one of the newest technologies for suppressing a specific gene product. Small interfering RNA (siRNA) are 21 nucleotides long, double stranded RNA fragments that are identical in sequence to the target mRNA. Silencing of specific gene targets, such as cancer-associated mutations in oncogenes and their amplification in tumors therefore represents promising therapeutic approach in treating cancer, and strategies focusing on classic oncogenes (such as *bcl-2*, *ras* and *HER-2/neu)* have been extensively explored in esophageal, breast, ovarian and other carcinomas. Other candidate targets may include genes associated with cell proliferation, metastasis, angiogenesis, and drug resistance. Very recently, Liang et al., (2011) showed that DNA vector-based Stat3-specific RNA interference (si-Stat3) blocks Stat3 signalling which was delivered by hydroxyapatite nanoparticles and suppresses mouse prostate tumor growth *in vivo*. In this study, the Stat3 downstream genes Bcl-2, VEGF and cyclin D1 were also strongly downregulated in the tumor tissues that also displayed significant increases in Bax expression and Caspase3 activity (Liang et al. 2011). While the application of antisense technology to the treatment of human cancer is conceptually straightforward, selection of appropriate gene targets is an important parameter in the potential success of siRNA cancer therapies (Ashihara 2010), and in practice there are many complicated, mechanistically based questions that must be considered. Importantly, folding of target RNAs or their association with specific proteins in the cell often prevents the ON molecules from binding to their targets. In addition, silencing of such genes must not affect

The ability of solid tumors to grow locally, and subsequently disseminate to distant organs, is dependent upon the formation of new blood vessels (Azad et al. 2008) and involves angiogenic factors such as vascular endothelial growth factor (VEGF). VEGF plays an important role in the proliferation of cancer cells, angiogenesis and vascular permeability in peritoneal dissemination. BevacizumAb, the only United States Food and Drug Administration (FDA)-approved anti-VEGF agent, is a monoclonal antibody that inhibits the binding of VEGF to VEGF receptors. CBO-P11, a cyclo-peptide, has proven to specifically bind to receptors of VEGF and may be used as targeting ligand for tumor angiogenesis. Deshayes et al., (2011) investigated the conjugation of CBO-P11 on the surface of poly(vinylidene fluoride) nanoparticles using the copper(I)-catalyzed Huisgen 1,3-dipolar

**2.4.3 Oncogene inhibition** 

(Cheung et al. 2010).

the functions of normal cells.

**2.5 Anti-angiogenic therapy** 

cycloaddition known as a "click" reaction. Nanoparticles were found to be spherical, dense, monodisperse and stable. No cytotoxicity was observed after four days of incubation demonstrating the biocompatibility of nanoparticles. Fluorescence highlighted the specific interaction of these functionalized nanoparticles for VEGF receptors, suggesting that the targeting peptide bioactivity was retained (Deshayes et al. 2011). Therapies based on mAb targeting and blocking of vascular endothelial growth factor, such as that conferred by the monoclonal bevacizumAb, have clearly demonstrated remarkable antitumor efficacy, although the mechanism of action here is not well understood. Combined treatment of bevacizumAb with Sorafenib (a small molecular inhibitor of several tyrosine protein kinases such as VEGFR and PDGFR) resulted in partial response or disease stabilization for ≥ 4 months (median, 6 months; range, 4 to 22+ months) in 22 (59%) of 37 assessable patients with advanced solid tumors .

Matrix metalloproteases (MMPs) are a family of more than 20 zinc and calcium dependent enzymes that degrade all major basement membrane and extracellular matrix components. These enzymes also promote tumor invasion and metastasis, regulating host defense mechanisms and normal cell function. They are well adapted to serve as signaling conduits in the tumor-stromal microenvironment given their crucial roles in the complex processes of tissue invasion, blood vessel homeostasis, and metastasis. Specifically, MMP-1, MMP-2 and MMP-9 mRNA expression in multivariate analyses has been correlated with a wellcharacterized clinical significance in solid tumors but have no such role in effusions, suggesting the clinical role of MMP is limited to solid lesions. MMP inhibitors (MMPIs) are expected to be useful for the treatment of diseases such as cancer, osteoarthritis, and rheumatoid arthritis. A vast number of MMPIs have been developed in recent years. With the failure of these inhibitors in clinical trials, more efforts have been directed to the design of specific inhibitors with different Zn-binding groups (Tu et al. 2008).

#### **2.6 Multidrug resistance inhibition/gene transfer strategies**

Cancer cells counter the influx of a chemotherapeutic by draining the drug from cells, deactivating the protein that transports the drug across cell walls, restoring DNA breaks, or developing some other mechanism to deactivate the drug. Multidrug resistance continues to be a major problem in the management of cancer and knockdown of drug resistance associated genes such as MDR1 (ABCB1), survivin, and pre-mRNA processing factor-4 (PRP-4) has been attempted in cancer cell lines. PRP-4 belongs to the serine/threonine protein kinase family, plays a role in pre-mRNA splicing and cell mitosis whereas survivin, a member of the inhibitor of apoptosis family of proteins, is implicated in both apoptosis inhibition and cell cycle control. Recently, Chen et al., (2010) developed two nanoparticle formulations, cationic liposome-polycation-DNA (Whitmore et al. 1999) and anionic liposome-polycation-DNA (LPD-II), for systemic co-delivery of doxorubicin (Dox) and a therapeutic small interfering RNA (siRNA) to multiple drug resistance (Hesdorffer et al. 1998) tumors. In this study, they have provided four strategies to overcome drug resistance. First, the investigators formed the LPD nanoparticles with a guanidinium-containing cationic lipid, i.e. N,N-distearyl-N-methyl-N-2-(N'-arginyl) aminoethyl ammonium chloride, which can induce reactive oxygen species, down-regulate MDR transporter expression, and increase Dox uptake. Second, to block angiogenesis and increase drug penetration, the authors have further formulated LPD nanoparticles to co-deliver vascular endothelial growth factor siRNA and Dox. An enhanced Dox uptake and a therapeutic effect were observed when combined with vascular endothelial growth factor siRNA in the

Nanomedicine Based Approaches to Cancer Diagonsis and Therapy 525

However, despite these advantages, there are currently major limitations to the use of viruses as gene delivery vectors. These include the limited carrying capacity of transgenic materials (i.e. size of plasmid) since the packaging capacity of viral vectors (usually up to 5 kb) is constrained; the potential for oncogenesis due to chromosomal integration or activation of oncogenes/inactivation of oncogene regulators; the generation of infectious viruses due to recombination; and concerns regarding instability that challenge

The potentially hazardous nature of viral vectors has been observed in a number of gene therapy-related patient mortalities in various clinical trials. Patient complications to date have included the rejection of DNA carriers, resulting in immune response that have led to already one death, Jesse Gelsinger, who died in 1999 from a rare metabolic disorder during a gene-therapy clinical trial at the University of Pennsylvania (Branca 2005;Ledford 2007;Stolberg 1999;Wilson 2010). DNA constructs need to be optimized to carry minimal immunogenic components without compromising both efficient and longterm transgene expression. In this context, technology employing minimal immunological defined gene expression technology (MIDGE) represents a promising future alternative to conventional plasmids in terms of biosafety, improved gene transfer, potential bioavailability, minimal size and low immunogenicity associated with these chemically engineered miniDNA vectors. MIDGEs are non-viral, lcc (linear covalently closed) miniplasmids synthesized *in vitro* via a patented chemical modification of linear open (lo). These plasmids confer the advantage of a minimal coding sequence that relieves complications from expression of additional unwanted genes and CpG motifs that have a 20-fold higher occurrence in bacterial cells. MIDGE lcc plasmid transfection expression was reported to increase luciferase transgene expression from 2.5 to 17 fold *ex vivo* compared to circular covalently closed (ccc), isogenic forms in a tissue-dependent manner (Schakowski et al. 2007) . In addition, the mean numbers of MIDGE vector molecules per cell was also found to be significantly higher, suggesting that linear lcc plasmids transfect cells more efficiently. MIDGE technology has already been applied, with promising results, to the development of a Leishmania DNA vaccine and a colon carcinoma treatment. Here, therapy was based on IL-2 delivery to specific tumor cell lines *ex vivo*, and revealed 2 to 4 fold higher relative transgene expression compared to the ccc control (Schakowski et al. 2001). In combination, the existing studies employing lcc DNA systems suggest that lcc DNA is superior to lo DNA in transfection efficiency, and to ccc DNA in transgene expression. Purified TelN (closely related to Tel) was previously reported to generate lcc plasmids *in vitro* in (*pal*-related) *telRL*-dependent manner and was shown to successfully deliver EGFP to human embryonal kidney cells *in vitro,* as well as IL-12 in an untargeted manner to inhibit metastasis formation in B16F10C57BL/6 melanoma model

At present, our laboratory is involved in design, and construction of bacteriophageencoded recombination systems to generate linear covalently closed (lcc) DNA minivectors. Conditional expression recombinase systems *in vivo* in *E. coli* provide a one step production system for step minivectors from specialized parental plasmids. The exploitation of this recombination system allows us to generate lcc miniplasmids that should have a preferential safety profile, preventing viable vector-chromosome, single recombination products in mammalian cells. In theory, the integration of covalently closed linear exogenous DNA into double-stranded (ds) DNA breaks is unlikely due to the unavailability of terminal ends, and a vector single recombination event into a target

production and storage.

mice *in vivo* (Heinrich et al. 2002).

nanoparticles. Third, to avoid P-glycoprotein-mediated drug efflux, they further designed another delivery vehicle, LPD-II, which showed much higher entrapment efficiency of Dox than LPD. Finally, the authors delivered a therapeutic siRNA to inhibit MDR transporter. Three daily intravenous injections of therapeutic siRNA and Dox (1.2 mg/kg) co-formulated in either LPD or LPD-II nanoparticles showed a significant improvement in tumor growth inhibition (Chen et al. 2010a). Numerous other similar studies highlights a potential clinical use for these multifunctional nanoparticles with an effective delivery property and a function to overcome drug resistance in cancer.
