**4. Challenges in nanoparticle based gene therapy**

To facilitate efficient gene expression, delivered plasmid DNA must initially circumnavigate various barriers to cellular and nuclear entry as seen in Figure 3. The lipoplex/polyplex must first be internalized by the cell membrane,for which there are many different possible routes including receptor mediated endocytosis, pinocytosis and phagocytosis (Godbey and Mikos 2001). Receptor-mediated endocytosis or clathrindependent internalization is the most common of these and can be exploited to engineer polyplexes to express attached ligands to facilitate this process (Morille et al. 2008).

Nanomedicine Based Approaches to Cancer Diagonsis and Therapy 531

**Folate receptor- mediated** 

B, C

**endocytosis** 

**Uptake and degradation via Endosome/ Lysosome** 

E

**Antibody** 

D

**pDNA Release/ drug efflux** 

**Targeted liposome** 

> **PEGylated Folate**

**Antigen** 

**Low pH dependent destabilization of endosome membrane** 

F

Fig. 3. A schematic generalized diagram of folate receptor/ non-receptor mediated non-viral delivery system: The tethers connecting the folate to the lipid headgroups consist of 250 Å long polyethyleneglycol spacers. Condensation/ complexation of DNA-based therapeutics with DNA delivery vector; (B, C) Non-specific adsorption to cell membrane and cellular internalization via non-receptor mediated (passive entry) or folate-receptor (folate tittered PEGylated liposome's shown in yellow balls and encapsulated drugs as red stars) mediated endocytosis pathway; (D) Monoclonal antibody-antigen complex mediated endocytosis pathway; (E) Uptake and degradation via endosome. lysosome; (F) Low pH dependent destabilization of endosome membrane; (G) Cytoplasmic release of plasmid DNA and drug efflux; (H) Nuclear targeting via nuclear localization signals, transcription and transgene

DNA

**Drug** 

G

expression.

**Nucleus** 

A

**Golgi** 

**Passive Entry** 

**Cytoplasm** 

**Nuclear Targeting** 

H

Pinocytosis is the process by which cells internalize liquids which contain suspended or soluble particles. Untargeted polyplexes that interact with the cell membrane electrostatically may be internalized via this pathway . Phagocytosis is another possible method of internalization involving the ingestion of larger particles greater than 0.5 μm in diameter.

Targeting and internalization of microspheres to phagocytic cells *in vivo* can be achieved through size exclusion. It is important to note that internalization mechanisms may also be largely dependent on the cell type, the vector used, and the process parameters of that a particular vector system (Duncan et al. 2006). On the cellular level, many complexes become buried in the endolysosomal compartment or are degraded in the cytoplasm. After internalization occurs, the lipoplex/polyplex is believed to be most commonly contained within an endocytic vesicle after which it is transferred to late endosomes and lysosomes. In these compartments the pH rapidly changes to the range of 4.5–6, and in order for successful transfection to occur the DNA must find a way to escape from these structures and reach the nucleus as DNA that remains in these cellular compartments is readily degraded (Pack et al. 2005). The final obstacle in the DNA vector's journey is the double bilayer membrane surrounding the nucleus, or nuclear membrane. While small molecules may gain access to the nucleus directly through its network of pores, larger molecules must be internalized by specific nuclear import proteins. This process is largely dependent on the size of the DNA and its conformation. As such, in the absence of specialized enhancer sequences, nuclear import of plasmids is limited to actively dividing cells undergoing mitosis.

In gene therapy studies, nuclear localization signal (NLS) peptides have been investigated as facilitators of nuclear transport with the aim of enhancing transgene expression. In order to improve overall transfection specificity and efficiency it is necessary to optimize intracellular trafficking of the DNA complex as well as the performance after systemic administration (Schatzlein 2001). Properties of vector such as size, shape, and surface characteristics can also have a major impact on its pharmacokinetic properties and delivery efficiency. For most nanoparticles, it is unknown what size and/or charge of nanoparticles could lead to defects in DNA transcription and chromosomal damage and aberration. However, there are indications that certain types of nanoparticles are capable of causing DNA damage, where the composition and the coating of nanoparticles are likely the key factors imparting genotoxic effects.

With respect to systematic *in vivo* applications, nanocarrier approaches face additional hurdles. First, despite advances in using PEG or other hydrophilic polymers for extracellular stability to prolong their circulation in the blood stream, a large fraction of the injected dose of nanoparticles accumulates in the liver and is taken up by hepatic phagocytes. Second, due to the vascular endothelial barrier, nanoparticles can only reach certain tissues such as the liver, spleen, and some types of tumors as a result of enhanced permeability and retention (an effect due to the presence of fenestrated endothelium in tumor blood vessels i.e passive targeting) effect, where the nanoparticles tend to accumulate in tumor tissues much more than in normal tissues. However, nanoparticles cannot, or rarely access, parenchymal cells in most normal tissues as they are simply excluded by the endothelial barrier. Thus, many potential disease targets cannot at present be addressed by existing nanocarrier approaches.

Pinocytosis is the process by which cells internalize liquids which contain suspended or soluble particles. Untargeted polyplexes that interact with the cell membrane electrostatically may be internalized via this pathway . Phagocytosis is another possible method of internalization involving the ingestion of larger particles greater than 0.5 μm in

Targeting and internalization of microspheres to phagocytic cells *in vivo* can be achieved through size exclusion. It is important to note that internalization mechanisms may also be largely dependent on the cell type, the vector used, and the process parameters of that a particular vector system (Duncan et al. 2006). On the cellular level, many complexes become buried in the endolysosomal compartment or are degraded in the cytoplasm. After internalization occurs, the lipoplex/polyplex is believed to be most commonly contained within an endocytic vesicle after which it is transferred to late endosomes and lysosomes. In these compartments the pH rapidly changes to the range of 4.5–6, and in order for successful transfection to occur the DNA must find a way to escape from these structures and reach the nucleus as DNA that remains in these cellular compartments is readily degraded (Pack et al. 2005). The final obstacle in the DNA vector's journey is the double bilayer membrane surrounding the nucleus, or nuclear membrane. While small molecules may gain access to the nucleus directly through its network of pores, larger molecules must be internalized by specific nuclear import proteins. This process is largely dependent on the size of the DNA and its conformation. As such, in the absence of specialized enhancer sequences, nuclear import of plasmids is limited to actively dividing

In gene therapy studies, nuclear localization signal (NLS) peptides have been investigated as facilitators of nuclear transport with the aim of enhancing transgene expression. In order to improve overall transfection specificity and efficiency it is necessary to optimize intracellular trafficking of the DNA complex as well as the performance after systemic administration (Schatzlein 2001). Properties of vector such as size, shape, and surface characteristics can also have a major impact on its pharmacokinetic properties and delivery efficiency. For most nanoparticles, it is unknown what size and/or charge of nanoparticles could lead to defects in DNA transcription and chromosomal damage and aberration. However, there are indications that certain types of nanoparticles are capable of causing DNA damage, where the composition and the coating of nanoparticles are likely the key

With respect to systematic *in vivo* applications, nanocarrier approaches face additional hurdles. First, despite advances in using PEG or other hydrophilic polymers for extracellular stability to prolong their circulation in the blood stream, a large fraction of the injected dose of nanoparticles accumulates in the liver and is taken up by hepatic phagocytes. Second, due to the vascular endothelial barrier, nanoparticles can only reach certain tissues such as the liver, spleen, and some types of tumors as a result of enhanced permeability and retention (an effect due to the presence of fenestrated endothelium in tumor blood vessels i.e passive targeting) effect, where the nanoparticles tend to accumulate in tumor tissues much more than in normal tissues. However, nanoparticles cannot, or rarely access, parenchymal cells in most normal tissues as they are simply excluded by the endothelial barrier. Thus, many potential disease targets cannot at present be addressed by existing nanocarrier

diameter.

cells undergoing mitosis.

factors imparting genotoxic effects.

approaches.

Fig. 3. A schematic generalized diagram of folate receptor/ non-receptor mediated non-viral delivery system: The tethers connecting the folate to the lipid headgroups consist of 250 Å long polyethyleneglycol spacers. Condensation/ complexation of DNA-based therapeutics with DNA delivery vector; (B, C) Non-specific adsorption to cell membrane and cellular internalization via non-receptor mediated (passive entry) or folate-receptor (folate tittered PEGylated liposome's shown in yellow balls and encapsulated drugs as red stars) mediated endocytosis pathway; (D) Monoclonal antibody-antigen complex mediated endocytosis pathway; (E) Uptake and degradation via endosome. lysosome; (F) Low pH dependent destabilization of endosome membrane; (G) Cytoplasmic release of plasmid DNA and drug efflux; (H) Nuclear targeting via nuclear localization signals, transcription and transgene expression.

Nanomedicine Based Approaches to Cancer Diagonsis and Therapy 533

technologies, used for diagnostic purposes (See Figure 4). Compared to conventional materials, inorganic nanomaterials provide several advantages such as simple preparative processes and precise control over their shape, composition and size. These systems provide promising potential not only in diagnostics, but also as delivery systems for therapeutic

B. Nanoparticles in clinical trials or FDA approved

Quantum Dots Carbon nanotubes

Fullerene

Fig. 4. Nanoparticles used in cancer diagnosis and treatment. Liposomes contain

hydrophilic coatings of PEG or multiple carboxylate groups.

amphiphilic molecules, which have hydrophobic and hydrophilic groups that self-assemble in water. Gold nanoparticles are solid metal particles that are conventionally coated with drug molecules, proteins, or oligonucleotides. Quantum dots consist of a core-and-shell structure (e.g., CdSe coated with zinc and sulfide with a stabilizing molecule and a polymer layer coated with a protein). Fullerenes (typically called "buckyballs" because they resemble Buckminster Fuller's geodesic dome) and carbon nanotubes have only carbon-to-carbon

A. Nanoparticles in proof of concept/ research stages

Dendrimer

Gold Nanoparticles

Quantum Dots (QDs) are a unique class of light emitting semiconductor nanoparticles ranging from 2-10 nanometers in diameter and are becoming highly popular for biological imaging due to their high intensity and stable fluorescence profile (most QDs are approximately 10–20× brighter than organic dyes). QDs usually consist of a CdSe core surrounded by a inorganic shell composed of ZnS (Pinaud et al. 2010). For biological imaging applications, they are given

agents and are discussed in detail below.

 

Liposomes

bonds.

**5.1 Quantum dots** 

### **4.1 Targeted nanomedicine**

Construction of organ-targeted gene delivery vectors is a promising route to improve the safety and efficacy of nanomedicine based cancer gene therapy. There are a variety of 'vector targeting' strategies, that can be accomplished using transcriptional targeting, transductional targeting, or ideally, a combination of these. While transcriptional targeting refers to the use of gene regulatory elements (promoters and enhancers) to restrict gene expression to specific cells, transductional targeting refers to the delivery of DNA to specific cells. Targeted gene expression has been analyzed using tissue-specific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). The addition of a ligand (i.e., folate, transferrin, RGD peptide, among others) to the nanoparticle surface, thus targeting the DNA to cells *in vivo* has been demonstrated quite successfully*.* Folate-receptor-targeted liposomes have proven effective in delivering doxorubicin *in vivo* and have been found to bypass multidrug resistance in cultured tumor cells (Immordino et al. 2006). Hong et al., (2010) exploited the possibility of combination of the functions of passive and active targeting by transferring-PEGlyated nanoparticles (Tf-PEG-NP), as well as sustained drug release in tumor by PEGylated drug for most efficient tumor targeting and anti-tumor effects enhancement. Such Tf-PEG-NP loaded with PEGylated drug conjugates could be one of the promising strategies in nanomedicine to deliver anti-tumor drugs to tumor (Hong et al. 2010). Further enhancement of the therapeutic index may also be achieved by overcoming barriers both at cellular and nuclear levels. In gene therapy studies, nuclear localization signal peptides have been investigated as facilitators of nuclear transport with the aim of enhancing transgene expression. Selective tumor targeting with minimal toxicity using folate modified, incorporating nuclear localization signal represents a popular approach. In recent years, Poly(εcaprolactone)/poly(ethylene glycol) (PCL/PEG) copolymers which are biodegradable and amphiphilic, are also emerging as a potential nanoplatform for anticancer agent delivery (Gou et al. 2011).
