**3.1 Targeted drug delivery**

One promising biomedical application of MFMNPs is as carriers for site-specific drug delivery. Many therapeutic agents, while pharmacologically effective, also exhibit sideeffects because of their toxicities. For example, cytotoxic compounds used in cancer therapy kill not only target cells but also normal cells in the body, resulting in undesired side-effects. Meanwhile, many barriers to the delivery of therapeutic agents are presented, including renal clearance of small molecular therapeutic agents and overexpressed membraneassociated multi-drug resistance developed by tumor cells. Therefore, many therapeutic agents are limited in their clinical application. As widely used nanocarriers, MFMNPs have been considered as alternatives for the target-specific delivery of drugs to different sites in the body. These engineered nanoparticulate carriers offer some advantages, including passive targeting due to the enhanced permeability and retention (EPR) effect and functionalized surface features for target-specific localization. It also may be possible to develop nanocarriers that respond to physiological stimuli, or to combine drugs with energy (heat, light, and sound) delivery for synergistic therapeutic effects.

### **3.1.1 Passive targeting**

Passive targeting relies on the properties of the delivery system and the disease pathology to accumulate the drug preferentially at the site of interest and avoid nonspecific distribution. Long-circulating nanoparticles of 20-200 nm in diameter containing surface PEG or poly(ethylene oxide) (PEO) blocks can accumulate at sites of disease such as tumors, infection, or inflammation through passive targeting via the EPR effect. Maeda and colleagues (2001) first described the EPR effect in their study of vascular abnormalities of solid tumors. Blood vessels in most solid tumors possess unique characteristics that are not usually observed in normal blood vessels, including: active angiogenesis and high vascular density; extensive production of vascular mediators that facilitate extravasation; defective vascular architecture (lack of smooth muscle layer cells, lack of receptors for angiotensin, large gap in endothelial cell-cell junctions, and anomalous conformations); and impaired lymphatic clearance of macromolecules and lipids from interstitial tissue.

Due to the EPR effect, nanopharmaceuticals (macromolecular drugs and drug-loaded nanoparticles) accumulate in tumor tissues with remarkable selectivity as schematically illustrated in Fig. 2. For example, the administration of polymer-drug conjugates results in 10-100 fold higher drug concentrations in the tumor compared to the administration of free drug (van Vlerken et al., 2007). This selective drug targeting to solid tumors results in substantial therapeutic benefits due to the higher drug accumulation in the tumor tissue, as well as fewer side effects. The EPR effect also has been observed in inflammatory and infectious tissues. Thus, the application of nanocarriers is expected to have therapeutic benefits for treating these diseases as well (Allen & Cullis, 2004).

Fig. 2. Schematic illustration for passive targeting using the EPR effect.

Another approach for passive targeting involves the tendency of nanoparticles to localize in the RES. This phenomenon provides an opportunity for nanoparticles to accumulate at high concentrations in the liver or spleen, where many macrophages are present. Overall, the passive targeting strategy provides a means of delivering MFMNPs (as contrast agents or drug carriers) or other nanoparticles to the targeted organs or tissues.

### **3.1.2 Active targeting**

Another promising approach towards increasing the local accumulation of nanoparticles in diseased tissue is known as active (or specific) targeting. Active targeting involves the

Passive targeting relies on the properties of the delivery system and the disease pathology to accumulate the drug preferentially at the site of interest and avoid nonspecific distribution. Long-circulating nanoparticles of 20-200 nm in diameter containing surface PEG or poly(ethylene oxide) (PEO) blocks can accumulate at sites of disease such as tumors, infection, or inflammation through passive targeting via the EPR effect. Maeda and colleagues (2001) first described the EPR effect in their study of vascular abnormalities of solid tumors. Blood vessels in most solid tumors possess unique characteristics that are not usually observed in normal blood vessels, including: active angiogenesis and high vascular density; extensive production of vascular mediators that facilitate extravasation; defective vascular architecture (lack of smooth muscle layer cells, lack of receptors for angiotensin, large gap in endothelial cell-cell junctions, and anomalous conformations); and impaired

Due to the EPR effect, nanopharmaceuticals (macromolecular drugs and drug-loaded nanoparticles) accumulate in tumor tissues with remarkable selectivity as schematically illustrated in Fig. 2. For example, the administration of polymer-drug conjugates results in 10-100 fold higher drug concentrations in the tumor compared to the administration of free drug (van Vlerken et al., 2007). This selective drug targeting to solid tumors results in substantial therapeutic benefits due to the higher drug accumulation in the tumor tissue, as well as fewer side effects. The EPR effect also has been observed in inflammatory and infectious tissues. Thus, the application of nanocarriers is expected to have therapeutic

lymphatic clearance of macromolecules and lipids from interstitial tissue.

benefits for treating these diseases as well (Allen & Cullis, 2004).

Fig. 2. Schematic illustration for passive targeting using the EPR effect.

drug carriers) or other nanoparticles to the targeted organs or tissues.

**3.1.2 Active targeting** 

Another approach for passive targeting involves the tendency of nanoparticles to localize in the RES. This phenomenon provides an opportunity for nanoparticles to accumulate at high concentrations in the liver or spleen, where many macrophages are present. Overall, the passive targeting strategy provides a means of delivering MFMNPs (as contrast agents or

Another promising approach towards increasing the local accumulation of nanoparticles in diseased tissue is known as active (or specific) targeting. Active targeting involves the

**3.1.1 Passive targeting** 

conjugation of targeting molecules that possess high affinity toward unique molecular signatures found on malignant cells. Targeting ligands, such as proteins, peptides, aptamers and small molecules, have been investigated to increase the site-specific accumulation of MFMNPs. For example, there are certain receptors that are overexpressed on the surface of solid tumor cells, such as antigens, integrin receptors, and folate receptors (Table 1). By bonding with these targeting molecules, MFMNPs can be targeted to the corresponding tumor cells and internalized by receptor- or antigen-mediated endocytosis.

Monoclonal antibodies (mAbs) were the first targeting agents to exploit molecular recognition to deliver MNPs; mAbs continue to be used widely, due to their high specificity. For instance, Herceptin®, an FDA-approved mAb to the HER2/neu (erbB2) receptor, has been used to modify DMSA-coated magnetite nanoparticles. When these MFMNPs were used as contrast enhancement agents, the MR imaging of mice bearing xenograft tumors showed a T2 decrease of ~20% due to the specific accumulation of the nanoprobe in the tumor (Huh et al., 2005). Nanoparticles modified with an HER2-specific antibody (Trastuzumab® or Herceptin®) also are able to localize and deliver the therapeutic payload specifically in HER2-expressing tumor cells (Kirpotin et al., 2006). Certain tumor cells express specific integrin receptors, such as αvβ5 or αvβ3 that can bind to the arginine-glycineaspartic acid (RGD) peptide sequence. The RGD peptide has been utilized for the delivery of MNPs to a variety of neoplastic tissues, including breast tumors, malignant melanomas, and squamous cell carcinomas (Montet et al., 2006).

Among the small targeting molecules, folate has been used to modify nanoparticles for targeted delivery to tumor cells that overexpress folate receptors. Recently, our group reported multilayer MFMNPs with a folate-modified surface and doxorubicin (an anticancer chemotherapeutic agent) loaded in the inner shell (Fig. 3) (Guo et al., 2011). The folateconjugated MFMNPs displayed a much greater cellular uptake than nonfolate-conjugated MFMNPs by a folate receptor–mediated endocytosis process (Fig. 4). Folate conjugation significantly increased nanoparticle cytotoxicity against human cervical carcinoma HeLa cells (Guo et al., 2011).

Fig. 3. Schematic illustration of multilayer MFMNPs with folate as the targeting ligand and loaded doxorubicin as the anticancer chemotherapeutic agent in the inner shell.

Fig. 4. Confocal microscopic images of HeLa cells incubated with (a, b) doxorubicin-loaded MFMNPs without folate conjugation or (c, d) folate–conjugated and doxorubicin-loaded MFMNPs in (a, c) folate-containing or (b, d) folate-free media (Guo et al., 2011).
