and the associated side effects are expected to be minimal. Thus, functionalized nanoparticles are of potential value in improving therapeutic index of drugs intended for the back of the eye diseases.

11.3.5  Intracellular Trafficking

Nanosystems might offer unique opportunities in targeting subcellular organelles, in addition to cell surface receptors as discussed above. In this respect, nanosystems are expected to be superior to other delivery systems including microparticles and implants, which have a dimension that is close to or larger than cell size. Intracellular targeting is particularly relevant for poorly permeable molecules including protein and nucleic acid drugs. Protein drugs might have different targets within a cell beyond cell surface receptors. For instance, antioxidant proteins might be most desirable in the mitochondria of the cell, while transcription factors might exert their effects in the cell nucleus. With respect to nucleic acid drugs, while delivery of siRNAs is desired in the cytoplasm of the cell, delivery of genes for protein overexpression is desired in the nucleus. Due to their large size and susceptibility to enzymatic degradation, intracellular targeting of macromolecules requires special delivery systems. Due to their small size and amenability for surface functionalization, nanosystems can potentially enhance cell entry as well as intracellular targeting of macromolecules. In cultured retinal pigment epithelial cells, the mass, number, and surface area uptake of carboxylate-modified polystyrene particles increases with a decrease in particle size in the range of 2,000–20 nm, with the uptake being 19% of the dose in 3 h for 20 nm particles (Aukunuru and Kompella 2002). The percent uptake for nanoparticles in this study remained about the same in the concentration range of 50–500 mg/ml. Similarly, in conjunctival epithelial cells, nanoparticle uptake increases with a decrease in particle size (Qaddoumi et al. 2004). Nanosystems may enter the cells through various mechanisms including adsorptive endocytosis, fluid phase endocytosis, phagocytosis, and receptor-mediated endocytosis.

PLGA nanoparticles (~100 nm) are endocytosed conjunctival epithelial cells largely by mechanisms independent of clathrin and caveolin-1-mediated pathways (Qaddoumi et al. 2003). Nanoparticles that enter the cell by clathrin-mediated endocytosis form early endosomes (pH 6.3–6.8), which later become late endosomes (Le Roy and Wrana 2005). Eventually, the endosomes and the nanoparticles reach the more acidic lysosomes for degradation. This mechanism of uptake will likely degrade and inactivate the drug or nanoparticles, unless they are resistant to lysosomal enzymes or escape endosomes at an early stage. Some evidence exists for the ability of PLGA nanoparticles to escape endosomes (Prabha and Labhasetwar 2004). Alternatively, nanoparticles that enter the cell by a caveolae-mediated mechanism form caveosomes, which are neutral in pH and may not destroy drug/ nanoparticles. Caveosomes traffic their contents to microtubules for transport to the golgi and endoplasmic reticulum instead of lysosomes. Interestingly, albumin nanoparticles enter retinal pigment epithelial cells via caveolae-mediated endocytosis

(Mo et al. 2007). Chitosan nanoparticles of ~200 nm and a positive surface charge were shown to bind to the outside of the cell membrane of alveolar epithelial (A549) cells and internalize primarily via adsorptive endocytosis and in part by clathrinmediated endocytosis (Huang et al. 2002).

Gene delivery by nanoparticles requires entry into the nucleus. Polyethylenimineamine DNA (PEI-DNA) nanocomplexes of ~150 nm in size were able to enter microtubules by an active motor-protein-driven transport on microtubules for transport toward the nucleus (Suh et al. 2004). These nanoparticles entered the perinuclear space within minutes after transfection. Nanoparticles fabricated with different materials appear to undergo different intracellular trafficking mechanisms. For instance, cationic liposomes behave differently than PEI–DNA complexes. Cationic liposomes synthesized with dioleoylphosphatidylethanolamine (DOPE) and 3b(N- (2-hydroxyethylaminoethane) carbamoyl)-cholestene (HyC-Chol) lipids were transported along microtubules toward lysosomes after entering the cell and were primarily destroyed within the lysosome before the drug could enter the cytoplasm (Hasegawa et al. 2001). The size of these liposomes was not given in the above study, which may also be a major contributor to intracellular trafficking.

Small cationic DNA–protamine complexes of ~120 nm behaved similarly to the HIV-TAT protein that is responsible for the cellular entry of the virus, HIV (Park et al. 2003). The protamine–DNA nanocomplexes had efficient intracellular uptake in the nucleus and the cytoplasm on a similar timescale to that of HIV-TAT protein. The mechanism by which the TAT-HIV protein allows cellular and nuclear entry is unknown, but there is evidence that cationic charge is a major player in uptake. Incorporation of nuclear localization signals on nanoparticle surface is a useful approach for nuclear targeting. Gold nanoparticles were functionalized with nuclear localization signal peptides derived from the SV40 virus T protein (M1), HIV-TAT protein (M2), adenoviral NLS protein (M3), and a synthetic peptide with a nuclear binding site and lysine amino acids (M4) (Tkachenko et al. 2004). The peptideconjugated gold nanoparticles were fabricated by ligating commercially available 20 nm gold nanoparticles with BSA conjugated to one of three peptides, resulting in a final size of 24 nm. The intracellular location of these four conjugated gold nanoshells was investigated in three cell lines: HeLa, 3T3/NIH, and HepG2. In vitro, the M1-conjugated gold nanoparticles were found in clusters in the cytoplasm (most likely in endosomes) and they accumulated on the outside of the nuclear membrane of all cell types after 3 h. Gold nanoparticles conjugated with the M2 peptide were found only in the cytoplasm of HeLa and HepG2 cells after 3 h, but were not found in any compartment in the 3T3/NIH cells. The M3 peptide-conjugated gold nanoparticles were found in the nucleus of HeLa cells and were able to escape the endosome, but were only found in the cytoplasm of 3T3/NIH cells and were absent in HepG2 cells after 3 h. The M4 peptide-conjugated nanoparticles were found in the nucleus of HeLa and HepG2 cells, but only in the cytoplasm of 3T3/NIH cells after 3 h. Cytoplasm and nuclear trafficking of these gold nanoshells is largely dictated by the structure of the peptide and cell type. However, cationic charge is playing a role in cytoplasm and nuclear uptake. Thus, by careful selection of nanosystems and functionalizing ligands, intracellular trafficking and organelle targeting can be controlled.