Critical Review. Delivery of Nanoparticle-complexed Drugs across the Vascular Endothelial Barrier via Caveolae

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1 IUBMB Life, 63(8): , August 2011 Critical Review Delivery of Nanoparticle-complexed Drugs across the Vascular Endothelial Barrier via Caveolae Zhenjia Wang, Chinnaswamy Tiruppathi, Jaehyung Cho, Richard D. Minshall, and Asrar B. Malik Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, IL Summary The endothelial cell monolayer lining the vessel wall forms a size-selective, semi-permeable barrier between the blood and tissue that must be crossed by blood borne therapeutic agents to reach diseased extravascular tissue. Nanoparticles engineered to carry drugs present an opportunity to enhance the specificity and efficacy of drug delivery. Therefore, an understanding of how these engineered nanoparticles are transported across the vessel wall will help us to more fully exploit this powerful therapeutic technology. Vascular endothelial cells are rich in caveolae, cell surface invaginations nm in diameter that mediate endocytosis of lipids, proteins, and viruses. Caveolar invaginations pinch off to form intracellular vesicles that can transport cargo across the cell and release the cargo into the extravascular space via exocytosis. Here, we will review the current concepts and state of development for delivering engineered nanoparticles across the endothelium via the caveolae-mediated pathway. Ó 2011 IUBMB IUBMB Life, 63(8): , 2011 Keywords drug design; membrane permeability; membrane proteins; signal transduction; vesicular exocytosis. Abbreviations SNARE, soluble N-ethylamaleimide-sensitive factor attachment protein receptor; NSF, N-ethylmaleimidesensitive factor; Cav-1-GFP, caveolin-1-green fluorescent protein; ALI, acute lung injury; AJs, adherens junctions; VE, vascular endothelial; BLMVEC, bovine lung microvascular endothelial cell; PEI, poly(ethylene imine); PLGA, poly(d, L-lactide-co-glycolide); GTPase, guanosine triphosphatase; ER, endoplasmic reticulum; NEM, N-ethylmaleimide; MHC, major histocompatibility complex-class I; BSA, bovine serum albumin; FDA, Food and Drug Administration; AIDS, acquired immunodeficiency syndrome; sirna, small interfering RNA; mbcd, Methyl-b-cyclodextrin. Received 31 March 2011; accepted 1 April 2011 Address correspondence to: Dr. Asrar B. Malik, Department of Pharmacology, University of Illinois, Chicago, IL abmalik@uic.edu INTRODUCTION The monolayer of endothelial cells lining the vessel wall forms a size-selective and semipermeable barrier between the blood and tissue thereby controlling the passage of macromolecules (such as plasma proteins) and fluid between the blood and interstitial spaces of tissue (1 3). Loss of this barrier function results in tissue inflammation, the hallmark of the inflammatory disease, acute lung injury (ALI) (4, 5). Two major pathways have been identified in endothelial cells, which regulate the transport of macromolecules and fluid across the endothelial barrier (1 3): the paracellular pathway regulates transport through interendothelial junctions, whereas the transcellular pathway regulates transport through the cell. The junctional structures (6) that are regulated by the paracellular pathway connect adjacent endothelial cells into a monolayer and restrict the movement of plasma macromolecules from the luminal side of the vessel to the abluminal side of the vessel. Two types of interendothelial junctions are present in the endothelium, tight junctions and adherens junctions (AJs), both contributing to the maintenance of the endothelial barrier. AJs, composed of vascular endothelial (VE)-cadherin bound to catenins, are dominant in most vascular beds. The integrity of AJs is critical in regulating paracellular permeability, as disruption of VEcadherin intercellular adhesion leads to excessive accumulation of fluid in the interstitium and is associated with pathological processes such as inflammation that in lung may lead to ALI. The transcellular pathway is the dominant pathway of transporting macromolecules in endothelial cells (1). The caveola is the major vesicular transporter in endothelial cells, occupying as much as 70% of the endothelial membrane in blood capillaries (7, 8), and selectively endocytoses macromolecules across the cells (such as, their size [3 nm) (1). Caveolae are membrane invaginations nm in diameter that pinch off to form intracellular vesicles and can move macromolecules across the endothelial barrier. Albumin, with a hydrodynamic size of 7 nm (9), for example, has been shown to be transported from the luminal to the abluminal side of endothelial cells via the caveo- ISSN print/issn online DOI: /iub.485

2 660 WANG ET AL. lae-mediated pathway (1, 2). To efficiently deliver macromolecular therapeutic drugs, such as a humanized monoclonal antibody (e.g., Herceptin), to extravascular sites of cancer from the bloodstream, the antibody was conjugated to peptides that bind the plasma protein albumin. This peptide-conjugated antibody was found to cross the endothelial barrier via the caveolaemediated pathway (10). Most small molecule drugs currently used are poorly water soluble and are not specifically targeted to diseased tissue resulting in inefficient drug delivery. However, the use of carefully engineered nanoparticles as carriers to deliver these small therapeutic molecules has recently been shown to increase both drug specificity and efficacy (11). Therefore, the application of nanotechnology to the delivery of therapeutics is widely expected to change the paradigm of pharmaceutical development for the foreseeable future. Nanotechnology refers to the engineering of matter at the atomic, molecular, or macromolecular level, at the length scale of nanometers (12). By using nanotechnology in the biomedical sciences (11, 13), it is believed that it will be possible to achieve 1) improved delivery of poorly water-soluble drugs; 2) targeted delivery of drugs in a cell- or tissue-specific manner; 3) improved transcytosis of drugs across tight epithelial and endothelial barriers (such as with peptide-conjugated Herceptin); 4) delivery of larger macromolecule drugs to intracellular sites of action; 5) codelivery of two or more drugs or therapeutic modalities for combination therapy; 6) visualization of sites of drug delivery by combining therapeutic agents with imaging modalities; and 7) real-time readout of the in vivo efficacy of specific therapeutic drugs. Although the utilization of nanotechnology for enhanced drug delivery has the potential to greatly improve drug design in the growing field of personalized medicine and enhance therapeutic efficacy with less toxicity, little attention has been given to the molecular pathways that control the transport of nanoparticles from blood to tissue. For example, how do therapeutic nanoparticles interact with the endothelial cells of the endothelial barrier? What molecular mechanisms regulate the transport of engineered nanoparticles across the endothelium and their deposition in diseased tissue? Developing a clearer understanding of the mechanisms of transport of therapeutic nanoparticles across the endothelium will enhance our ability to develop novel approaches for delivery across the vessel wall efficiently and will establish the scientific underpinnings for exploiting this pathway for therapeutic nanoparticle delivery. As caveolae are nanoscale vesicles that have been found to be central to endocytosis and transcytosis in endothelial cells, we explored whether the caveolae-mediated pathway might be used to deliver therapeutic nanoparticles across the endothelial barrier. We recently reported that caveolae are able to internalize nanoparticles and to transport them across the endothelial monolayer (14). This finding strongly suggests that the caveolae-mediated pathway might be exploited for efficient delivery of therapeutic nanoparticles (e.g., those carrying genes, drugs, or biologics). Here, we will review the current concepts related to the delivery of therapeutic nanoparticles via the caveolaemediated pathway. CHARACTERISTICS OF CAVEOLAE Caveolar Structure Caveolae were first identified by electron microscopy and defined as flask-shaped invaginations in the plasma membrane that range from 50 to 100 nm in diameter (3). Caveolae exhibited no obvious coat. They are abundant on specific cell types, such as endothelial cells, smooth muscle cells, fibroblasts, and adipocytes. The main structural feature of caveolae is the caveolin-1 protein. Roughly, molecules of caveolin-1 are present in a single caveolar structure as quantified by fluorescence of caveolin-1-green fluorescent protein (GFP) (15, 16). The relative amount of cholesterol in caveolae is estimated to be more than 100 times greater than that of caveolin-1, 20,000 molecules in an isolated caveola (17). Specific glycosphingolipids, such as, monosialotetrahexosylganglioside, and sphingomylin are also enriched in caveolae relative to the bulk plasma membrane. Therefore, caveolae represent specialized, morphologically distinct, sphingolipid-cholesterol microdomains, stabilized by caveolin-1 (17). After photobleaching of caveolin-1-gfp, fluorescence recovery has shown that caveolin- 1 proteins in caveolae are relatively immobile (18), indicating that caveolae maintain their identity during trafficking. Additionally, studies of caveolae trafficking in fused cells expressing caveolin-1-gfp and caveolin-1-red fluorescent protein showed little exchange between fluorescently labeled caveolae, supporting the view that caveolae are highly stable (18). Recent studies (19) using comparative proteomics identified polymerase I and transcript release factor/cavin-1 as a putative caveolar coat protein. Cavin-1 selectively associates with mature caveolae at the plasma membrane but not Golgi-localized caveolin. Live cell imaging demonstrated (20) that cavin-1 was recruited to the caveolar domains and bound these domains to induce the membrane curvature that produced the flask shape of caveolae. Loss of cavin-1 on caveolae leads to degradation of caveolae via lysosomes (20). Caveolar Endocytosis Caveolae-mediated endocytosis is an important cellular process that transports membrane-associated components including receptors bound to ligands and cell surface proteins bound to viruses into or across the cell. This process has been shown to be finely controlled by a series of signaling molecules (21 23). Caveolae-mediated endocytosis has been shown in some cases to be initiated by a ligand binding to its receptor that is localized in caveolar invaginations on the cell surface. One example of this is caveolae-mediated endocytosis of albumin molecules by endothelial cells. Albumin binds a 60-kDa glycoprotein (gp60) on the endothelial cell surface (24, 25) activating Src kinase and the clus-

3 CAVEOLAE-MEDIATED DELIVERY OF THERAPEUTIC NANOPARTICLES 661 tering of gp60 that result in a physical interaction of gp60 with caveolin-1 on the endothelial surface (26). Activated Src phosphorylates caveolin-1, gp60, and dynamin-2 (a pinchase associated with the neck of the caveolar invagination) initiating the intracellular budding of caveolae from the plasma membrane that results in intracellular vesicle formation (27, 28). Inside the cell, caveolar vesicles are seen continuously docking and fusing with early endosomes then dissociating (29). During this transient interaction with endosomes, caveolar vesicles maintain their identity because they dock, fuse, and detach from the endosomal membrane without disassembling their caveolin-1 protein structures in the caveolae (18). This kiss-and-run type of interaction explains why there is only a limited amount of caveolin-1 on the endosomal membrane at any given time (16). This kiss-and-run interaction can be enhanced by the expression of a dominant-active mutant of Rab5, a small guanosine triphosphatase (GTPase) that regulates membrane trafficking from the plasma membrane to the early endosome (30). Expression of a dominantactive mutant of Arfl, a small GTPase, known to regulate membrane trafficking to and from the transitional endoplasmic reticulum (ER) and cis-golgi complex, resulted in entrapment of stable caveolar domains on an enlarged Golgi complex (15). Caveolar Exocytosis Exocytosis is the process in which intracellular vesicles fuse with the plasmalemma on the abluminal side of the endothelium and discharge their vesicular content into the extracellular space. During caveolar transcytosis in endothelial cells, caveolae are internalized and move across the cell and when in apposition to the abluminal plasma membrane, the caveolae fuse exposing their intravesicular contents to the extracellular space. Caveolar exocytosis is regulated by the soluble N-ethylamaleimide-sensitive factor attachment protein receptor (SNARE) machinery (2, 31). There are two types of SNAREs, syntaxin and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein-25 (SNAP-25) family members localized on target plasma membranes (t-snares) and vesicle-associated membrane protein (VAMP) localized on vesicle membranes (v-snares). Before exocytosis, when caveolar vesicles dock with the basolateral membrane of the endothelium, v-snares bind t-snares resulting in a SNARE complex. NSF, a hexameric ATPase, induces disassembly of the v-and t-snare complex, thereby, recycling SNARE monomers for subsequent membrane fusion events. NSF binds the SNARE complex through soluble a- or b-snap. Treatment with N-ethylmaleimide (NEM) (32), an inhibitor of NSF, has been shown to inhibit transendothelial transport of albumin both in situ and in cultured cells, consistent with the role of NSF in controlling SNARE assembly. Caveolar Size and Dynamics Assessed by Nanoparticles Caveolar structures have generally been characterized using electron microscopy (33, 34). As the nm-sized caveolae are smaller than the optical diffraction limit, it is difficult to measure caveolar size using an optical microscope. To enable the study of the dynamics of caveolar trafficking in live cells, optical microscopy is the tool of choice. Recent advances in super-resolution optical microscopy have made it possible to visualize and localize single fluorescent molecules and nanoparticles at a spatial resolution below the optical diffraction limit. Huang et al. (35) recently used three-dimensional high-resolution imaging by stochastic optical reconstruction microscopy to visualize the size of clathrin-coated pits. We recently developed a methodology using dual-color nanoparticle pairs to measure caveolar size and assess the dynamics of caveolar assembly in living endothelial cells. An advantage of using nanoparticles for this assessment is that they can be made in various sizes, and can exhibit novel optical properties such as a wide range of fluorescence spectra (e.g., multicolor imaging) (36). Before the measurement of caveolar size, we found that caveolae were able to internalize albuminconjugated spherical nanoparticles ranging from 20 to 100 nm in diameter but the smaller (i.e., 20 and 40 nm in diameter) nanoparticles were internalized more efficiently. This is consistent with the size limitations of caveolae that limit the internalization of albumin-conjugated nanoparticles larger than the size of an individual caveola. Our studies on the colocalization of internalized fluorescent albumin-conjugated nanoparticles with caveolin-1 in endothelial cells showed most nm-sized nanoparticles were internalized by caveolar vesicles (14). Figure 1A demonstrates the concept of the optical ruler based on the use of a dual-color nanoparticle pair. Two nanoparticles with the same fluorescent color are separated at a distance D of less than half of the emission wavelength, and, thus, the same fluorescent profile of the nanoparticles are detected on the image plane (left of Fig. 1A) due to the optical diffraction limit (e.g., optically spatial resolution is determined by half of the emission wavelength of object), and, therefore, the nanoparticles cannot be distinguished. However, the individual nanoparticles of a dual-color nanoparticle pair (i.e., two particles are internalized in single caveolae) can be optically distinguished, as, for example, red and green nanoparticle images are recognizable due to the emission difference. Locating each color-coded diffraction limit image provides a measure of the distance between the two nanoparticles, even though the distance is below the optical diffraction limit. Figure 1B shows the result of internalization of a mixture of BSA-conjugated 40-nmgreen and 40-nm-red fluorescent nanoparticles in endothelial cells. The merged yellow objects showed a spatial separation between the centers of the diffraction limit spot of green and red nanoparticles; in this example, the separation distance is 80 nm. Combining the size of 40-nm nanoparticles, the measured caveolar diameter might be 120 nm. We used the dual-color nanoparticle pair methodology to measure the size of individual caveolae in endothelial cells. We incubated a mixture of bovine serum albumin (BSA)-conjugated spherical nanoparticles of different sizes with bovine lung microvascular endothelial cells (BLMVECs). After their internal-

4 662 WANG ET AL. Figure 1. Two populations of caveolae in endothelial cells detected by a nanoruler using dual-color nanoparticle pairs. (A) The principle of a nanoruler using dual-color nanoparticle pairs. Left: Two nanoparticles (green dots) with similar fluorescence spectrum are separated at a distance D of less than half the emission wavelength. The use of separate detectors will measure the same fluorescent profile at the optical diffraction limit (large green spot in the image plane). Right: Two nanoparticles having different fluorescence spectrums (red and green). Red and green nanoparticles detected using appropriate filters will appear as two circular diffraction limited spots in the image (as indicated by red and green spots) and measuring the distance between the two centers gives the linear distance between particles. (B) Merged images of dual-color pairs of 40 nm BSA-coated nanoparticles in endothelial cells (green particle emission at 515 nm and red particle emission at 605 nm). The image of the nanoparticles was acquired at 488 nm for green and 543 nm for red. Left: Showing co-localization of red and green nanoparticles (red line on the image shows the cross-section of the image representing the image size profile on the right); Right: image size profile of red and green nanoparticles showing the separation between two nanoparticles obtained by measuring the distance between centers of diffraction images of red and green nanoparticles; for example, in this case, the separation is 80 nm, i.e., less than the diffraction limit. (C) Diagram shows caveolae and aggregates of caveolae sharing dual-color albumin-conjugated nanoparticles. Using different sizes of albuminconjugated nanoparticles allows the measurement of the size of caveolae and of caveolae aggregates. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] ization, we determined the linear size of individual caveola by measuring the separation distance between the two color diffraction limit spots of the nanoparticles and incorporating the size of nanoparticles. We used different sizes of green and red fluorescent nanoparticles (14), such as, 20 nm (green) and 40 nm (red), 40 nm (green) and 40 nm (red), and 100 nm (green) and 40 nm (red). We found two sizes of vesicles in endothelial cells, one size of 100 nm were thought to represent individual caveolae, whereas the second size of 250 nm were thought to represent aggregates of multiple caveolae (as shown in Fig. 1C) as both sizes of vesicles colocalized with Cav-1. Using this, dual-color nanoparticle pairs approach also allowed us to assess the dynamics of caveola vesicle formation in live endothelial cells. Studies on time-lapse trajectories of photoluminescence of 40 nm (green) and 40 nm (red) albumin-conjugated nanoparticles internalized in live endothelial cells suggest two types of vesicles (14), the immotile vesicle that might be associated with aggregates of multiple caveola and the motile vesicle that might be a single caveola. The comigration of dualcolor nanoparticle pairs indicates that the nanoparticle pairs share the same intracellular compartment and the calculated size is similar to that independently determined for a single caveola (unpublished data). This result is consistent with previously reported properties of caveolae in endothelial cells (1 3, 14). CAVEOLAE-MEDIATED TRANSPORT OF NANOPARTICLES Gold Nanoparticles Immunogold nanoparticles have been used in electron microscopy for more than 50 years, however, the terms nanoparticle and nanotechnology were not in use. As colloidal gold nanoparticles are electron dense, they can be easily observed using transmission electron microscopy. Gold nanoparticles coated with antibodies or enzymes have been utilized to detect antigens on cell surfaces, for example, the heterogeneous expression of protein antigens on neuronal cells can be distinguished using different sized immunogold nanoparticles (37). Figure 2. Transcytosis of gold-labeled albumin in perfused lung microvessels. Vesicles 1, 2, and 3 (V1, V2, and V3) represent caveolae-mediated internalization of gold nanoparticles. V3 additionally shows gp60 receptor-bound gold nanoparticles from binding at the cell surface. The release of gold nanoparticles from vesicles by exocytosis at the abluminal surface is indicated by V4 and V5. (Reproduced from Ref. 1, with permission from the Annual Reviews of Physiology, Volume 72, 2010 by Annual Reviews, [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

5 CAVEOLAE-MEDIATED DELIVERY OF THERAPEUTIC NANOPARTICLES 663 To study albumin transcytosis in pulmonary microvessels via the caveolae-mediated pathway, albumin, the ligand of the gp60 receptor that localizes to caveolae, was adsorbed to the surface of a 20-nm-sized gold nanoparticle. Binding of the resulting albumin-gold nanoparticle to gp60 on the luminal surface of the endothelium induced caveolae-mediated endocytosis (1, 3) (Fig.2). The albumin-gold-nanoparticle-loaded caveolae traveled across the cell, docked on the abluminal side of the endothelium, and discharged the albumin-adsorbed gold nanoparticles into the interstitial tissue. We also observed that a single caveola contained multiple 20-nm-sized gold nanoparticles. This finding was the first demonstration that caveolae are able to actively transport engineered nanoparticles across an endothelial barrier. Simian Virus 40 (SV40) Simian virus 40, a simple nonenveloped DNA virus that replicates in the nucleus, was the first virus shown to enter cells via the caveolae-mediated pathway (17). The diameter of an SV40 virus is 50 nm. Electron microscopy studies showed the virus was internalized by invaginations that budded into the cell to form vesicles each containing a single viral particle (15). The invaginations were later shown to be caveolae (17). After binding to major histocompatibility complex (MHC) class I molecules on the cell surface, the SV40 virus moves laterally along the plasma membrane until localized in a caveolar invagination (29). When associated with a latent caveola, the SV40 virus activates a signal that induces local tyrosine phosphorylation, depolymerization of the cortical actin cytoskeleton, and local production of phosphatidylinositol 4,5-bisphosphate (15). Subsequently, both actin and the pinchase, dynamin-2 are recruited to the virus-loaded caveola. Actin forms dynamic tail-like structures radiating from the virusloaded caveola, and dynamin-2 transiently associates with the caveolar neck leading to the pinching off of the caveola into the cytoplasm (29). These events lead to efficient vesicle formation, internalization of the cell surface caveolar microdomains and release into the cytosol of the caveolar virus-containing vesicles. Polystyrene Nanoparticles We have investigated the size-dependence of caveolae-mediated endocytosis of engineered nanoparticles using albumin-conjugated fluorescently labeled polystyrene nanoparticles ranging from 20 to 100 nm in size (14). Measurement of absorption of albumin conjugated to nanoparticles demonstrated that the conjugation reaction via amine-carboxyl bond formation between albumin and nanoparticles had occurred (14). Incubation of the albumin-conjugated fluorescent polystyrene nanoparticles with a monolayer of BLMVECs showed, using confocal microscopy, that the cells internalized the albumin-conjugated nanoparticles. Further, we observed that internalization of the engineered nanoparticles by the endothelial cells was dependent on nanoparticle size and elapsed time. Internalization of 20- and 40-nm nanoparticles was 5 10 times greater than that seen with the 100-nm particles over the same time period, reflecting the sizerestrictive nature of caveolar internalization (14). Moreover, albumin conjugation to the nanoparticles facilitated efficient nanoparticle endocytosis, because uptake of polyethylene glycol (PEG)-conjugated nanoparticles was significantly lower than albumin-conjugated nanoparticle uptake (14). We, further, addressed the role of caveolae-mediated endocytosis of albumin-conjugated nanoparticles in endothelial cells. We assessed whether caveolin-1, the major protein of the caveolar vesicle colocalized with the internalized albumin-conjugated fluorescently labeled polystyrene nanoparticles. The results showed that at least 70% of the albumin-nanoparticles colocalized with caveolin-1, suggesting that albumin-conjugated polystyrene nanoparticles are mostly internalized in caveolae in endothelial cells (14). To address the rate of caveolae-mediated transcytosis across a monolayer of endothelial cells, we recently developed a method by which we can, in real time, measure the rate of transcytosis of albumin-conjugated fluorescently labeled polystyrene nanoparticles across a monolayer of cultured endothelial cells using Transwell filters (unpublished data). We cultured BLMVECs to confluence on a Transwell membrane. As the Transwell membrane does not transmit light (38), this allowed us to selectively quantify the fluorescent nanoparticles that crossed the endothelial monolayer to the bottom of the Transwell measuring the photoluminescence using a multiwell reader. We calculated the transytosis rate of the nanoparticles that moved across the endothelial cells via the transcellular pathway by determing the percentage of cells that moved across the monolayer at a series of time points. Using this method, we additionally demonstrated the size dependence of transcytosis of the albumin-conjugated nanoparticles across a monolayer of endothelial cells. These experiments showed that transcytosis of 20-nm-sized nanoparticles is more efficient than that of 100-nm nanoparticles. This finding is consistent with our previous data supporting caveolar size as a major determinant in the uptake of albumin-conjugated fluorescent nanoparticles in endothelial cells (14). Polyplex Polyplex is a complex of poly(ethylene imine) (PEI) and small interfering RNA (sirna) used for gene delivery (39). sirna, base pairs of double-stranded RNA that can interfere with the expression of target genes, holds great potential for gene therapy. PEI is an efficient gene-delivery polymer due to its ability to act as a proton sponge and buffer endosomal acidification (39). Recently, Gabrielson et al. (40) reported the conjugation of folic acid to PEI for targeted delivery of polyplexes to caveolae in HeLa cells. Folic acid is a ligand, which binds folate receptors that are known to be localized to caveolar invaginations on the cell surface that subsequently bud to form intracellular caveolae. Inhibition of the caveolar pathway using drugs [i.e., methyl-b-cyclodextrin (mbcd) and genistein] or interference RNA against caveolin-1 reduced gene delivery efficiency, suggesting that the polyplexes were delivered via the caveolae-mediated pathway. mbcd destroys the caveolar struc-

6 664 WANG ET AL. Figure 3. Caveolae-mediated pathways of nanoparticle transport. The red arrows represent caveolae-mediated transport of nanoparticles across endothelial cells (transcytosis), and the blue arrows respresent the interactions between nanoparticle-loaded caveolae and intracellular compartments, such as the endosomal and lysosomal compartments. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] ture by depleting cholesterol from caveolae, whereas genistein, a tyrosine kinase inhibitor, blocks caveolar internalization by preventing the required phosphorylation of caveolin-1. In addition, Gabrielson et al. (40) also studied intracellular trafficking of the polyplexes and found that caveolar vesicles loaded with polyplexes avoided intersection with lysosomes and subsequent degradation. These results further suggest that the caveolar pathway might be an efficient means to deliver therapeutics. Signaling of Caveolae-mediated Nanoparticle Transport Based on the studies of albumin-coated gold nanoparticles and conjugated polymer nanoparticles (1, 14), we show a trafficking model for nanoparticles in endothleial cells and the molecular signals regulating the caveolae-mediated transport of nanoparticles (Fig. 3). When the albumin moiety of the albumin-conjugated nanoparticles binds gp60 (albumin-binding protein) localized in caveolar invaginations on the cell membrane, ligand-bound gp60 and caveolin-1 proteins cluster due to multiple albumin ligands conjugated to individual nanoparticles, following the Src activation. The Src kinase is activated by phosphorylation (41) at Tyr 416 and dephosphorylation at Tyr 527 after the albumin nanoparticle binding occurs. Activated Src, in turn, phosphorylates caveolin-1, gp60, and dynamin-2 signaling the fission of caveolar invaginations from the internal side of the plasma membrane. The complete molecular processes are still unclear. Some of the caveolar vesicles loaded with nanoparticles travel directly to the basal side of the endothelial cells, and dock on and fuse with the basal membrane and undergo exocytosis, a process that is believed to be regulated by SNAREs (31). The caveolar vesicles might also interact with each other to form aggregates. Studies of the internalization of dual-nanoparticle pairs in lung endothelial cells have shown the formation of caveolar aggragates in endothelial cells (14). In addition, caveolar vesicles have been shown to interact with endosomes and lysosomes (15), and eventually nanoparticles are degradated in lysosomes. The model of caveolae-mediated transport of engineered nanoparticles shows a series of signaling moelcules that regulate the trafficking of nanoparticles, such as Src kinase and dynamin-2. To efficiently deliver therapeutic nanoparticles across the endothelial barrier via the caveolaemediated pathway, we must investigate more about the molecular mechanisms of caveolae-mediated trafficking of engineered nanoparticles in live cells and intact microvessels using advanced optical microscopy, such as intravital microscopy. TARGETING OF THERAPEUTIC NANOPARTICLES TO CAVEOLAR PATHWAY Therapeutic Nanoparticles In the past two decades, there has been a progressive increase in the number of commercially available, particle-based therapeutic products (13). Among these products, liposomal drugs and polymer-based drugs are the two dominant classes, accounting for more than 80% of the total. Liposomes are self-assembling, spherical lipid vesicles comprising a membrane bilayer of natural or synthetic amphiphilic lipid molecules. Liposomes have been widely used as pharmaceutical carriers because of their unique properties: 1) encapsulation of both hydrophilic and hydrophobic therapeutic agents with high efficiency; 2) protection of the encapsulated drugs from undesired effects of external conditions; and 3) capability to be functionalized using specific ligands that can target specific cells, tissues and organ of interest. For example, Doxil was the first Food and Drug Administration (FDA)-approved liposomal drug formulation and was used for the treatment Kaposi s sarcoma in acquired immunodeficiency syndrome (AIDS) patients (42). Polymer-based drug

7 CAVEOLAE-MEDIATED DELIVERY OF THERAPEUTIC NANOPARTICLES 665 Figure 4. Multifunctional nanoparticles for targeting using caveolar pathway. The multifunctional engineered nanoparticle has the capability of simultaneously carrying therapeutic agents, targeting molecules and imaging agents. Therapeutic agents include sirna, proteins and small drug molecules, and targeting molecules (specific antibodies and recognition peptides) while imaging agents include fluorescent probes and magnetic contrast agents. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] delivery systems in oncology have grown exponentially with the advent of biodegradable polymers (43). In these systems, drugs are either physically dissolved, entrapped, encapsulated, or covalently attached to the polymer matrix. For example, biodegradable polymers (poly-l-lactide, poly-[l-glutamate], poly-(d,l-lactide-co-glycolide) (PLGA), and PEG are currently being exploited as drug delivery systems. Recently, Langer s group (44, 45) developed multifunctional and biodegradable core-shell nanoparticles of PLGA-lecithin-PEG for controlled drug delivery and release. These nanoparticles contain a hydrophobic PLGA core, a soybean lecithin monolayer outside the core, and hydrophilic PEG to increase water solubility and link target ligands of the nanoparticles. Design of Multifunctional Nanoparticles Although these two types of engineered targeted nanoparticle delivery systems (liposomal and polymer-based nanoparticles) have been widely used in preclinical and clinical trials, little attention has been paid to interactions that occur between therapeutic nanoparticles and the endothelium. This interaction is of critical importance as it greatly affects the efficiency of delivery of the therapeutic nanoparticles to target tissues. The size of the two types of nanoparticle-based therapeutics varies from tens to thousands of nanometers (42), and most of them are not targeted to specific cell types due to the lack of targeting agents to conjugate to the nanoparticle surfaces. Because caveolae in endothelial cells are endogenous efficient transporters of nanoparticles, the design of multifunctional nanoparticles to target caveolae and subsequent tissue sites is important for developing novel approaches to efficiently deliver therapeutic nanoparticles. Figure 4 describes multifunctional nanoparticles. Nanoparticles have the capability to simultaneously carry multiple therapeutic agents, targeting molecules, such as conjugated antibodies or other recognition agents, and agents to allow imaging. Potential therapeutic agents include sirna, inhibitory proteins and antibodies, whereas imaging agents such as infrared fluorescent molecules embedded in nanoparticles allow the visualization and tracking of nanoparticles in tissue real time. For treatment specifically of ALI, engineered nanoparticles introduced into the blood stream might carry anti-inflammatory agents, such as dexamethasone to extravascular sites of inflammation. The nanoparticles could be conjugated to albumin or folic acid as targeting agents and be delivered to the lung extravascular tissue via the caveolar pathway. We would also be able to label nanoparticles with infrared dyes for in vivo tracking of drugs to determine the efficiency of targeting of the nanoparticles. CONCLUSIONS AND PERSPECTIVES We have reviewed the current concepts for the development of engineered nanoparticle delivery via the caveolae-mediated pathway. Data from our group and others have shown that caveolae are able to transport a variety of engineered nanoparticles across the endothelium, for example, viruses (natural nanoparticles), as well as metallic (gold) and polymer (polystyrene and PEI) nanoparticles. Most importantly, we have shown that caveolae-mediated transport is highly dependent on nanoparticle size, thus, this fact must be considered in the rational design of therapeutic nanoparticles. Nanoparticles, smaller than caveolae are more easily internalized by caveolae and transported across the endothelium. In addition, smaller nanoparticles have a larger surface area to volume ratio, thus, in general, delivery using smaller nanoparticles is predicted to enhance delivery of therapeutic agents. Exploiting caveolae-mediated delivery of therapeutic nanoparticles is an exciting research area, not yet fully explored. Many questions have yet to be addressed, for example, the role of cavoelae in mediating the transport of engineering nanoparticles in vivo. It is unclear whether Src activation is required for caveolae-mediated transport of nanoparticles. Recent advances in tracking individual fluorescent nanoparticles have enabled the investigation of specific molecular mechanisms of nanoparticle transport via caveolae using genetic approaches, such as the transfection of caveolin-1-gfp into cells to visualize individual caveolae, and the knockdown of caveolin-1 and Src to inhibit transcytosis of nanoparticles in endothelium. Future studies should address the role of caveolae in mediating the transport of engineered nanoparticles in microvessels by evaluating transcytosis in live Cav-1 knockout versus wild-type mice. To address these questions, the field will greatly benefit from advanced optical microscopic methods (46) (e.g., intravital microscopy) together with the careful design of multifunctional nanoparticles, that take into account variations in nanoparticle size, shape, and biofunction. On the basis of results of these future studies, we will be in a better position to effectively

8 666 WANG ET AL. design therapeutic nanoparticles that can be utilized for the efficient delivery through the caveolar pathway of therapeutic agents to diseased tissue. These therapeutic nanoparticles may well benefit treatment of cancer and lung injury patients. ACKNOWLEDGEMENTS The authors thank Dr. Laura K. Price for critical reading of this manuscript. This work was partly supported by UIC/CCTS, UL1RR from the National Center for Research Resources. This work was supported by NIH grants P01HL060678, P01HL077806, and R01HL REFERENCES 1. Komarova, Y. and Malik, A. B. (2010) Regulation of endothelial permeability via paracellular and transcellular transport pathways. Annu. Rev. Physiol. 72, Mehta, D. and Malik, A. B. (2006) Signaling mechanisms regulating endothelial permeability. Physiol. Rev. 86, Predescu, S. A., Predescu, D. N., and Malik, A. B. (2007) Molecular determinants of endothelial transcytosis and their role in endothelial permeability. Am. J. Physiol. Lung Cell. Mol. 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