Synthetic Cell Surface Receptors for Delivery of Therapeutics and Probes☆

Advanced Drug Delivery Reviews 64 (2012) 797–810

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Advanced Drug Delivery Reviews

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Synthetic cell surface receptors for delivery of therapeutics and probes☆

David Hymel, Blake R. Peterson ⁎

Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045, USA article info abstract

Article history: Receptor-mediated endocytosis is a highly efficient mechanism for cellular uptake of membrane-impermeant Received 23 December 2011 ligands. Cells use this process to acquire nutrients, initiate signal transduction, promote development, regu- Accepted 20 February 2012 late neurotransmission, and maintain homeostasis. Natural receptors that participate in receptor-mediated Available online 25 February 2012 endocytosis are structurally diverse, ranging from large transmembrane proteins to small glycolipids embedded in the outer leaflet of cellular plasma membranes. Despite their vast structural differences, these recep- Keywords: tors share common features of binding to extracellular ligands, clustering in dynamic membrane regions that Receptors ligands pinch off to yield intracellular vesicles, and accumulation of receptor-ligand complexes in membrane-sealed cholesterol endosomes. Receptors typically dissociate from ligands in endosomes and cycle back to the cell surface, lipids whereas internalized ligands are usually delivered into lysosomes, where they are degraded, but some can endocytosis escape and penetrate into the cytosol. Here, we review efforts to develop synthetic cell surface receptors, de- trafficking fined as nonnatural compounds, exemplified by mimics of cholesterol, that insert into plasma membranes, recycling bind extracellular ligands including therapeutics, probes, and endogenous proteins, and engage endocytic delivery membrane trafficking pathways. By mimicking natural mechanisms of receptor-mediated endocytosis, syn- endosomes thetic cell surface receptors have the potential to function as prosthetic molecules capable of seamlessly aug- membranes fluorescence menting the endocytic uptake machinery of living mammalian cells. vancomycin © 2012 Elsevier B.V. All rights reserved.

Contents

1. Receptor-mediated endocytosis ...... 797 2. Cellular uptake and trafﬁcking of cholesterol-mimetic membrane anchors ...... 799 3. Synthetic cell surface receptors comprising binding motifs linked to cholesterylamine ...... 802 3.1. Fluorophores and other small protein-binding motifs ...... 802 3.2. A synthetic Fc receptor...... 802 3.3. A synthetic vancomycin receptor ...... 805 3.4. Related synthetic compounds that escape from endosomes...... 806 4. Other approaches ...... 806 5. Conclusions and perspectives ...... 808 Acknowledgments...... 808 References ...... 808

1. Receptor-mediated endocytosis amino acids, sugars and ions must interact with speciﬁc membrane- bound protein pumps or channels to access the cell interior. Other The plasma membrane of eukaryotic cells represents a universal cell-impermeable small molecules, macromolecules, and particles, barrier that protects fragile intracellular structures from toxic or detri- must be actively taken up by cells, with regions of the plasma mem- mental extracellular materials. Whereas small hydrophobic molecules brane functioning to capture solutes by invaginating and pinching off can often penetrate this lipid bilayer through passive diffusion, polar to form intracellular vesicles. This process, termed endocytosis, represents multiple related mechanisms for the internalization of extracellular molecules [1-3]. These mechanisms include phagocytosis, where ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on speciﬁc cell types engulf large particles, pinocytosis, where small re- "Approaches to drug delivery based on the principles of supramolecular chemistry". ⁎ Corresponding author. gions of cellular plasma membranes are actively invaginated to capture E-mail address: [email protected] (B.R. Peterson). solutes, and receptor-mediated endocytosis (RME). In RME,

0169-409X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2012.02.007 798 D. Hymel, B.R. Peterson / Advanced Drug Delivery Reviews 64 (2012) 797–810 internalizing receptors on the cell surface bind cell-impermeable li- with clathrin requires interactions between cytosolic residues of this re- gands to concentrate these ligands in the cell, providing a mechanism ceptor with adapter proteins such as ARH,[15,16] which associates with of cellular uptake that is thousands of times more efficient than non- phosphatidylinositol-4,5-bisphosphate at the cytofacial leaflet of the specific pinocytosis. plasma membrane. These interactions allow the LDLR to constitutively Receptors involved in RME comprise a structurally diverse group deliver LDL into membrane-sealed vesicles that fuse to form endosomes of biomolecules. These receptors range from macromolecular pro- (Fig. 2). In this process, clathrin polymerizes to form a lattice of clathrin teins that span the plasma membrane to small glycolipids, anchored hexagons and pentagons that surround vesicles as they bud from the only to the plasma membrane outer leaflet. Cell-impermeable small plasma membrane. Changes in membrane curvature required for vesi- molecules, lipids, peptides, proteins, nucleic acids, and carbohydrates cle budding are stabilized both by clathrin and a number of clathrin- can be internalized by RME, enabling the consumption of nutrients, binding proteins including FCHO proteins that drive flat plasma mem- elimination of pathogens, and termination of signals initiated by ex- brane segments into highly curved endocytic vesicles loaded with tracellular stimuli. By exploiting RME, certain viruses, protein toxins, cargo [1]. The pH of these internalized vesicles drops as a consequence and other pathogens invade cells and cause disease [4]. These patho- of activation of proton pumps and opening of chloride channels, and gens include simian virus-40 (SV40), which enters cells through RME these vesicles fuse with relatively short-lived acidic (pH~6) sorting upon binding to the small glycolipid ganglioside GM1 (M.W. 1603 g/ endosomes that accept receptor ligand complexes [17,18]. This de- mol). Fig. 1 shows structures of the protein capsid of this virus[5] and crease in pH, and the intrinsic tubular-vesicular geometry of sorting a side view of the VP1 pentamer subunit[6] that binds the pentasac- endosomes, facilitates the dissociation of receptors from ligands [19]. charide head group of ganglioside GM1. This figure also shows struc- Many free receptors subsequently traffic to the endocytic-recycling tures of extracellular regions and representations of other receptors compartment (ERC), a long-lived organelle, before cycling back to the involved in RME such as the membrane-spanning low-density lipo- cell surface. In this way, the LDLR can be reused up to several hundred protein (LDL) receptor,[7] the transferrin receptor (TFR),[8] and the times during its ~20-hour lifespan. Sorting endosomes containing free lipid-anchored receptor FcγRIIIB (CD16)[9] bound to or below the LDL mature to form more acidic late endosomes (pH~5.5), and these cognate ligands LDL,[10] transferrin, and the Fc fragment of human compartments subsequently fuse with lysosomes, more acidic organelles IgG. (pH~5) containing highly hydrolytic enzymes. Hydrolysis in lysosomes RME of cholesterol-laden LDL particles by the LDL receptor (LDLR) of cholesteryl esters, protein, and other components of LDL, liberates has been extensively characterized,[7,11-14] and key features of this these nutrients for utilization by the cell [11,12]. Because LDL is effi- process are illustrated in Fig. 2. The LDLR binds LDL particles defined ciently delivered to late endosomes and lysosomes (within~30 min), by a core of ~1500 molecules of fatty acid-esterified cholesterol that is fluorescent complexes of LDL[20] are often used as markers for these encapsulated by a monolayer of free cholesterol, phospholipids, triglyc- compartments at the terminal end of the endosomal pathway. erides, and a single protein termed apolipoprotein B-100. By recogniz- Clathrin-dependent endocytosis is thought to be responsible for ing the protein component of LDL, the LDLR provides a major the uptake of about half of all ligands of cell surface receptors. These mechanism by which vertebrate animals internalize exogenous choles- ligands include LDL, epidermal growth factor (EGF), and transferrin terol, a key building block required for the biosynthesis of steroid hor- (TF), a carrier of the essential nutrient iron in serum of vertebrate ani- mones and bile acids, and the assembly of cellular membranes. By mals [1]. The TF protein binds the transferrin receptor (TFR),[8] and in- binding the cytosolic protein clathrin, the LDLR clusters in clathrin- ternalization of TF by RME results in release of iron in acidic endosomal coated pits on the cellular plasma membrane. Association of the LDLR compartments for utilization by the cell. However, unlike LDL, the apo-

Fig. 1. Natural receptors and ligands involved in receptor-mediated endocytosis. From left to right, X-ray crystal structures of the extracellular domains of the human low-density lipoprotein (LDL) receptor, the human transferrin receptor, and FcγRIIIB are shown illustrating the nature of association with the plasma membrane. A structure of the ligand LDL determined by electron cryomicroscopy is at the upper left (reduced in scale; image courtesy of Dr. Dr. Wah Chiu, Baylor College of Medicine). Other ligands shown from left to right include receptor-bound transferrin, receptor-bound Fc region of human IgG, the SV40 capsid (reduced in scale; image courtesy of Dr. Ten Feizi, Imperial College, London), and the VP1 pentamer of the SV40 capsid bound to the head group of ganglioside GM1. D. Hymel, B.R. Peterson / Advanced Drug Delivery Reviews 64 (2012) 797–810 799

Fig. 2. Endocytic trafficking of receptors and ligands. This model illustrates uptake of low-density lipoprotein (LDL) mediated by the LDL receptor (LDLR), diferric transferrin (TF-Fe3+) internalized by the transferrin receptor (TFR), entry of cholera toxin (CTX) and simian virus-40 (SV40) after binding to ganglioside GM1, and trafficking of glycosylphosphatidylinositol- anchored proteins (GPI-AP). Ligand-bound LDLR and TFR undergo clathrin-mediated endocytosis, whereas binding of CTX and SV40 to GM1 primarily results in endocytic uptake via clathrin-independent carriers (CLIC) and trafficking through GPI-enriched endocytic compartments (GEEC). The receptors undergo plasma membrane recycling, LDL is degraded in lysosomes, and transferrin remains bound to its receptor, releasing iron in sorting endosomes. CTX and SV40 traffic to the ER and penetrate into the cytosol, resulting in toxicity or infection respectively. The t1/2 and pH values are approximate and cell-type dependent.

TF remains bound to its receptor, the receptor-ligand complex cycles pathways, and can affect the endocytosis of specific receptors. Many back to the plasma membrane, and apo-TF is released from the TFR at lipid-linked proteins undergo endocytosis through clathrin- the neutral pH of the extracellular environment. For this reason, fluores- independent carriers (CLIC) and traffic through GPI-anchored protein- cent transferrin conjugates are often used to label early endosomes in enriched early endosomal compartments (GEEC) [30]. Other mecha- studies of endocytic processes. Some receptors including LDLR and nisms of clathrin-independent endocytosis have also been reported, TFR are internalized via a constitutive clathrin-dependent pathway and this field was recently reviewed [3,31]. that does require ligand binding. In contrast, binding of EGF to the epider- Protein toxins and viruses commonly exploit receptor-mediated mal growth factor receptor (EGFR) and subsequent receptor dimerization endocytosis to penetrate into the cell interior [4]. In some cases, mul- is required to trigger clathrin-mediated endocytosis of this receptor tiple distinct endocytic mechanisms of cellular uptake have been si- [21]. multaneously observed [32]. In contrast to most protein ligands, Some cell surface receptors are attached to the plasma membrane viruses and other pathogens internalized by RME typically avoid deg- by lipids inserted into the exofacial leaflet of the bilayer. Posttransla- radation in lysosomes, either by engaging trafficking itineraries that tional modification of proteins with glycosylphosphatidylinositol bypass these organelles, by fusion of viral envelopes with endosomal (GPI)-lipids is used to anchor receptors such as folate receptor-2 membranes to escape from endosomes, or by production of proteins and FcγRIIIB (CD16) on the cell surface. These receptors bind 5- or peptides that disrupt endosomal or phagosomal membranes at methyltetrahydrofolate[22,23] and the invariant Fc region of low pH [33]. Here, we review recent efforts to develop synthetic mole- immunoglobulin-G[9,24] to promote RME of these ligands [25]. Similar- cules with trafficking properties similar to natural surface receptors. By ly, small glycolipids function as receptors involved in RME. Ganglioside inserting into cellular plasma membranes, these synthetic cell surface GM1 enables the protein cholera toxin[26] and the non-enveloped virus receptors are designed to promote the endocytosis of endogenous li- SV40 to enter cells upon binding to its pentasaccharide head group gands, therapeutics, and molecular probes, and in some cases have [27,28]. Because of the lack of a direct connection to the highly efficient been shown to enable escape of internalized cargo from endosomal clathrin machinery via a cytoplasmic region, the endocytosis of GPI- compartments into the cytoplasm and nucleus of living cells. linked proteins and other lipid-linked receptors is slower than the uptake of most transmembrane proteins, and is thought to involve multi- 2. Cellular uptake and trafficking of cholesterol-mimetic membrane ple, possibly simultaneous, endocytic uptake mechanisms [28]. Uptake anchors of lipid-bound ligands has been reported to involve lipid rafts, membrane domains enriched in cholesterol and sphingolipids, and in some Natural receptors are typically anchored to cell surfaces through cell types include flask shaped invaginations termed caveolae [29]. either transmembrane protein segments or attached lipids such as Many proteins covalently or noncovalently associated with cholesterol, GPI anchors and glycosphingolipids that insert into the exofacial leaf- sphingolipids, or saturated lipids associate with lipid rafts that segre- let of the plasma membrane. In contrast, cytoplasmic proteins are gate and concentrate membrane proteins, regulate signal transduction commonly anchored to intracellular membranes via other lipids 800 D. Hymel, B.R. Peterson / Advanced Drug Delivery Reviews 64 (2012) 797–810 such as palmitoyl thioesters, myristoyl amides, farnesyl and related enriched in the plasma membrane and ERC compared to other organ- thioethers, and cholesterol esters [34,35]. Because of its high abun- elles [36]. dance in cellular plasma membranes, and its role in cycling between The trafficking of cholesterol between the plasma membrane and the cell surface and early/recycling endosomes,[36] we have focused the ERC has been proposed to occur through both vesicular (via endo- on using analogues of cholesterol, particularly derivatives of choles- cytosis) and non-vesicular mechanisms [44]. However, a recent com- terylamine (Fig. 3), to construct synthetic compounds that mimic cel- parison of dehydroergosterol and fluorescent BODIPY cholesterol lular uptake and trafficking properties of natural cell surface (Fig. 3), in BHK cells engineered to express antisense mRNA against receptors. A wide variety of synthetic mimics of other natural lipids the heavy chain of clathrin, suggests that sterol internalization from [37-41] and peptides[42] capable of insertion into membranes of living the cell surface to the ERC occurs predominantly through vesicular cells have also been reported. uptake dependent on clathrin-mediated endocytosis [48]. Subsequent Cholesterol has been termed the central lipid of mammalian cells cycling of cholesterol from the ERC back to the plasma membrane is [43]. Among its many biological roles, this sterol is critically impor- thought to involve vesicular transport similar to plasma membrane tant for stabilization of animal cell membranes. To estimate the num- recycling of natural cell surface receptors [44]. Despite bearing a ber of cholesterol molecules in cellular membranes, the plasma fluorophore in the tail of the sterol, BODIPY cholesterol appears to membrane of a typical mammalian cell was modeled by Maxfield closely mimic the membrane trafficking and cellular efflux properties [44] as a sphere of 10 μm3 with a surface area of 3×108 nm2. This of natural cholesterol [48-50]. In contrast, other fluorescent sterols membrane is composed of approximately 2/3 lipid and 1/3 protein, with modified tails such as 22-NBD cholesterol[51] and 25-NBD cho- and based on the average surface area of a lipid of 0.6 nm2,[45] con- lesterol[52] do not exhibit cellular trafficking properties similar to tains about 109 lipid molecules in both leaflets [44]. Because choles- cholesterol. The use of fluorescent sterols to study cholesterol traf- terol represents about 30% of the lipids in the plasma membrane, ficking in living cells was recently reviewed [53]. the plasma membrane contains roughly 3×108 cholesterol mole- A wide variety of mimics and derivatives of cholesterol have been cules, corresponding to about half of the total abundance of cholester- synthesized and investigated [54]. A comparison of the biophysical ol (about 6×108 molecules) in all membranes of mammalian cells. properties of the nonnatural compounds 3β-amino-5-cholestene This sterol rapidly flips between the two leaflets of this membrane (also known as cholesterylamine or aminocholesterol, Fig. 3) and with a half-time of approximately 1 second,[46] but the distribution of 3α-amino-5-cholestene was reported by Bittman in 1992 [55]. cholesterol between leaflets of the plasma membrane is asymmetric, These studies revealed that the 3β-isomer exhibits much slower bi- 8 with a smaller fraction residing in the exofacial leaflet (~ 0.75×10 phasic vesicle exchange kinetics (t1/2 =118.9 h and 9.3 h) compared 8 molecules), compared to the cytofacial leaflet (~ 2.25 ×10 mole- with the 3α-isomer (t1/2 =393.1 min and 16.1 min) and free cholester- cules) [36]. Within cells, the ERC represents the second largest reser- ol (monophasic t1/2 =256 min). These results suggest the possibility of voir of cholesterol, containing about 2 ×108 molecules of this sterol. unique interactions between the 3β-isomer of cholesterylamine and Photobleaching studies of the fluorescent natural product dehy- phospholipids. Intrigued by these observations, we developed a practi- droergosterol (Fig. 3), a close structural and functional mimic of cho- cal method to synthesize multigram quantities of 3β-cholesterylamine, lesterol, indicate that cholesterol dynamically cycles between the [56] and we have investigated the biological properties of a variety of its plasma membrane and ERC. In this rapid process, it has been estimated derivatives. Some of our early work in this area[57-62] has been that 1×106 molecules of cholesterol enter and leave the ERC per second reviewed [63]. [47]. The cholesterol content of other mammalian organelles such as the Recent biophysical studies have investigated cholesterylamine in endoplasmic reticulum (containing~5% of cellular cholesterol), the model bilayer membranes [64]. This synthetic steroid was found to Golgi apparatus, lysosomes, and mitochondria is much lower than the form a liquid ordered phase very similar to cholesterol with saturated plasma membrane and ERC. This heterogeneous distribution of choles- lipids, suggesting that cholesterylamine could be a good substitute for terol among biological membranes is thought to relate to the affinity of cholesterol. Consistent with this interpretation, fluorescent N-alkyl- this sterol for saturated and monounsaturated lipids that are highly cholesterylamines bearing neutral or negatively charged head groups such as Pennsylvania Green (PG)-Glu-β-Ala-cholesterylamine (1, Fig. 3) are avidly taken up by living mammalian cells, associate with the cellular plasma membrane, and accumulate in transferrin- positive early/recycling endosomes (Fig. 4, panels A-D). Although partitioning between the plasma membrane and early/recycling endosomes can differ depending on the structure of the head group and linker attached to cholesterylamine,[61] many of these compounds rapidly cycle between the plasma membrane and early/recycling endosomes. Some fluorescent cholesterylamine derivatives exhibit plasma membrane recycling kinetics similar to cycling of the LDL receptor, making a round trip between these two compartments in less than 10 minutes [61]. Structurally optimized fluorescent cholesterylamines such as 1 associate avidly with the exofacial leaflet of cellular plasma membranes and become localized in the ERC (see Fig. 4, panels A-B), showing a overall distribution similar to the fluorescent cholesterol-binding macrolide filipin [52]. Many other fluorescent analogues of cholesterol are not stably incorporated in cellular plasma membranes. For example, confocal microscopy of Jur- kat lymphocytes treated with 22-NBD cholesterol reveals rapid trafficking of this compound to the Golgi apparatus and nuclear membrane (see Fig. 4, panels E-F). By cycling between the plasma membrane and ERC of mammalian cells, cholesterylamines mimic some of the major membrane traffick- Fig. 3. Structures of cholesterol, cholesterylamine, and representative fluorescent ing properties of natural cholesterol. However, the charged amino analogues. group of cholesterylamine also confers some significant biological D. Hymel, B.R. Peterson / Advanced Drug Delivery Reviews 64 (2012) 797–810 801

Fig. 4. Confocal laser scanning and differential interference contrast microscopy of living Jurkat lymphocytes treated with fluorescent probes. These micrographs illustrate the rapid binding of the green fluorescent cholesterol mimic 1 to cellular plasma membranes (panel A) and the accumulation of this compound in early/recycling endosomes (panels B and D). A comparison of the divergent trafficking of green fluorescent transferrin-AlexaFluor-488 and red fluorescent DiI-LDL is shown in panel C. In panel D, extensive colocalization of green fluorescent 1 with red fluorescent transferrin-AlexaFluor-647 is observed. In contrast, green fluorescent 22-NBD cholesterol does not remain associated with cellular plasma membranes and traffics to the Golgi apparatus and nuclear membrane (panels E-F). The Leica SPE microscope settings in panels A, B, E, and F are identical (high resolution 3×scans). The images shown in panels C and D were acquired as identical rapid single scans for examination of colocalization of the red and green channels. Scale bar: 10 μm. differences compared with the alcohol of cholesterol in living cells. membranes,[46] whereas desolvation of the protonated amino Whereas cholesterol can be esterified by fatty acids derived from group of cholesterylamine engenders a substantial energetic penalty phosphatidylcholine mediated by lecithin cholesterol acyltransferase against equilibration across the leaflet of vesicles [55]. Consistent (LCAT) for storage in lipoproteins and lipid droplets,[65] the predomi- with this observation, studies of living Jurkat lymphocyte cells treated nantly protonated amino group of cholesterylamine and N-alkyl- with N-alkyl-cholesterylamines linked the pH-sensitive hydrophobic cholesterylamine derivatives is highly unlikely to be a substrate of Tokyo Green fluorophore[66] indicate that cholesterylamines do not this enzyme. Cholesterol also rapidly flips between leaflets of cellular flip between membrane leaflets. In these experiments, cellular 802 D. Hymel, B.R. Peterson / Advanced Drug Delivery Reviews 64 (2012) 797–810

fluorescence imaged by confocal microscopy was compared in the pres- derivatives of N-alkyl-cholesterylamine are shown adjacent to the ence and absence of Bafilomycin A1,[67] a natural product inhibitor of structure of ganglioside GM1 in Fig. 5. Structurally similar vacuolar ATPases that raises endosomal pH and allows assessment of cholesterylamines-linked to dinitrophenyl (DNP), 7-nitrobenz-2-oxa- the abundance of the acid-sensitive Tokyo Green fluorophore in endo- 1,3-diazole (NBD), biotin, and nickel2+ chelated by the NTA head somes of treated cells. If this cholesterylamine had flipped to the cytofa- group of 2 exhibit receptor-like properties, enabling synthetic cial leaflet of endosomes, the linked Tokyo Green fluorophore would be receptor-mediated endocytosis of antibodies and other proteins, in- exposed to the neutral pH of the cytoplasm, resulting in strong endoso- cluding streptavidin and oligohistidine-tagged proteins that bind to mal fluorescence. These experiments indicated that despite achieving a these small head groups on cell surfaces [57,60-62,70]. To illustrate high relative abundance in the ERC, fluorescent cholesterylamines do the subcellular distribution and receptor-like activity of green fluores- not flip from the exofacial leaflet to the cytofacial leaflet of endosomal cent 1 in a suspension cell line, confocal laser scanning micrographs of membranes of living cells [66]. As a consequence, cholesterylamines human Jurkat cells treated with this compound followed by addition show dynamic membrane trafficking properties that are similar to of a red fluorescent antifluorescein antibody at two time points are many cell surface receptors, projecting N-linked groups either from shown in Fig. 6. These images show the localization of 1 on cell surfaces the cell surface into the extracellular environment or from the exofacial and in early endosomes, and time-dependent endocytic cellular uptake leaflet of early/recycling endosomes into the lumen of these intracellu- of this IgG dependent on treatment of cells with 1. lar compartments during plasma membrane recycling [61]. This proper- The structure of the linker region and head group of ty allows the use of N-alkyl-cholesterylamines and related compounds cholesterylamine-derived synthetic receptors affects the ability of as a platform for the construction of mimics of cell surface receptors. these compounds to bind cell surfaces, partition between the plasma membrane and early/recycling endosomes, and promote the uptake 3. Synthetic cell surface receptors comprising binding motifs of impermeable ligands [61]. Correspondingly, treatment of cells linked to cholesterylamine with optimized synthetic receptors can enhance the uptake of proteins and other ligands by over 100-fold as quantified by flow cytometry 3.1. Fluorophores and other small protein-binding motifs [61,62]. Ligands internalized in this way are delivered to late endosomes and lysosomes as evidenced by colocalization of fluorescent de- Natural receptors involved in RME share a unifying structural ar- rivatives with EGFP-lgp120[61] or DiI-LDL [72]. Additionally, chitecture of projection of ligand-binding motifs from lipids or pro- optimized synthetic receptors exhibit recycling half-lives of approxi- teins embedded in the cellular plasma membrane. The smallest of mately 3 min at 37 °C in living Jurkat lymphocytes [61]. This value is these receptors associate with the exofacial leaflet of cellular plasma similar to the recycling kinetics of free cholesterol,[44] C6-NBD sphingo- membranes through linked GPI-anchors and sphingolipids. Structures myelin,[73] and the LDL receptor[12] in other mammalian cell lines. of the GPI anchor of human erythrocyte acetylcholinesterase[68] and the much smaller glycosphingolipid ganglioside GM1[69] are illustrated 3.2. A synthetic Fc receptor in Fig. 5. To create synthetic cell surface receptors that are similar in size to ganglioside GM1, we have synthesized N-alkyl-cholesterylamines Cholesterylamines have been shown to engage major endocytic linked to a variety of small protein-binding motifs including fluoro- membrane trafficking pathways occupied by many natural cell surface phores,[57,61] peptides,[62] biotin,[60] and metal chelators [70]. Repre- receptors. To probe the ability of these compounds to mimic natural Fc sentative examples of fluorescent (1)[71] and metal-chelating (2)[70] receptors (FcR) such as GPI-linked human FcγRIIIB (CD16),[9] our

Fig. 5. Structures of the GPI-anchor of human erythrocyte acetylcholinesterase as a prototypical membrane anchor found in GPI-linked receptors, the small natural receptor ganglioside GM1, and examples of small synthetic cell surface receptors (1, 2). D. Hymel, B.R. Peterson / Advanced Drug Delivery Reviews 64 (2012) 797–810 803

Fig. 6. Confocal laser scanning and DIC micrographs of Jurkat lymphocytes treated with the green fluorescent synthetic receptor PG-Glu-β-Ala-cholesterylamine (1) followed by rabbit antifluorescein IgG conjugated to the red fluorescent Cy5 fluorophore. laboratory previously synthesized a derivative of the FcIII cyclic peptide, compound resided in early/recycling endosomes, and rapid cycling

[74] a compound with high affinity and specificity (Ki =25 nM) for the between these subcellular locations was observed [72]. Under these Fc region of human IgG, linked to a close analogue of cholesterylamine, conditions, the abundance of sFcR on Jurkat cell surfaces was substan- 3β-amino-5α-cholestane (dihydrocholesterylamine) [72]. The struc- tially higher than the natural Fc receptors FcγRI (~1.1×104 / cell) and ture of a minimalistic synthetic Fc receptor (sFcR) and a model of this FcγRII (~3×104 / cell) found on THP-1 monocyte cells. However, sFcR bound to the Fc region of human IgG are shown in Fig. 7 (panels treatment of Jurkat cells with sFcR followed by a single wash with A-B). Human Jurkat lymphocytes do not express FcRs, and treatment media resulted in rapid shedding of this compound from cell surfaces of this cell line with fluorescent human IgG alone does not result in ap- with a half-life of 0.6 hours. Based on recent studies of cholesteryla- preciable uptake of the antibody (Fig. 7, panel C). However, Jurkat cells mines as substrates for reverse cholesterol transport,[71] shedding treated with the sFcR and fluorescent human IgG showed dose- of the sFcR from cells likely involves this natural mechanism of cho- dependent uptake of this fluorescent antibody (Fig. 7, panel D). When lesterol efflux. these cells were compared with human THP-1 cells that express the In some autoimmune diseases, circulating autoreactive antibodies natural FcRs FcγRI and FcγRII, Jurkat cells treated with one micromolar cause tissue damage and inflammation. As a consequence, it has been sFcR revealed greater uptake of human IgG (Fig. 7, panel E) [72]. proposed that sFcRs may have therapeutic utility by promoting the The sFcR rapidly inserted into and readily effluxed from cellular cellular uptake and lysosomal degradation of human IgG in vivo plasma membranes. Comparison with fluorescent bead standards [72]. In this novel therapeutic approach, injection of sFcRs might en- and analysis by flow cytometry revealed that treatment of Jurkat able these compounds to insert into plasma membranes of diverse cells with one micromolar sFcR for 1 hour installed about 6.2×105 mammalian cells remove circulating IgG from the extracellular envi- molecules of sFcR per cell surface. Another major population of this ronment to reduce the concentration of these ligands. To support 804 D. Hymel, B.R. Peterson / Advanced Drug Delivery Reviews 64 (2012) 797–810

Fig. 7. Panel A: Structure of a synthetic Fc receptor (sFcR). Panel B: Model of sFcR embedded in the plasma membrane and bound to the Fc region of human IgG. Panels C-D: confocal laser scanning (left) and differential interference contrast (right) micrographs of Jurkat lymphocytes treated for 4 hours as shown. Scale bar: 10 μm. Panel E: Cellular uptake of fluorescent human IgG (0.5 µM, 4 hours) by THP-1 cells and Jurkat cells treated with sFcR as quantified by flow cytometry. this idea, studies of a simple cell culture model showed that addition therapeutic applications of sFcRs are worthy of further exploration of the sFcR (Fig. 7) to media containing Jurkat lymphocyte cells de- because IVIG is beneficial for treating a number of autoimmune disor- pletes extracellular IgG as a consequence of synthetic receptor- ders including multiple sclerosis, myasthenia gravis, pemphigus, mediated endocytosis [72]. This approach is conceptually similar to Wegener's granulomatosis, Churg-Strauss syndrome, and chronic in- the effective but costly clinical treatment of autoimmune disease flammatory demyelinating polyneuropathy, among other diseases. with intravenous immunoglobulin-G (IVIG), which promotes the ca- Although the development of synthetic cell surface receptors as tabolism of antibodies in vivo including pathogenic IgG [75]. Potential agents for depleting endogenous ligands is an intriguing new concept, D. Hymel, B.R. Peterson / Advanced Drug Delivery Reviews 64 (2012) 797–810 805 additional preclinical studies are necessary to validate this approach mediated transcytosis to access natural pathways for delivery of nu- in vivo. trients across membranes such as the blood–brain barrier [77]. To explore the potential utility of synthetic receptor-mediated 3.3. A synthetic vancomycin receptor drug delivery, termed synthetic receptor targeting, our laboratory designed[78] a cholesterylamine linked to the peptide sequence D- To promote receptor-mediated delivery, a wide variety of drugs Phe-D-Ala. This dipeptide binds[79] the glycopeptide antibiotic vanco- and probes have been linked to ligands of natural cell surface recep- mycin, a drug of last resort against gram positive pathogens such as tors. These ligands include LDL, transferrin, folate, vitamin B12, and methicillin-resistant Staphylococcus aureus [80]. As shown in Fig. 8 many others [76]. By coupling drugs to these ligands, cells expressing (panel A), this synthetic vancomycin receptor (sVancoR) was proposed cognate receptors accumulate these drug-ligand complexes in late to bind cellular plasma membranes, project the vancomycin-binding endosomes and lysosomes. Hydrolysis of functional groups or cleavage motif from cell surfaces, and promote endocytic delivery of vancomycin of disulfides linking drugs to ligands can release therapeutic agents. This into mammalian cells. Treatment of normal mammalian cells with approach has also been used to target receptors involved in receptor- sVancoR and the vancomycin ligand was proposed to deliver this ligand

Fig. 8. Panel A: Structure of the synthetic vancomycin receptor (sVancoR) and a model illustrating synthetic receptor-mediated delivery of the antibiotic vancomycin. Panels B-C: Confocal laser scanning (left) and differential interference contrast (right) microscopy of living J-774 macrophages alone (Panel B) and infected with L. monocytogenes (Panel C). Prior to microscopy, sVancoR (10 μM) was added to cells for 1 hour at 37 °C, cells were washed, and fluorescent vancomycin conjugate (3.6 μM) was added for 2 hours at 37 °C. Scale bar: 10 μm. Panel D: Eradication of intracellular L. monocytogenes in HeLa cells dependent on the concentration of sVancoR as determined by subculture of bacteria. Panel E: Treat- ment with sVancoR enables vancomycin to prevent toxicity of L. monocytogenes to HeLa host cells. Panel F: Distribution of fluorescent vancomycin in mice 8 hours after intraper- itoneal injection as shown. Fluorescence of cells isolated from tissues was analyzed by flow cytometry. 806 D. Hymel, B.R. Peterson / Advanced Drug Delivery Reviews 64 (2012) 797–810 into lysosomes, where the antibiotic would be degraded. In contrast, endosomes enhances hydrophobicity to promote interaction with this delivery of vancomycin into mammalian cells infected with the and destabilization of membranes [86]. Alternatively, weakly basic gram-positive intracellular pathogen Listeria monocytogenes was hy- endosomolytic monoamines that become increasingly protonated pothesized to release vancomycin from endosomes. Expression of the in endosomes, as well as pH-labile covalent bonds integrated into membrane-lytic protein listeriolysin O (LLO)[81,82] by L. monocytogenes delivery systems, have also been used to disrupt the structure and was proposed to trigger release of the antibiotic because LLO enables function of endosomes. These and related approaches have been re- this pathogen to escape from phagosomes and enter the cytoplasm cently reviewed [87-89]. [83]. Consistent with this hypothesis, sVancoR was shown by confocal We have investigated the delivery of synthetic membrane-active laser scanning microscopy of normal J-774 mouse macrophage cells to peptides into early endosomes using cholesterylamines. The incorpo- promote endocytosis of a fluorescent conjugate of vancomycin, result- ration of glutamic acid residues proximal to N-alkyl-3β-cholesteryla- ing in its encapsulation in late endosomes/lysosomes (Fig. 8, panel B). mine can be used to preferentially localize these compounds in early However, under the same conditions, in cells infected with L. monocyto- endosomes compared with the plasma membrane of living cells, pro- genes, fluorescent vancomycin was observed to escape from endosomes viding a unique platform for targeting molecules to these intracellular and diffuse throughout the cytosol and nucleus (Fig. 8, panel C). compartments [90]. By linking a membrane-lytic peptide termed PC- In human HeLa cells infected with L. monocytogenes, treatment 4[91] to an N-alkyl-cholesterylamine, we demonstrated that a with vancomycin and sVancoR eradicated this intracellular pathogen disulfide-linked fluorescent probe can be released from early endo- (Fig. 8, panel D). Moreover, sVancoR and vancomycin rescued HeLa somes into the cytoplasm and nucleus of living mammalian cells cells from the lethal effects of this pathogen (Fig. 8, panel E). Control [90]. This novel two-component delivery system employed the endo- compounds that do not bind vancomycin or that exhibit lower affinity some disruptor (3) shown in Fig. 9 (panel A). This cholesterylamine- for cellular plasma membranes did not show these beneficial effects, linked endosome disruptor (3) was proposed to trigger cleavage of further supporting the hypothesis that sVancoR does not simply per- the disulfide of the fluorescent cholesterylamine 4 and release fluoro- meabilize cells, but rather binds vancomycin on cell surfaces and pro- phore 5 (Fig. 9, panel B) into the cytosol and nucleus of animal cells motes endocytosis by mimicking the trafficking of natural cell surface through the mechanism shown in Fig. 9 (Panel C). This mechanism is receptors [78]. based on the observation that, like the extracellular environment, endo- Studies of sVancoR in vivo in mice examined the ability of this somes are oxidizing[92] and disruption of these compartments could compound to affect the tissue distribution of a fluorescent vancomy- allow reduced glutathione (GSH), present at high concentrations in cin conjugate [78]. In these experiments, mice were injected intraper- the cytosol, to cleave disulfides targeted to the lumen of these itoneally with the fluorescent vancomycin alone or the fluorescent organelles. vancomycin and the sVancoR at 50 mg/kg. After eight hours, the tis- Confocal laser scanning microscopy of living Jurkat lymphocyte sues shown in Fig. 8 (panel F) were harvested, single cell populations cells treated with the cholesterylamine-linked fluorescent disulfide were prepared, and cellular fluorescence was analyzed by flow cy- (4) revealed that this compound accumulates to high levels in early tometry. These studies revealed that sVancoR substantially enhances endosomes (Fig. 9, panel D). In contrast, when the cholesterylamine- the accumulation of fluorescent vancomycin in a wide range of tis- linked endosome disruptor (3) is coadministered with 4, the disulfide sues, particularly in skeletal muscle, pancreas, spleen, liver and of 4 is cleaved within 14 hours, and the fluorescence of 5 can be ob- brain. The synthetic receptor-mediated accumulation of fluorescent served throughout the cytoplasm and nucleus (Fig. 9, panel E). This vancomycin in the brain is of particular interest given that less than method for disruption of early endosomes appears to be effective in a 2% of all drugs are capable of penetrating the blood–brain barrier. wide variety of mammalian cells. In Jurkat lymphocytes, the efficacy The brain studies of sVancoR analyzed all cells isolated from this of endosome disruption by 4 was quantified by flow cytometry as organ by flow cytometry, and the distribution among different cell EC50=1.6 μM. This value was obtained by measuring the enhancement types within the brain was not determined. Consequently, more de- of fluorescence associated with release of the pH-sensitive carboxy- tailed studies are needed to determine the magnitude of delivery fluorescein group of 5 from acidic endosomes into the neutral cytosol. into different cell types. If cholesterylamine-derived synthetic recep- Toxicity of 3 to Jurkat lymphocytes after treatment for 48 hours required tors can be demonstrated to efficiently engage receptor-mediated higher concentrations (IC50 =9.0μM), indicating useful selectivity of re- transcytosis pathways into the CNS in vivo, this approach could pro- lease of the probe over toxic effects to cells. This selectivity likely relates vide important new tools for accessing this poorly accessible tissue. to the pH-dependent membrane-lytic activity of the PC4 peptide[91] and effective targeting of this peptide to early endosomes by the linked 3.4. Related synthetic compounds that escape from endosomes cholesterylamine. Although a number of synthetic agents have been shown to disrupt endosomes of mammalian cells,[87-89] the ability of Receptor-mediated endocytosis is a highly efficient cellular uptake cholesterylamine to target less hydrolytic early/recycling endosomes mechanism. Ligands internalized through this process are encapsulated and release cargo from these compartments may be beneficial for the in membrane-sealed endosomes and are typically shuttled into lyso- delivery of sensitive materials such as nucleic acids and proteins into somes containing highly hydrolytic enzymes. As a consequence, many the cytosol. therapeutics and probes internalized through this route remain trapped in these compartments and do not reach the cytosol or nucleus in high 4. Other approaches concentrations. Although many viruses enter cells through endocytic mechanisms by mimicking the uptake of natural ligands, these patho- A number of diverse methods for cell surface engineering have gens have evolved to avoid degradation in lysosomes. For example, been reported, and a comprehensive review on this topic was recent- SV40 accesses alternative membrane trafficking pathways to bypass ly- ly published [93]. Methods that mimic natural cell surface receptors sosomes, enveloped viruses such as influenza escape from endosomes include a technique termed "protein painting"[94-97] in which pro- by fusion of viral envelopes with endosomal membranes,[84] and teins linked to glycoinositol phospholipids are added to cells and be- non-enveloped viruses produce peptides or proteins that disrupt mem- come incorporated into cellular plasma membranes. This approach branes at low pH [33]. Previously reported studies of endosome disrup- has been used to study cellular signaling, plasma membrane organi- tive peptides, proteins, and polymers have extensively focused on zation, and recognition of modified cell surfaces by the immune sys- pH-dependent fragments and mimics of hydrophobic/acidic fuso- tem. Related recombinant GPI-linked proteins have been used to genic proteins of enveloped viruses such as influenza. Protonation protect vasculature from immune responses to transplanted organs of glutamic acids and other acidic residues[85] of these peptides in [98]. Because GPI-linked proteins undergo clathrin-independent D. Hymel, B.R. Peterson / Advanced Drug Delivery Reviews 64 (2012) 797–810 807

Fig. 9. Structures of the cholesterylamine-PC4 endosome disruptor (3, panel A), a fluorescent disulfide-linked cholesterylamine (4, panel B), and products of cleavage of 4 by reduced glutathione (GSH, panel B). Panel C: Proposed mechanism of release of fluorescent probe 5 upon disruption of early endosomes. Panels D-E: Confocal fluorescence (left) and DIC (right) micrographs of living Jurkat lymphocytes treated with 4 (2.5 μM) for 12 hours. In Panel E, cells were additionally treated with endosome disruptor 3 (2 μM). Scale bars=10 μm. Panel F: efficacy of release of fluorescent probe 5 as analyzed by flow cytometry. Panel G: Toxicity to Jurkat cells after incubation with 3 for 48 hours at 37 °C as analyzed by flow cytometry. 808 D. Hymel, B.R. Peterson / Advanced Drug Delivery Reviews 64 (2012) 797–810 endocytosis, Jurkat lymphocytes treated with a GPI-linked variant of promising, vancomycin is known to have intrinsic liabilities associated the immunoglobulin Fc receptor FcγRIII will endocytose ligands that with toxicity,[80] and other cell-permeable antibiotics are effective bind this receptor [99]. Single-chain antibodies covalently linked to against intracellular pathogenic bacteria. Consequently, the delivery of lipids have been used to construct related cell surface receptors vancomycin using this approach may be of limited clinical utility. How- [96]. Similarly, palmitoylated Protein A and Protein G can be incorpo- ever, this proof-of-concept that a synthetic receptor that binds vanco- rated into cellular plasma membranes and have been used to create mycin can be used to extend the range of its intracellular antibiotic artificial cell surface receptors that bind antibodies or other proteins activity to the cytoplasm of mammalian cells should encourage studies fused to antibody Fc regions [100-103]. Similarly, recombinant diph- of other drug delivery systems based on this approach. theria toxin T domain has been used to anchor the IgG-binding pro- Synthetic receptor-mediated endocytosis of vancomycin by mam- tein ZZ to cellular plasma membranes [104,105]. Using synthetic malian cells infected with L. monocytogenes resulted in escape of this glycopolymers, Bertozzi and coworkers created mimics of cell surface antibiotic from endosomes into the cytoplasm and nucleus. This mucins that recognize glycan-binding proteins and become internal- change in subcellular distribution in the presence of this pathogen is ized into early endosomes [39]. This chemical approach was used to likely due to expression of the bacterial protein LLO, which enables study cell surface phenomena with a level of molecular control that this pathogen to escape entrapment in intracellular membrane- cannot be obtained using conventional biological approaches. Other sealed phagosomes [81-83]. Based on this observation, membrane- polymers have also been used to display binding groups from cell sur- lytic peptides linked to cholesterylamines have been investigated. faces,[106] and promote the uptake of impermeable ligands [107].A These studies led to the discovery that delivery of the membrane- driving force behind some of these strategies has been to enhance lytic peptide PC4 into early/recycling endosomes by a cholesteryla- the immunogenicity of treated tumor cells as an approach to create mine could effectively release a disulfide-linked fluorophore into potential cancer vaccines. Coating of cells with receptor-like proteins the cytoplasm and nucleus of a variety of mammalian cell lines [90]. has also been used as a strategy to promote cell-cell interactions such Further development of this strategy could focus on improving the as targeting of stem cells to sites of inflammation or injury. potency and efficacy of these endosome disruptive agents, establish the range of cargo sizes that can be released by these compounds, 5. Conclusions and perspectives and work to overcome challenges in therapeutic delivery of biomolecules such as RNAi. The delivery of short interfering RNA (siRNA) with The chemical biology approach of mimicry of membrane- this approach is especially promising given that cholesterol- associated biomolecules such as cholesterol can be used to design conjugated siRNAs can silence genes both in vitro and in vivo, particularly prosthetic molecules that become seamlessly incorporated into cellu- in the liver [110-113].Examinationofthebiologicalconsequencesofen- lar plasma membranes. Although many membrane-associated bio- dosome disruption may be useful for optimization of these compounds. molecules including proteins, lipids, and carbohydrates[108,109] all For example, silica crystals and aluminum salts have been shown to have the potential to be mimicked in this way, our laboratory has fo- disrupt lysosomes and releasecathepsinB,activatinginflammasomes cused on constructing synthetic cell surface receptors from deriva- that function as sensors of lysosomal damage [114].Thedevelopment tives of cholesterylamine, a cholesterol analogue easily prepared of potent synthetic compounds that efficiently release cargo by targeting from cholesterol [56]. These mimics of cholesterol are a particularly and selectively disrupting early/recycling endosomes without triggering useful platform for these studies because they can be readily linked inflammasomes or other detrimental biological responses could provide to ligand binding motifs, many of these compounds become selectively important new tools for the delivery of therapeutics and probes. inserted into the exofacial leaflet of plasma membranes of living mammalian cells, and these compounds can rapidly cycle between the Acknowledgments cell surface and early/recycling endosomes. Because cholesterylamines can be designed to not only deliver ligands into endosomes, but can We thank the NIH (R01 CA83831) for financial support. DH thanks also be linked to membrane-lytic peptides that disrupt these compart- the American Chemical Society Division of Medicinal Chemistry for a ments and release linked cargo into the cytoplasm and nucleus,[90] pre-doctoral fellowship. this platform may be useful for the development of improved methods for intracellular delivery of a wide variety of poorly permeable agents. Therapeutic applications of synthetic cell surface receptors are at References an early stage of development. Treatment of cancer cells with [1] H.T. McMahon, E. Boucrot, Molecular mechanism and physiological functions of receptor-like molecules has been proposed as a strategy to enhance clathrin-mediated endocytosis, Nat. Rev. Mol. Cell Biol. 12 (2011) 517–533. immunogenicity for construction of cancer vaccines[96,104] and to [2] G.J. Doherty, H.T. McMahon, Mechanisms of endocytosis, Annu. Rev. Biochem. fl 78 (2009) 857–902. target stem cells to sites of in ammation and injury [100,102]. Another [3] K. 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