Molecular physiology of Arc/Arg3.1: The oligomeric state hypothesis of synaptic plasticity

Abstract The immediate early gene, Arc, is a pivotal regulator of synaptic plasticity, memory, and cognitive flexibility. But what is Arc protein? How does it work? Inside the neuron, Arc is a protein interaction hub and dynamic regulator of intra‐cellular signaling in synaptic plasticity. In remarkable contrast, Arc can also self‐assemble into retrovirus‐like capsids that are released in extracellular vesicles and capable of intercellular transfer of RNA. Elucidation of the molecular basis of Arc hub and capsid functions, and the relationship between them, is vital for progress. Here, we discuss recent findings on Arc structure–function and regulation of oligomerization that are giving insight into the molecular physiology of Arc. The unique features of mammalian Arc are emphasized, while drawing comparisons with Drosophila Arc and retroviral Gag. The Arc N‐terminal domain, found only in mammals, is proposed to play a key role in regulating Arc hub signaling, oligomerization, and formation of capsids. Bringing together several lines of evidence, we hypothesize that Arc function in synaptic plasticity—long‐term potentiation (LTP) and long‐term depression (LTD)—are dictated by different oligomeric forms of Arc. Specifically, monomer/dimer function in LTP, tetramer function in basic LTD, and 32‐unit oligomer function in enhanced LTD. The role of mammalian Arc capsids is unclear but likely depends on the cross‐section of captured neuronal activity‐induced RNAs. As the functional states of Arc are revealed, it may be possible to selectively manipulate specific forms of Arc‐dependent plasticity and intercellular communication involved in brain function and dysfunction.

| INTRODUCTION
Understanding brain mechanisms in learning and memory is one of the great challenges of neuroscience. Representation of information in neural networks is widely thought to rely on, in part, synaptic plasticity-the ability of the synapse to change in strength in response to use or disuse. [1][2][3] The generation of long-term functional changes in synaptic transmission requires neuronal activity-dependent alterations in gene transcription and regulation of protein synthesis. Synaptic plasticity, like cell differentiation and growth, is a complex cellular response orchestrated by numerous and functionally diverse genes. [4][5][6][7][8][9] As briefly summarized below, the activityregulated cytoskeleton-associated protein (Arc; aka activity-regulated gene Arg3.1) has emerged as a pivotal regulator of synaptic plasticity with important functions in memory formation and post-natal cortical maturation.

| Arc gene expression dynamics and function in synaptic plasticity
Arc was discovered due to robust enhancement of its mRNA expression in the rat hippocampus following electricallyinduced seizures in vivo. 10,11 In mammals, Arc is predominantly expressed in excitatory glutamatergic neurons of the central nervous system. Arc belongs to a class of immediate early genes in which RNA polymerase II docks near the transcriptional start site, allowing transcription within 2-5 minutes of cellular or behavioral stimulus. 12,13 The Arc pre-mRNA has a single coding-exon and a 3′UTR harboring two introns that are spliced to generate the mature Arc mRNA. No alternative splice variants are known. After the stimulus, Arc mRNA shows a wide somatodendritic distribution, with transport along the dendritic arbour at rates up to 70 μm/min. 14 Arc mRNA accumulates beneath stimulated post-synaptic sites where it undergoes activity-dependent translation. [14][15][16][17][18] Arc mRNA is subject to translation-dependent degradation, due to the presence of the exon junction complex on spliced Arc mRNA which recruits machinery for RNA decay. 19 Stimulus-evoked translation of Arc in neuronal dendrites occurs in short bursts, which aligns well with translation-dependent RNA decay. 20,21 The newly synthesized protein also undergoes rapid ubiquitination and proteasomal degradation with an estimated half-life between 30 and 60 min. [22][23][24][25] Together these studies indicate rapid induction and tight spatial and temporal control of Arc expression.
Various forms of long-term plasticity are impaired when the Arc gene is ablated, or the RNA is knocked down. Arc is causally implicated in the neuronal activity-induced enhancement of synaptic transmission in long-term potentiation (LTP), decreased transmission in long-term depression (LTD), as well as homeostatic synaptic scaling. [25][26][27][28][29][30][31][32][33][34][35] Arc is also required for LTP induced by exogenous application of brain-derived neurotrophic factor (BDNF) in the dentate gyrus, 25,36 and LTD induced by application of the group I metabotropic glutamate receptor (mGluR) agonist, DHPG, in the hippocampal CA1 region. 31 All these Arc-dependent forms of plasticity are associated with enhanced Arc expression in the post-synaptic compartment of excitatory glutamatergic neurons.

| Arc function in memory and postnatal cortical maturation
Numerous studies have demonstrated learning taskspecific expression of Arc mRNA in subpopulations of cells engaged in memory encoding. [37][38][39][40][41][42][43][44][45] Loss of function approaches using constitutive Arc knockout, conditional gene deletion, or knockdown by antisense RNA support a role for Arc in long-term memory formation, whereas learning (acquisition) itself does not require Arc. 26,27,46 Studies employing local, brain region-specific Arc knockdown in the adult brain support a casual role for Arc expression in hippocampal-dependent spatial memory formation, 26,46 while Arc expression in the amygdala is necessary for Pavlovian fear memory. 47 Memory processes triggered by a reminder cue (reconsolidation and extinction) are similarly dependent on brain region-specific Arc expression. [48][49][50] Arc knockin mice harboring mutations in the two predominant ubiquitination sites have impaired degradation of Arc in the ubiquitin proteasomal system. 51 These mice learn and remember a spatial task normally and have a lower threshold for hippocampal mGluR-dependent LTD, but they are impaired in a reversal-learning task which requires switching to a new learning strategy. This suggests that the Arc protein turnover rate influences plasticity and contributes to cognitive flexibility. 51 Arc also has important functions in neocortical circuits. During motor learning, Arc expression in neuronal ensembles of the secondary motor cortex is necessary for the consolidation of task-specific motor responses. 42,[52][53][54] During post-natal maturation of the visual cortex in rodents, Arc expression supports ocular dominance column formation and orientation tuning of visual inputs, as well as receptive field plasticity. 33,[55][56][57][58] Collectively, research indicates a role for Arc in post-natal maturation of the visual cortex as well as consolidation of hippocampal-dependent memory (spatial learning and contextual fear) and hippocampalindependent motor learning. However, causal roles for Arc-dependent forms of plasticity in behavior remain to be established.

| Arc as a protein interaction hub
At the molecular level, Arc is proposed to act as a protein interaction hub. 59 Arc has more than 30 validated interaction partners, as shown by affinity purification methods or by direct binding assays such as surface plasmon resonance. According to this model, Arc regulates intra-cellular signaling and plasticity by binding to different effector proteins. For example, Arc interaction with the clathrin adaptor protein, AP2, dynamin 2, and endophilin, facilitates clathrin-mediated endocytosis of AMPA-type glutamate receptors and promotes decreases in synaptic strength in LTD and synaptic scaling 28,31,60,61 ( Figure 1A). Arc can also be targeted to inactive synapses, by binding to inactive calcium/calmodulin-dependent protein kinase-β (CaMKII-β), which enables a selective weakening of inactive synapses. 35,62 During LTP at the medial perforant path input to the dentate gyrus, stabilization of nascent actin filaments and consolidation of LTP depends on Arc synthesis. 25,63 Newly synthesized Arc binds to the actin cross-linking protein, drebrin A, 64 and regulates the activity of the actin-severing protein, cofilin 25 ( Figure 1B). Arc also enters the nucleus and associates with the histone acetylases, TIP60, and CREB binding protein. 65,66 Nuclear Arc was shown to downregulate transcription of AMPA receptor GluA1 subunits to support a dendrite-wide synaptic downscaling, 66 while recent work implicates Arc in the regulation of chromatin state 65,67,68 ( Figure 1C). In sum, convergent evidence supports a role for Arc as a neuronal activity-induced hub that regulates both synaptic and nuclear mechanisms in the post-synaptic neuron ( Figure 1A-C).

| Arc as a retrovirus-like capsid
In a bioinformatic analysis from 2006, Arc was identified as one of 103 human protein isoforms with conserved homology to retroviral Group-specific antigen (Gag) polyprotein. 77 The work suggested that Arc protein and modern retroviruses originated from ancient Ty3/Gypsy retrotransposon elements. Current evidence indicates that the Arc gene has been repurposed in separate evolutionary events towards mammalian Arc and Drosophila , which has two Arc genes (dArc1 and dArc2). [78][79][80][81] In an exciting new development, Arc protein from mammals and Drosophila were shown to self-assemble into retrovirus-like capsid structures that contain Arc mRNA. Arc capsids are released from neurons in extracellular vesicles (EVs), which transmit the capsid and release the RNA into neighboring cells 80,81 (Figure 1D). In primary mouse hippocampal neuronal cultures, immunogold electron microscopic (immuno-EM) labelling showed native Arc protein inside a subpopulation of EVs isolated from the culture medium. 80 Depolarization of cultures by KCl treatment increased Arc-EV release into the medium. When neuronal cultures from Arc knockout mice were treated with EVs isolated from a wildtype (WT) neuron culture medium, somatodendritic expression of Arc was observed. Treatment with DHPG (a mGluR agonist) further enhanced translation, indicating activitydependent translation of internalized Arc mRNA. At the Drosophila larval neuromuscular junction, dArc1 protein and mRNA are enriched in exosome-like EVs produced in the pre-synaptic bouton. 81 Immuno-EM analysis of lysed EVs showed capsid structures that contained Arc. Using selective pre-and post-synaptic genetic manipulations, Ashley and colleagues provide evidence that dArc1 capsids bearing dArc1 RNA are transferred to postsynaptic muscle fibers, and that this transfer is required for Arc protein expression in muscle. Functionally, the Drosophila study provides evidence that Arc capsids/ EVs are required for synapse maturation and activitydependent synapse formation. 81,82 Thus, Arc capsid transmission in EVs represents a novel form of intercellular communication, with Arc mRNA as one of the cargos ( Figure 1D).

| Hub versus capsid: A tantalizing dichotomy
A tantalizing dichotomy now exists between Arc as an intra-cellular signaling hub on one hand 59 and a capsid vehicle for intercellular RNA transfer on the other 82,83 ( Figure 1). Resolving the relationship between these seemingly disparate functional states is vital for progress. Here, we discuss recent discoveries on Arc structurefunction and regulation of oligomerization that are yielding exciting new insights into the molecular physiology of the protein and the path towards applications in systems neuroscience. The unique features of mammalian Arc are emphasized while drawing comparisons with dArc and retroviral Gag. Finally, we provide working models of mammalian Arc function in plasticity related to its oligomeric state: monomer/dimer, tetramer, large oligomer, and capsid.   (Figure 2A). Arc has a loose tertiary structure within and between its domains, suggesting a floppy protein with conformational flexibility.
The predicted evolutionary relationship to Gag was borne out by crystal structure analysis of the rat Arc CT, revealing 3D homology to the Gag capsid (CA) domain of human immunodeficiency virus (HIV) and Rous sarcoma virus. 79 Based on this homology the Arc CT is also known as the CA domain. Gag CA is comprised of two domains, the CA-NTD and CA-CTD, which self-assemble to form a capsid shell. 85 Arc CA also has two domains, the N-lobe and C-lobe, both of which are homologous to Gag CA-CTD. 79 Notably, the Arc N-lobe has evolved a ligand binding pocket that does not exist in retroviral Gag. The crystal  structure of the isolated N-lobe was obtained in complex with peptides of TARP γ-2 (a.k.a. stargazin), a transmembrane auxiliary subunit of AMPA-type glutamate receptors, and CaMKII-α. Identification of the N-lobe binding motif led to the validation of additional ligands: the Nmethyl-d-aspartate glutamate receptor (NMDAR) subunit 2A (GluN2A), the scaffolding protein guanylate kinase anchoring protein (GKAP), and the actin-binding protein WAVE1. 79,86 As a multiligand binding site, the N-lobe provides a compelling structural basis for hub signaling along with other established interaction sites on Arc. Arc N-lobe binding to stargazin is implicated in AMPAR endocytic trafficking. 79 However, the mechanisms regulated by Arc interaction with stargazin and other N-lobe ligands remain to be defined. An atomic resolution structure of a full-length Arc has yet to be achieved. However, lower resolution structural information of full-length Arc and subregions was obtained by small angle X-ray scattering (SAXS) analysis alongside synchrotron radiation circular dichroism (SRCD) spectroscopy. 84 The insolubility of Arc is caused by the NT region as all fragments lacking the region are monomeric. For SAXS analysis, the soluble full-length monomer was obtained by short exposure to high pH, which neutralizes the positive charge on the NT. In a hybrid model of Arc monomer ( Figure 2B), the oppositely charged domains interact, making a compact "closed" structure that stabilizes the linker region. Based on SRCD analysis and structural predictions, the Arc NT domain was proposed to be an antiparallel alpha-helical coiled coil. The compact state of Arc and relative size of the domains were further validated by intra-molecular fluorescence lifetime FRET imaging in CA1 pyramidal neurons in hippocampal slices. 84 Given the flexibility of the protein, 76 it is possible that Arc also has an "open" conformational state with an extended linker and different orientations of the domains. Such conformational changes might serve to activate or inhibit domain-specific functions of Arc in response to cellular factors and stimuli.
A major question is whether ligand binding to the Nlobe induces structural and functional changes. Using SAXS to assess conformational changes, Hallin and colleagues mixed partner peptides (GluN2A, stargazin, GKAP, and WAVE1) with the isolated CA, the separate N-and C-lobes, or CA flanked by the linker region and Cterminus. The peptides bound selectively to the N-lobe but did not elicit a major conformational change. 84 However, a subsequent molecular dynamic simulation of the N-lobe crystal structure, with and without bound stargazin peptide, showed greater flexibility in the absence of ligand. 86 The change in flexibility occurs in the N-terminal strand of the N-lobe adjoining the linker region. Upon binding of stargazin to the N-lobe pocket, the N-terminal strand folds back on the peptide to form a beta-sheet which stabilizes the structure. This observation from molecular dynamic simulations leaves open the possibility that ligand binding impacts the flexibility of the linker and orientation between domains.
In studies using pure phospholipid liposomes, the highly basic NT region was found to mediate Arc interaction with phospholipid membrane. 84,87 Palmitoylation of cysteine residues (C95, C96, and C98) in the NT domain allowed insertion of Arc into the hydrophobic core of the bilayer, and mutation of residues impaired Arc-dependent LTD in hippocampal neuronal cultures. 87 Because of the insolubility of full-length Arc, no data are available on the effects of ligand binding or membrane binding on protein conformation.
Given the evolutionary link with retroviruses, it is useful to contrast and compare Arc structure-function with that of HIV Gag. The relationships are summarized in Figure 3. Retroviral capsids consist of large lattices of hexamers that assemble into approximately spherical structures of variable size. 88 The capsid shell harbors and protects the viral genome which enters the host cell during infection. In the case of HIV, capsids are assembled by the Gag polyprotein precursor, which contains four major domains: matrix (MA), capsid (CA), nucleocapsid (NC), and p6. Each domain plays a distinct and crucial role in the formation of the immature virion. 89,90

OLIGOMERIZATION AND CAPSID FORMATION
Many proteins form oligomers where two or more monomers of a protein interact to form a larger complex. Smaller oligomers like dimers and tetramers are the most prevalent, and a survey of Escherichia coli proteins suggests that only one-fifth of proteins are solely monomeric. 91 Oligomeric forms can provide increased stability of the protein, create new interfaces for binding partners and regulate protein activity. Importantly, many proteins form reversible oligomers, in which the various oligomeric forms exist in equilibrium. The equilibrium may shift according to the intra-cellular environment, protein concentration, partner availability, and post-translational modifications.
Recombinant Arc has been shown to form reversible oligomers, 76,92 consistent with a physiological role. This differs from uncontrolled aggregation, where monomers assemble into irreversibly bound clusters, as seen in βamyloid deposits. 93 Myrum and colleagues monitored the size of human Arc by dynamic light scattering (DLS), showing monomers, dimers, and oligomers ranging up to 40 units, depending on the buffer conditions. In physiological phosphate buffer, a 12-unit oligomer predominated. 76 Negative-stain transmission EM analysis of purified protein in salt buffer shows irregular-shaped particles. However, subsequent work on the purified protein and functional studies demonstrated the existence of Arc capsids. 80,81 These discoveries sparked a further investigation of the mechanism of Arc self-association and the function of oligomers.

| Regulation of oligomerization by an Arc N-terminal helical coil motif
The isolated Gag CA self-associates through dimerization motifs, 94 which are also predicted in Arc CA. 95 Surprisingly, however, the isolated Arc CA is monomeric, 84,95,96 and the NT-region is needed for oligomerization and capsid formation. 80,84,97 A recent study identified a critical role for Coil-2 in the antiparallel coiled coil. 97 Employing affinity purification of ectopically expressed Arc in HEK293 cells, the isolated Coil-2 region undergoes self-association, whereas Coil-1 and other segments of the Arc protein do not have this property. Coil-2 harbors a 28-amino-acid stretch, the oligomerization region, that is necessary for self-association of full-length Arc (illustrated in Figures 2 and 3). Alanine substitution scanning further identified a 7-amino acid oligomerization motif ( 113 MHVWREV 119 ), critical for the self-association of fulllength Arc. Interestingly, protein cross-linking treatment of HEK293 cells transfected with the alanine substituted oligomerization motif mutant (Arc s113-119A ) or Arc WT revealed a low abundant dimer along with monomer for both proteins. Analysis of purified Arc s113-119A by different methods (DLS, SAXS, size exclusion chromatography, and transmission EM) showed a nearly homogenous dimer population, with no higher-order species. Both transmission and cryoEM analysis showed the presence of capsids of approx. 30 nm in diameter in Arc WT samples, whereas no capsids were found in samples of Arc s113-119A . 97 Seeking to identify the molecular structure of the Arc-Arc interface, a peptide of the oligomerization region was synthesized, and an atomic resolution crystal structure was obtained, showing a dimer arranged into an anti-parallel coiled coil. Point mutations of critical residues in the interaction surface (M113 and W116) inhibited self-association of full-length Arc. The work suggested that Arc self-association above the dimer stage and capsid formation requires NT-NT interactions, mediated by the Coil-2 interface (Figure 4, step 2). Structural analysis of Arc CA by nuclear magnetic resonance (NMR) spectroscopy demonstrated a monomeric bilobar structure. 96 The authors proposed that, in fulllength Arc, the CA might obtain a different orientation that enables oligomerization, possibly as a function of ligand binding to the N-lobe. When the isolated CA was exposed to high temperatures, structural rearrangement of the N-lobe was observed by CD spectrum analysis and NMR, while DLS showed the irreversible formation of a larger oligomeric population. Strikingly, these changes were abolished in the presence of the GluN2A peptide, suggesting that ligand binding to the N-lobe initiates a conformational change that inhibits temperature-induced oligomerization of Arc CA (Figure 4, step 4). However, whether ligand binding impacts full-length Arc oligomerization in vitro or in vivo is unknown.

| Regulation of oligomerization by phosphorylation of Arc CA
CaMKII is a major regulator of signal transduction for plasticity in the post-synaptic compartment and a crucial molecule for the structural organization of the postsynaptic density (PSD). 98 The Arc CA is phosphorylated by CaMKIIα at S260 in the N-lobe, close to the border with the C-lobe. 95 Using DLS to monitor oligomerization, purified full-length Arc formed a stable tetramer whereas constructs lacking the N-terminal half of Arc were monomeric. Reversible temperature-dependent oligomerization was shown, in agreement with a previous report. 76 Further experiments were done under conditions that resulted in 1.8 MDa particles, corresponding to approx. 32 Arc units (an octamer of tetramers). The introduction of the phosphomimetic S260D mutation blocked the generation of oligomers larger than tetramers (Figure 4, step 3). 95 Interestingly, similar inhibition of higher-order oligomerization was observed upon mutation of Gag CA-like dimerization motifs in the N-lobe and C-lobe. Coupling these observations, the authors propose that phosphorylation at S260 promotes the formation of an intra-molecular salt bridge that prevents the CA-CA intermolecular interactions needed for oligomerization above the tetramer stage. 95 CaMKIIα catalyzed phosphorylation at S260 was further implicated in adaptive learning and synaptic plasticity. Arc knockin mice harboring non-phosphorylatable S260A exhibited impaired learning during fear conditioning training. 95 In the analysis of DHPG-induced LTD in the CA1 region of hippocampal slices, LTD magnitude was enhanced in knockin mice relative to WT, whereas LTP induction was not affected. In Purkinje cells in cerebellar slices, ectopic expression of Arc WT protein induced synaptic depression which occluded with LTD induced by treatment with the protein kinase C activator phorbol-12,13-diacetate (PDA), while expression of Arc S260D phosphomimetic failed to induce synaptic depression. Combining biochemistry and electrophysiology, the work suggests that oligomers larger than tetramers are necessary for full expression of LTD, and phosphorylation of S260 inhibits oligomerization and dampens LTD. Eriksen and colleagues 97 reported that disruption of the oligomerization motif in Coil-2 blocks Arc-induced enhancement of transferrin endocytosis in HEK293 cells. Notably, mutation of the adjacent endophilin binding site in Coil-2 disrupts endocytosis without disrupting Arc selfassociation. Thus, current evidence suggests that higherorder Arc oligomers facilitate endocytosis and LTD.
Biochemical analysis of Arc protein-protein interactions using GST-pulldown showed that mutations that inhibit high-order oligomerization and dampen LTD are not associated with loss of binding to endophilin or stargazin, two binding partners implicated in AMPAR endocytosis and LTD. 28,79 The enhancement of LTD in oligomerization-competent Arc could be due to the recruitment of additional binding partners and/or more efficient endocytosis due to the concentration of binding partners in a multivalent oligomeric scaffold.
A recent study showed that Arc is phosphorylated by the serine/threonine kinase TNIK (Traf2 and NCKinteracting kinase) at Ser67 and T278. 70 Ser67 is located in a previously identified nuclear retention domain in NT Coil-1. 66 Studies with overexpression of Arc-Myc-FLAG in Neuro2a cells indicate a role for S67 phosphorylation in the nuclear export of Arc, allowing accumulation of Arc in an F-actin fraction. T278 is located in the short linear hinge between the N-lobe and C-lobe. Transmission EM analysis of recombinant Arc WT protein showed round capsid-like structures, whereas samples of ArcT278D phosphomimetic showed aggregates but were devoid of capsids. 70 DLS analysis also showed large diameter aggregates and a smaller diameter subpopulation. In the study of Zhang and colleagues, 95 T278 was identified as a protein kinase C (PKC) phosphorylation site in Arc transfected HEK293 cells. While evidence for PKC phosphorylation of Arc in neurons is lacking, DLS analysis showed that recombinant ArcT278D is able to form tetramers but not higherorder oligomers. 95 The discrepancy between studies in the DLS results for ArcT278D may reflect differences in the buffers used, 70 yet the studies converge in indicating a potential role for T278 phosphorylation in inhibiting capsid formation. Finally, it is interesting to note that in Gag CA, the flexibility of the short hinge segment between lobes is important for capsid assembly. 85 In Arc CA, glycine 277 is predicted to confer flexibility between lobes, and substitution with alanine at this site again results in a tetramer peak by DLS. 95 It has therefore been proposed that phosphorylation of adjacent T278 may impair oligomerization by restricting flexibility between lobes. 95

Arc interaction with mRNA
Pastuzyn and colleagues found that bacterially expressed Arc copurifies with RNA. 80 Fewer capsids were obtained after an RNA stripping procedure, while the addition of purified mRNA of enhanced green fluorescent protein (EGFP) increased the yield of regular-shaped capsids. This suggested that the interaction of Arc with RNA can promote capsid assembly. In brain lysates, Arc mRNA coimmunoprecipitated with Arc protein. 80 Hence, Arc interaction with its cognate RNA may be physiologically relevant.
The impact of RNA on the size of Arc oligomers was investigated in vitro using single-molecule total internal reflection fluorescence (smTIRF) microscopy. 97 In this method, a self-labelling SNAP tag on the Arc N-terminus was covalently labeled with a fluorescent dye, while biotinylation of the Arc C-terminus allowed tethering to a glass coverslip. The size of each Arc oligomer was determined by photobleaching, where the number of photobleaching steps corresponds to the number of dye molecules. Under these conditions, Arc WT and Arc s113-119A were mainly dimeric or trimeric. However, the addition of purified EGFP mRNA to Arc WT induced the formation of oligomers containing 40-70 Arc molecules, while adding the purified Arc mRNA coding region induced larger assembles in the range of 70-100 units, with the largest containing up to 170 molecules. This mRNA-induced facilitation of higher-order oligomerization (Figure 4, step 4) was almost completely abolished in the Arc s113-119A mutant. While it is still unknown whether these large oligomers measured by photobleaching represent capsids or subcapsid structures, the size range fits with the estimate of 130 units per Arc capsid. 97 In HIV, recognition of viral genomic RNA is mediated by trans-acting zinc finger motifs in the Gag NC domain. 90,99 Additionally, non-specific binding to host RNA is thought to function as a scaffold for Gag-Gag interactions and oligomerization. 90,[100][101][102] This interaction is mediated by a patch of basic residues at the NC N-terminus. 90,100 Although Arc does not have amino acid sequence similarity to Gag NC and lacks a zinc finger, the NT oligomerization motif is flanked by positively charged patches that might interact with polyanionic RNA. The fact that mRNAs for both EGFP and Arc facilitate oligomerization points to an electrostatic interaction rather than RNA sequence-specific binding to the protein. 97 Interestingly, the replacement of the NC domain with a leucine zipper coiled-coil is able to reinstate nucleic acidinduced Gag multimerization. 103,104 Thus, Arc Coil-2 has a similar function to Gag NC in mediating RNA-induced multimerization.
While the dimer is proposed as the building block for oligomeric assembly, the mechanism of Arc dimer formation is unknown. Dimer formation does not require the Coil-2 oligomerization motif or the predicted CA interaction motifs. However, results from smTIRF suggest that dimerization involves interaction with Arc mRNA 97 because protein samples treated with purified Arc mRNA contain dimers but are devoid of monomers ( Figure 4, step 1). As Arc monomers were abundant in samples treated with EGFP mRNA, dimerization may depend on binding to specific sequences in Arc mRNA.

RELEASE, UPTAKE, AND CARGO
Viral capsids are released from the host cell through the budding of the plasma membrane. The process of virion release depends on the type of virus, but in the case of HIV its release relies on different domains of the Gag protein.
The budding process is promoted by the interaction of the basic MA domain with the lipid bilayer, and by the p6 domain which recruits the endosomal sorting complex required for transport. Consequently, the viral capsid is packed within a membrane envelope which can be secreted from the host cell.
Arc capsids are packed into exosome-like EVs of cultured cortical neurons and released. 80 By transferring vesicle-containing medium to neuronal cultures from Arc knockout mice, it was shown that Arc capsids can be taken up by recipient cells by endocytosis, and the internalized Arc RNA is translated, with enhanced translation upon application of DHPG. Interestingly, Arc mRNA was also transmitted into cells after the addition of purified Arc containing non-enveloped capsids to the medium. This is surprising as membrane proteins are normally required for the transfer of mRNA across endosomal membranes.
The packaging and release of Arc capsids may be facilitated by the attachment of the Arc NT to the plasma membrane. 84,87 Recent experiments using giant unilamellar vesicles, a model system for the eukaryotic cell membrane, show that purified Arc promotes the formation of small vesicles that penetrate into the interior. 105 Such a mechanism may facilitate the formation of mRNAcontaining EVs from the plasma membrane. This study also demonstrates the intrinsic ability of Arc to change membrane curvature independently of associated proteins. Perhaps the intrinsic ability of Arc to curve membranes explains the ability of non-enveloped capsids to deliver RNA to the neuronal cytoplasm. 105,106 In the study of Pastuzyn et al., treatment of purified Arc with RNaseA did not affect the abundance of Arc mRNA or bacterial (Asn) mRNA, indicating that these transcripts are encapsulated and shielded from enzymatic degradation. 80 The abundance of Arc/Asn mRNA inside capsids was proportional to expression in bacterial lysates, consistent with a non-specific mechanism of encapsulation. A full characterization of cargo from endogenous Arc capsids has not been achieved but will be key to unraveling function. The small volume of Arc capsids is estimated to give an RNA carrying capacity of 10 kb, which is enough to accommodate only 2-3 copies of Arc mRNA. Even for microRNAs and other small RNAs, the carrying capacity would be low. However, low carrying capacity is a general feature of EVs. In various physiological and pathological processes involving intercellular RNA transfer by EVs, the transmission of RNA within vesicles is at low copy number. 107,108 Intercellular transfer of Arc protein and mRNA in the intact mammalian brain has not been shown. However, a recent study on dorsal root ganglion (DRG) responses to inflammation support a possible physiological role for Arc-containing EVs. 109 Arc is translated in axons of DRG neurons in response to inflammatory mediators. In Arc knockout mice, excessive skin vasodilation occurs in response to inflammatory challenge, and this response is inhibited by injection of a mixed EV pool that includes Arc-positive EVs but is not affected by control injection with Arc-negative EVs. It is not yet known whether endogenous Arc capsids are involved, as EVs were obtained by overexpression of Arc in a nociceptor cell line derived from DRG neurons.

AND CAPSID STRUCTURES
Drosophila has two Arc proteins, dArc1 and dArc2, encoded by separate genes. The mammalian and dArc proteins independently evolved from the domestication of different Ty3-Gypsy retrotransposons, resulting in proteins with distinct structures. 78 dArcs have a bilobar CA with N-lobe and C-lobe. However, the dArc N-lobe lacks the peptide ligand binding pocket of mammalian Arc. dArc proteins also lack the large NT domain of mammalian Arc (Figure 3). dArc1, but not dArc2, contains a structured zinc-finger pair at its C-terminus homologous to the NC domain of retroviruses and is predicted to be involved in RNA binding. This feature might explain why only dArc1 capsids are enriched in Arc mRNA. 81,110 A high-resolution crystal structure of the dArc1 CA reveals a homodimer and a dimerization interface in the C-lobe homologous to retroviral CA. 78 In another major advance, single particle cryo-EM was used to resolve the structure of dArc capsids. 110 The dArc proteins both assemble into icosahedral capsids, with the capsid shell formed by 240 copies of the dArc CA. While there is no NT domain, there is a short N-terminal stretch of 41 residues in dArc1 and 28 residues in dArc2. These predicted amphipathic αhelices form flexible spikes that extend outwards and inwards from the capsid shell. The spikes occlude openings in the shell and could be involved in membrane interactions and regulation of access to the capsid interior. 110 The interior of capsids is highly basic which could promote non-specific encapsulation of RNA, while the zinc fingers in dArc1 confer binding to dArc1 mRNA.
In mammals, the N-terminal strand of the N-lobe is exposed for ligand binding, whereas in Drosophila it tucks inside the N-lobe hydrophobic core. dArc does not bind stargazin peptide and lacks the binding site motif. 111 Interestingly, the mammalian Arc N-lobe in complex with ligand adopts a βstrand structure similar to dArc N-lobe. 78,111 As discussed in Hallin et al., 86 this raises the possibility that dArc N-lobe could bind peptide ligands under conditions that liberate the N-terminal strand, albeit with a binding specificity different from that of mammalian Arc N-lobe. 111 Another recent study performed structural characterization of the dArc lobe domains. 111 While the mammalian Arc lobes are monomeric, the isolated Nand C-lobes of dArc1 and dArc2 are all oligomeric in solution. Using constructs with a truncated N-terminal tail, the dArc2 N-lobe formed a novel domain-swapper dimer in the crystal structure. This contrasts with the penta/hexameric structures in dArc2 capsids, indicating a critical role for the N-terminal strand in capsid assembly. In HIV, the CA protein has multiple functions beyond its structural role in capsids. In the early phases of HIV-1 infection, CA contributes to viral trafficking and uncoating, recognition of host cellular proteins and nuclear import of the viral pre-integration complex. 112 Notably, the domain-swapped dArc2 dimer has structural homology to flavivirus C protein dimer, 111 with postulated roles in host cell transcription, mRNA splicing, and decay, 113 while one chain of the dArc2 dimer resembles histone core protein monomer. It will therefore be interesting to evaluate possible non-capsid roles of dArc2 in nuclear regulation of gene expression, for instance upon disassembly of dArc2 capsids in muscle at the neuromuscular junction.

DOMAIN IN OLIGOMERIZATION AND HUB FUNCTION
In the domestication of mammalian Arc from ancient retrotransposon elements, the NT domain has evolved functional properties of multiple Gag domains. Arc NT has only 18% sequence similarity to retroviral MA, from which it is predicted to have evolved, yet they share physicochemical properties. The MA domain is critical for targeting Gag to the plasma membrane. The targeting involves a patch of basic residues in the MA highly basic region and membrane anchoring by myristoylation. 90,114 Similarly, Arc interaction with the phospholipid membrane is mediated by electrostatic interactions with the Nterminal region 84 and palmitoylation of Coil-2 in the NT domain. 87 As discussed, the Coil-2 oligomerization motif also has functional similarity to Gag CA in mediating selfassociation, and to Gag NC in mediating RNA-facilitated oligomerization. These unique properties suggest a major, multifaceted role for the NT domain in regulating mammalian Arc function ( Figure 5).
The Arc dimer is proposed as the basic assembly unit and the addition of dimer units is supported by the Coil-2 oligomerization interface and facilitated by mRNA. The assembly from tetramers to higher-order oligomers further depends on CA-CA interactions. The most parsimonious model is that NT-NT interactions mediated by Coil-2 induce a conformational change that enables CA-CA interactions in further oligomerization. Functionally, evidence suggests that Arc oligomers larger than tetramers are necessary for the full expression of LTD. Biochemical data shows that the transition from tetramers to 32unit oligomers is arrested by phosphorylation of CA by CaMKII, TNIK, and PKC, and possibly by ligand binding to the N-lobe (Section 3). In this model, the NT serves to activate the capacity for CA oligomerization while phosphorylation of CA inactivates it.
The long, disordered linker region between the NT and CA also warrants mention as a target for regulation. Extracellular signal-regulated kinase (ERK), downstream of NMDAR and BDNF activation of TrkB receptors, drives Arc transcription and translation. 21,[115][116][117][118] The Arc linker region has a linear binding motif for ERK and is phosphorylated by ERK in vitro and in vivo on S206, at the border with the N-lobe. 69 In hippocampal neuronal cultures, S206 phosphorylation promotes cytosolic localization relative to nuclear localization of activity-induced Arc. Following LTP induction in the dentate gyrus of live rats, endogenous Arc undergoes enhanced S206 phosphorylation in the cytoskeletal fraction. Interestingly, the distal end of the linker (residues 195-206) binds AP2μ and dynamin 2 critical for clathrin-mediated endocytosis of AMPARs, as well as calnexin, an integral transmembrane protein of the endoplasmic reticulum. Calnexin is a negative regulator of clathrin-mediated endocytosis in F I G U R E 5 Pivotal role of NT domain in mammalian Arc function. Mammalian Arc has evolved functional properties of multiple HIV Gag domains (see Figure 3 for comparison). The large NT domain is unique to mammals and confers properties of membrane binding, self-association, and RNA-induced oligomerization. The NT domain is proposed to regulate the CA domain function of higher-order oligomerization and N-lobe ligand binding. The central disordered linker between mArc domains is also a prospective target of regulation as it binds several Arc protein partners and undergoes phosphorylation during in vivo LTP.

NT domain CA domain
Regulation -Membrane binding -Self-association -RNA-induced oligomerization -Ligand binding -Higher-order oligomerization Linker neurons, in addition to its canonical role as a chaperone in protein quality control. 119 Arc binds to the cytosolic tail of calnexin in BDNF-treated neurons in vitro and after LTP induction in vivo. 120 Notably, binding of the HIV-1 protein Nef to the C-tail of calnexin disrupts chaperoning function. 121 While the relationships between the binding partners, phosphorylation, and oligomerization remain to be explored, the linker segment closest to the N-lobe appears to be a nexus for regulation.

FUNCTION IN MAMMALIAN SYNAPTIC PLASTICITY
Arc is known as a dynamic regulator of intra-cellular mechanisms of synaptic plasticity with assumed cellautonomous functions, be they synaptic mechanisms or nuclear mechanisms. An outstanding issue that has puzzled researchers for a long time is how stimulus-induced Arc subserves opposite modifications of synaptic efficacy in various forms of LTD, LTP, and homeostatic synaptic scaling. 122 With recent advances, it seems clear that understanding the relationship between oligomerization and hub protein-protein interactions is key to resolving Arc's molecular functions. We propose that oligomerization occurs in discrete stages, with each stage representing a distinct functional state of the Arc hub. The cell biological function is determined by the specific set of binding partners, the concentration of the binding partners, and subcellular localization (dendrites/spines and soma/nucleus) including targeting to membranes, actin cytoskeleton, and multiprotein complexes such as the PSD.
We propose that Arc has five distinct states in the neuron (1) a nascent pool of Arc monomer and dimer at sites of translation, (2) a dimer that regulates actin cytoskeleton dynamics in LTP, (3) a tetramer that facilitates AMPAR endocytosis and supports basic LTD function of low amplitude, (4) a larger oligomer (32 units) that amplifies LTD, and (5) a capsid (130 units) that functions in intercellular signaling. Regulation of oligomeric state would occur by post-translational modifications, availability of binding partners, and local concentration of Arc. Below we outline a working hypothesis of bidirectional regulation of synaptic plasticity based on the Arc oligomeric state ( Figure 6).

| Working model of Arc monomer/ dimer function in LTP
Several lines of evidence suggest rapid turnover of newly synthesized Arc to support a time-window of LTP consolidation at the perforant input to the dentate gyrus. 122 Local infusion of Arc antisense oligodeoxynucleotides during the LTP maintenance phase results in reversion of LTP to baseline by 30 minutes along with marked inhibition of Arc protein synthesis. 25 Arc synthesis is required for the stabilization of nascent F-actin filaments in the perforant path termination zone on granule cell dendrites, 25 which in turn is required for stable structural enlargement of dendritic spines. 63 The dynamic role of the new Arc in actin filament reorganization fits with the function of a monomer or dimer, rather than a more metabolically stable oligomer ( Figure 6, state 2). Although Arc does not bind F-actin directly, it associates with the Factin-binding protein drebrin A. 64,70 Drebrin A is a major regulator of F-actin stability and spine stability. Arc is SUMOylated during LTP, which favors interaction with drebrin A. 64 Thus, selective targeting of F-actin regulation in LTP could occur through SUMOylation and association with drebrin A. ERK could also target Arc for function in LTP; ERK activity promotes local dendritic accumulation of Arc mRNA at stimulated perforant path synapses, 18,123 controls Arc translation in the synaptic compartment during LTP maintenance, 116 and phosphorylates Arc S206 in the cytoskeletal fraction. 69

| Working model of Arc tetramer and large oligomer in LTD
Arc associates with components of the endocytic machinery (AP2, dynamin 2, and endophilin 3) to enhance internalization of AMPARs during LTD. 60 Recombinant Arc has been shown to facilitate the polymerization of dynamin 2 and stimulate its GTPase activity in vitro. 124 We propose the following sequence of events. Arc initially interacts with AP2 in the process of recruiting clathrin to the membrane and selecting cargo. Arc then dissociates from AP2, oligomerizes, and serves as a scaffold for dynamin 2 and endophilin 3, which in turn results in the scission of endocytic vesicles containing receptors for internalization ( Figure 6, state 3). The smallest unit of the oligomer supporting endocytosis and LTD could be a tetramer, which based on available evidence is stabilized by CA phosphorylation. 95,97 The full expression of LTD requires larger oligomers, potentially a 32-unit octamer of tetramers 95 ( Figure 6, state 4). The enhanced LTD could be due to the concentration of binding partners on a multivalent Arc scaffold or to the recruitment of new partners.
We speculate that new partners are recruited to 32mer oligomeric Arc by selective exposure of the N-lobe ligand binding site. Arc interacts with the auxiliary subunit of AMPARs, TARP γ-2 (orstargazin). Stargazin and other TARP family members play an important role in regulating trafficking and plasma membrane surface mobility of AMPARs by interacting with PSD-95 and related scaffolding proteins. 125,126 The binding of the stargazin cytoplasmic tail to PSD95 serves to tether AMPARs in the post-synaptic membrane, across from pre-synaptic sites of glutamate release. Competitive binding of the Arc Nlobe to the stargazin cytoplasmic tail might disrupt tethering, resulting in increased lateral diffusion of AMPARs away from the PSD and increased endocytosis. 122 The Arc N-lobe is also well-suited for structural remodeling of the PSD through regulation of liquid-liquid phase separation. Interaction of the stargazin cytoplasmic tail with the PSD95 N-terminus regulates phase separation at the synapse. 127 Interaction of GKAP (aka DLGAP1) with the guanylate kinase domain at the PSD95 C-terminus is also crucial for PSD structure. 128,129 GKAP binds PSD95 through its five GKAP repeats, two of which are able to bind the Arc N-lobe. 79,86 Pulldown of Arc complexes from mouse forebrain shows that PSD-95 is the most abundant Arc interactor, with 72 proteins of 107 in the complex containing the Arc N-lobe consensus motif. 130 For full expression of LTD, we propose that higher-order Arc exposes N-lobe binding sites resulting in lateral movement and endocytosis of AMPARs along with structural remodeling of the PSD.

| On Arc capsid formation and function
The discovery of Arc capsids raises profound questions regarding mechanisms and subcellular sites of assembly, the capture of RNA cargo, and the impact of the released EVs at target sites. The Rous Sarcoma virus seems to start capsid formation by trafficking Gag proteins to the nucleus, where the association with RNA induces dimerization. 131,132 These early particles with RNA are then exported back to the cytosol for assembly to capsid structures. Arc also enters the nucleus where it can be retained or exported back to the cytoplasm through identified nuclear import, retention, and export sequences. 66 There is no data thus far on Arc oligomerization in the neuronal nucleus or evidence for binding to nuclear mRNA. However, fluorescence fluctuation spectroscopy of Arc-EGFP in transfected HeLa cells indicates that Arc is monomeric in the nucleus and monomeric or dimeric in the cytoplasm. 133 If this is the case in neurons, Arc mRNA-induced dimerization would occur in the cytoplasm rather than the nucleus. In HIV Gag, assembly starts in the cytosol before the immature capsid associates with the cellular membrane to complete its formation. Conceivably, pre-existing Arc protein resident in the PSD is utilized in capsids ( Figure 6, state 5). However, with an estimated 130 Arc units in a capsid, 97 assembly from newly synthesized protein is more likely.
The formation of Arc capsids in the cytoplasm following transcriptional activation would allow efficient capture of activity-induced RNAs. Current evidence indicates that Arc capsids encapsulate mRNA non-specifically. 80 However, Arc capsids could serve to capture a highly specific plasticity cargo depending on the location and time of capsid formation after the LTP or LTD induction event. LTP in the dentate gyrus is associated with different temporal patterns of expression of mRNA, microRNA, longnon-coding RNA, and differential expression of repeat elements including retrotransposons, simple DNA repeats, and tRNA. 6 The capsid function would therefore depend on the cross-section of RNA, which changes greatly during the first hours after LTP induction. Arc mRNA expression during LTP positively correlates with the expression of specific long-noncoding RNAs and retrotransposons including the endogenous retroviral sequence, MER21B, and the long-interspersed nuclear element-1, L1MD3. 6 It is difficult to speculate on functional outcomes without knowledge of capsid cargo. In the nervous system, EVs are implicated in several forms of neuron-glia cell interactions, while evidence for inter-neuronal signaling by EVs remains sparse. 83,[134][135][136][137] Arc capsids may represent a new form of communication specific to activity-dependent modification of neural circuits. The discovery of Arc capsid transfer between cells suggests that this form of Arc works through paracrine signaling, affecting multiple cells. Intercellular transfer of genetic information to neighboring cells may serve as a way of altering cellular state in response to network activity. The functional impact of Arc EVs will be specific to the cargo F I G U R E 6 Working hypothesis on Arc oligomer function in synaptic plasticity. Arc's ability to self-associate into different oligomeric states is likely to be a key mechanism in terms of its cellular functions, synaptic strengthening, and weakening. We propose that Arc has 5 states in the neuron: (1) Arc monomers and dimers at the site of Arc translation in the spine. (2) Arc dimer role in F-Actin cytoskeletal regulation during LTP. (3) Arc tetramers supporting AMPAR endocytosis and consequent LTD. (4) Arc 32-mers supporting enhanced (full) LTD through recruitment of PSD partners and enhanced lateral mobility of AMPARs (dotted arrows imply a shift between oligomeric states). (5) Large Arc capsids encapsulating mRNA and functioning in intra-cellular signaling. (6) Across all states of Arc, its function and oligomeric state are regulated by Arc protein concentration, availability of protein interaction partners and mRNA, post-translational modifications (PTMs) of the Arc protein, and its interaction/position at the cellular membrane. We refer to Sections 7.1-7.3 for a detailed description.
profile and the cellular targets (pre-synaptic neuron, adjacent post-synaptic neurons, and glial cell types) in the adult brain. Another interesting area to address is the identity of the target recipient cells for Arc capsids. If the capsid is released from a granule cell dendrite, would it go into another granule cell dendrite, to neighboring glia, or to the perforant path terminal? What effect may this have on plasticity in the hippocampus? At the neuromuscular junction in Drosophila larvae, transfer of Arc in capsids from the pre-synaptic boutons to muscle is critical for plasticity, yet the mechanism of Arc function in this context is unknown.

FUTURE PERSPECTIVES
Seminal advances in Arc structure and its biochemical and biophysical characterization provide a basis for understanding how Arc works. Current evidence suggests that the multifunctional nature of Arc relates to hub proteinprotein interactions and oligomeric state. Regulation of oligomerization by post-translational modifications and interactions with mRNA have been discovered, with surely much more to be revealed.
The data on oligomerization so far is almost exclusively from in vitro biochemical experiments on purified protein, and knowledge of endogenous Arc oligomerization and oligomeric states in vivo is lacking. Evidence of Arc capsids in mammalian brain tissue is lacking and no information is available on the dynamics of capsid formation and intercellular transfer following synaptic activation, plasticity, or learning. Given the multiple states of the protein and its complex regulation, insights gained from gene knockout and mRNA knockdown approaches are limited.
New tools are needed for imaging Arc activity-state (biosensors) and for probing domain-specific functions and regulatory mechanisms. Tools that would allow labeling or functional manipulation of endogenous Arc would be an advantage. To this end, single-domain antibodies (nanobodies) that bind the Arc CA domain have been developed. 138,139 The recombinant nanobodies bind with high affinity (low nM Kd) and are suitable for expression in mammalian cells as intra-bodies. One of the nanobodies, ArcNb H11, binds selectively to the N-lobe ligand pocket with 35 000 times higher affinity than stargazin, competitively inhibits stargazin binding, and allows affinity purification of intra-cellular Arc. As the molecular function of Arc continues to unfold, strategies for selective manipulation of Arc hub function, oligomerization, and capsid formation can be used to address numerous outstanding issues in systems neuroscience, including the contribution of Arc-dependent synaptic plasticity to neural circuit dynamics and information processing in learning and memory.

This work was supported by a Research Council of Norway
Toppforsk grant (249951) to CRB. MSE was supported by PhD fellowship from the University of Bergen, Faculty of Medicine.


s2fieldsofstudy: Biology Medicine

text:
Coacervate-mediated novel pancreatic cancer drug Aleuria Aurantia lectin delivery for augmented anticancer therapy

Background Pancreatic cancer, one of the cancers with the highest mortality rate, has very limited clinical treatment. Cancer cells express abnormal glycans on the surface, and some lectins with a high affinity for the glycans induce apoptosis in cancer. In this study, the efficacy of Aleuria Aurantia lectin (AAL) for the treatment of pancreatic cancer was evaluated and the efficacy improvement through AAL delivery with mPEGylated coacervate (mPEG-Coa) was investigated. Methods AAL was treated with pancreatic cancer cells, PANC-1, and the expression level of caspase-3 and subsequent apoptosis was analyzed. In particular, the anticancer efficacy of AAL was compared with that of concanavalin A, one of the representative anticancer lectins. Then, methoxypolyethylene glycol-poly(ethylene arginylaspartate diglyceride), a polycation, was synthesized, and an mPEG-Coa complex was prepared with polyanion heparin. The AAL was incorporated into the mPEG-Coa and the release kinetics of the AAL from the mPEG-Coa and the cargo protection capacity of the mPEG-Coa were evaluated. Finally, improved anticancer ability through Coa-mediated AAL delivery was assessed. Results These results indicated that AAL is a potential effective pancreatic cancer treatment. Moreover, mPEG-Coa rapidly released AAL at pH 6.5, an acidic condition in the cancer microenvironment. The initial rapid release of AAL effectively suppressed pancreatic cancer cells, and the continuous supply of AAL through the Coa transporter effectively inhibited proliferation recurrence of cancer cells. Conclusion AAL is a potential novel drug for the treatment of pancreatic cancer therapeutic agent. In addition, a continuous supply of drugs above the therapeutic threshold using mPEG-Coa could improve therapeutic efficacy. Supplementary Information The online version contains supplementary material available at 10.1186/s40824-022-00282-6.

Found ubiquitously in animals, plants, and fungi, lectins are carbohydrate-binding proteins that perform a variety of biological roles [5]. In particular, glycans on the surfaces of tumor cells influence tumor cell division, migration (metastasis), and recognition by the immune system [6]. Cancer cells exhibit a variety of altered glycosylation patterns in surface biomolecules such as glycolipids and glycoproteins, and the recognition of these aberrant changes may provide a basis for the discovery of biomarkers and therapeutic agents [7,8]. Because of their ability to discriminate these variances in carbohydrate structures, plant lectins are potential diagnostic reagents and promising candidates for medicinal and clinical applications in many research fields [9]. Moreover, it is known that lectin-induced cancer cell surface glycan binding induces caspasemediated apoptosis in cancer cells [10]. Consequently, several lectins with anticancer therapeutic properties have recently been spotlighted as natural anticancer agents. For instance, concanavalin A (Con A), a wellknown anticancer lectin that induces caspase-mediated apoptosis in cancer cells, is effective against a wide range of cancers, including melanoma and colon cancer [11]. In addition, Schwarz et al. showed the inhibitory effect of Con A on the proliferation of pancreatic cancer. In our previous study, Aleuria Aurantia lectin (AAL), a fucosetargeting lectin, was shown to have a high surface binding ability to pancreatic cancer cells compared to plant lectins Con A and CA19-9 antibody [12]. Hence, in this study, AAL was selected as a promising drug candidate for pancreatic cancer treatment and its therapeutic efficacy was analyzed.
Although biopharmaceuticals have exhibited excellent anticancer efficacy, a cargo drug directly administrated into a body could lose expected therapeutic functions due to short half-life, instability in blood circulation, enzymatic degradation, and immunogenicity [13]. Therefore, the development of a sustained-release exogenous delivery system that protects a therapeutic agent for a long period is necessary to increase therapeutic efficacy and efficiency. Polycation-based coacervates (Coa) can be excellent protein delivery carriers. A polycation methoxypolyethylene glycolpoly(ethylene arginylaspartate diglyceride) (mPEG-PEAD) forms a spherical droplet structure of mPEG-Coa in aqueous conditions through self-assembly with its polyanionic heparin counterpart via electrostatic interaction and following liquid-liquid phase separation [14,15]. Coa can protect the incorporated cargo protein, preserve its bioactivity, and induce a sustained release of cargo molecules. In our previous studies, the therapeutic efficacy of Coa-mediated exogenous protein delivery was demonstrated in various applications including bone regeneration [16,17], cartilage repair [18], angiogenesis [15], and skin wound healing [19,20]. Moreover, a simple chemical modification (i.e., mPEGylation) of the cation backbone effectively improved the colloidal stability of Coa structure [14,15]. Therefore, exogenous delivery of AAL using Coa could protect encapsulated cargo AAL from the harsh external environment while continuously releasing it, thereby improving their therapeutic efficacy (Fig. 1).
In the present study, we hypothesized that AAL lectin, which has a high affinity for pancreatic cancer surface glycans, is a superior treatment for pancreatic cancer, especially with the aid of mPEG-Coa for sustained AAL delivery and cargo protection. Consequently, a continuous supply of AAL with preserved bioactivity could achieve excellent anticancer effects. The current study investigated (1) anticancer efficacy of AAL, (2) Coa-mediated protective ability and sustained release kinetics of cargo AAL, and (3) enhanced anti-pancreatic cancer agents' efficacy through exogenous AAL delivery.

Comparison of anticancer efficacy between AAL and Con A
Pancreatic cancer cell line PANC-1 was cultured in a basal medium consisting of DMEM (89% (v/v)), penicillin-streptomycin solution (1% (v/v)), and FBS (10% (v/v)). When the confluency reached approximately 80%, PANC-1 cells were trypsinized, seeded at 100,000 cells/well on 24-well cell culture plates, and incubated at 37 °C, 5% CO 2 , and 95% humidity for 24 h. After 24 h, the old media was discarded and changed to fresh media containing 100 μg/mL of Con A or 100 μg/mL of AAL and incubated for 12 h. To compare the caspase-3 inducing ability between AAL and Con A, we performed a Caspase-3 assay according to the manufacturer's suggestion. In addition, to analyze the level of caspasemediated apoptosis, a TUNEL assay was carried out according to the manufacturer's protocol.

Pancreatic cancer-specific anticancer effects of AAL
PANC-1 cells were cultured as mentioned above and normal human pancreatic ductal epithelial cells H6C7 (Korean Cell Line Bank, Seoul, Republic of Korea, passage number 7) were cultured in keratinocyte-SFM (serum-free medium) with supplements. Each cell line was seeded at 10,000 cells/well on 96-well cell culture plates, and incubated at 37 °C, 5% CO 2 , and 95% humidity for 24 h. After 24 h, to analyze the pancreatic cancer-specific anticancer efficacy by the concentration of AAL, the old media was replaced with a medium containing 0-100 μg/mL of AAL and incubated for 24 h. After 24 h of incubation, cell viability was determined by WST-1 assay. WST-1 reagent was prepared by mixing EZ-Cytox with basal media at a 1:10 (v/v) ratio, treated to each well, and incubated for 3 h. Subsequently, optical density was measured at 440 nm by a microplate spectrophotometry.

Synthesis of mPEG-PEAD
First, 5 mmol of Fmoc-Asp, 5 mmol of EGDE, and 5 mg of TBAB were dissolved in 2.5 mL of 1,4-dioxane, and polymerization was done at 100 °C for 48 h. Obtained intermediate polymer, poly(ethylene Fmoc-aspartate diglyceride) (FPED), was washed using diethyl ether and dried under vacuum conditions. After that, the FPED was perfectly dissolved in 5 mL of DMF. Subsequently, 1 mmol of EDC, 1 mmol of NHS, and 5 mg of DMAP were mixed in FPED solution for 1 h. The number average molecular weight (Mn) and polydispersity index (PDI) of FPED were measured by Agilent gel permeation chromatography (GPC) system with Styragel GPC column (Waters) and 0.1% LiBr in DMF as a mobile phase at a flow rate of 1 mL/min. Then, 1 mmol of mPEG (MW 350) dissolved in 2 mL of DMF was applied to the mixture, and the reaction was continued for 48 h. After 48 h, 4 mL of piperidine was added and reacted for removing Fmoc

Fluorescence imaging of coacervates
For fluorescence imaging of Coa, a fluorescent dye, CellTracker Red, was incorporated into Coa. First, 4 μL of CellTracker Red was mixed with 50 μL of heparin solution (10 mg/mL), and then 400 μL of mPEG-PEAD was added. Supernatant was removed after centrifuging at 15,000 rpm for 15 min and resuspended with 200 μL of pH 7.4 PBS or pH 6.5 PBS. Coa complexes were observed using a fluorescence microscope (Nikon Ti-E) at different time intervals: 5-, 30-, and 60-min. Circularity (i.e., Colloidal stability) of Coa was estimated using randomly selected 30 particles by ImageJ software (http:// rsbweb. nih. gov/ ij/, National Institutes of Health, Bethesda, MD, USA).

Preparation of AAL loaded coacervates
10 mg/mL of mPEG-PEAD and heparin were separately prepared in PBS and sterilized using a 0.22 µm filter membrane. Then, 2 μL of AAL (0.5 mg/mL) was first mixed with 10 μL of heparin solution. Thereafter, 80 μL of mPEG-PEAD solution was added to the heparin:AAL mixture. The final mass ratio was 1:100:800 of AAL:heparin:mPEG-PEAD. An increase in the solution turbidity was immediately observed due to the instantaneous formation of Coa. Particle size of fabricated AAL loaded mPEG-Coa or empty mPEG-Coa (i.e., without AAL loading) was measured by Malvern Zetasizer.

In vitro AAL release kinetics
Herein, biotin-AAL was used to quantify unloaded and released AAL by reaction with streptavidin-HRP and substrate TMB. AAL loaded Coa was prepared as previously described. Each group containing 1 μg of biotin-AAL was placed in pH 7.4 PBS or pH 6.5 PBS and incubated at 37 °C for 21 days. After centrifuging at 15,000 xg for 15 min on days 0, 1, 2, 3, 5, 7, and 14, the supernatant was collected and replaced with PBS of the same pH. Day 0 sample was used to calculate AAL loading efficiency. Unloaded and released AAL in collected supernatants was measured using a custom ELISA-like assay. One hundred microliters of collected samples were transferred to a 96-well ELISA plate and incubated for 24 h. After adsorption of the lectins, the coated plates were blocked with 1% BSA, after washing with PBST. Then, 100 μL of avidin-HRP (25 ng/mL) was applied and incubated for 30 min. After incubation and washing, 100 μL of ABTS substrate solution was added to each well, incubated for 30 min, and the optical density was measured by a microplate spectrophotometry at 405 nm with wavelength correction set at 650 nm. AAL amount was calculated using a biotin-AAL standard curve.

Cargo AAL protection ability of coacervates in trypsin
Considering the loading efficiency, Coa containing 1 μg of AAL was prepared. AAL loaded Coa or bolus AAL was incubated in 100 μL of 500 ng/mL trypsin for 0, 12, or 24 h. At each predetermined time point, 100 μL of 1× protease inhibitor solution was added and incubated for 5 min to exclude further digestion by trypsin. Then, 50 μL of heparinase cocktail solution (1 μL of 4000 units/mL) was applied and incubated at 37 °C for 2 h for complete disruption of Coa complex. The remaining AAL was directly determined using ELISA-like lectin quantification method described above in the section 'In vitro AAL release kinetics' .

Statistical analysis
All quantitative data were expressed as mean standard deviation and analyzed by one-way analysis of variance (ANOVA) with Tukey's post-hoc method, using Graphpad Prism V 7.0 (Graphpad Software Inc., San Diego, CA, USA). Differences were considered statistically significant when the p-value < 0.05.

Anticancer efficacy of AAL
Caspase-3 induction by AAL treatment and corresponding apoptosis of pancreatic cancer cells were analyzed and compared to that of Con A (Fig. 2). When AAL was treated with pancreatic cancer cells PANC-1, the expression of caspase-3 was increased in the pancreatic cancer cells (Fig. 2A). In this regard, it could be carefully inferred that AAL, like other therapeutic lectins, triggered the downstream signaling pathways of caspase-3-mediated apoptosis including cytochrome C, caspase-9, and pro-caspase-3 [10,21]. Consequently, the expression of TUNEL, an indicator of apoptosis increased (Fig. 2B). In addition, AAL effectively Fig. 2 Anticancer mechanism of AAL against pancreatic cancer cell PANC-1. A Caspase-3 activity and B TUNEL expression (i.e., apoptosis) treated with AAL or Con A after 12 h Fig. 3 Biocompatibility of AAL to normal pancreas cell H6C7 and AAL concentration-dependent anticancer efficacy induced caspase-mediated pancreatic cancer apoptosis when compared with Con A.
The biocompatibility of AAL was also evaluated by investigating it's in vitro cytotoxicity in normal pancreas cells H6C7 (Fig. 3). Up to 100 μg/mL of AAL exhibited excellent biocompatibility toward H6C7 cells. On the other hand, concentration-dependent cytotoxicity was observed in PANC-1. When 100 μg/ mL of AAL was applied to the cells for 24 h, 99.2 ± 3.2% cell viability was observed in H6C7 cells, whereas only 19.3 ± 1.9% cell viability was detected in PANC-1.

Characterization of mPEG-PEAD
mPEG-PEAD polycation was synthesized according to the procedure described in Fig. 4A. Additionally, sequential chemical modification of the synthesized intermediates was analyzed using 1 H-NMR spectral techniques (Fig. 4B-D). 1 H-NMR spectrum of Fmoc-PED exhibited a signal at 5.0-5.4 (−CH proton of aspartate) and 3.2-4.6 ppm (methylene protons of glyceride moiety) (Fig. 4B). 1 H-NMR spectrum of mPEG-PED showed a signal at 3.2 ppm (−CH 3 group of mPEG) with 1.4-1.6 ppm (−NH 2 of in the PED polymer side chain) (Fig. 4C). In addition, 1 H-NMR spectrum of the final mPEG-PEAD polycation indicated signals at 1.3-1.8 ppm (conjugated arginine moiety) (Fig. 4D). Overall NMR spectral results revealed that the stepwise synthesis of mPEG-PEAD was successfully preceded. In addition, the number average molecular weight of FPED was 4559.3 g/mol with 1.5 PDI. Based on this results, degree of polymerization was calculated to approximately 8.6. The synthesized mPEG-PEAD polycation had a zeta potential of approximately 26.7 mV. In order to determine the neutral condition of Coa complex during electrostatic interaction and subsequent self-assembly, zeta potentials at varying weight ratios of mPEG-PEAD/heparin were measured (Fig. 5). 8:1 of mass ratio indicated a neutral charge property of fabricated mPEG-Coa. Moreover, the particle size of mPEG-Coa slightly increased from 304.3 nm in empty mPEG-Coa (i.e., without AAL loading) to 491.0 nm after AAL loading into the mPEG-Coa (Fig. S1).

Sustained release and cargo protection abilities of coacervate
Colloidal stability and pH-responsive structural deformation of mPEG-Coa were evaluated by morphological transformation. Fluorescence images revealed that the formed mPEG-Coa maintains a stable spherical structure at pH 7.4 ( Fig. 6A and B). On the other hand, an immediate deformation and random aggregation of mPEG-Coa structure were observed in an acidic environment (pH 6.4) (Fig. 6A and B).
Loading efficiency and release kinetics of cargo AAL in mPEG-Coa were evaluated by a custom ELISA-like lectin quantification assay. The initial loading efficiency of AAL into mPEG-Coa was over 99%. Although similar cargo AAL release rates were observed in both pH conditions on the first day, a burst release profile was observed in pH 6.5 thereafter (Fig. 6C). The mPEG-Coa in pH 7.4 exhibited a cumulative AAL release of 5.82 ± 0.15% for 14 days, whereas 27.68 ± 1.04% of release was detected in pH 6.5.
In addition to sustained delivery of cargo protein, mPEG-Coa mediated exogenous delivery offers effective cargo protection. To evaluate the cargo protection efficacy, AAL-loaded mPEG-Coa and naked bolus AAL were incubated in trypsin, a global protease. Significantly improved protection against a protein degradation enzyme in the surrounding environment was observed in encapsulated AAL in mPEG-Coa after 12 and 24 h at 37 °C (Fig. 6D). In contrast, in the naked AAL group, 67.33 ± 9.45% and 13.33 ± 6.11% of the initial AAL remained after 12 and 24 h of incubation, respectively.

Enhanced anticancer efficacy via coacervate mediated AAL delivery
Herein, PANC-1 cells were incubated with empty mPEG-Coa, bolus AAL, or AAL loaded mPEG-Coa for 5 days (Fig. 7). Treatment with empty mPEG-Coa did not induce any toxicity to PANC-1 for 5 days, indicating that the electrolyte components in mPEG-Coa had good biocompatibility. In addition, 77.2% of cell viability was observed on day 1 when PANC-1 was directly treated with bolus AAL. Although there was a higher cancer cell death with the treatment of bolus AAL than the control in all-time points, cancer cells slightly regrowth from day 1. Specifically, the viability of PANC-1 cells increased to 82.1 and 93.6% on days 3 and 5, respectively. On the

Discussion
Several plant lectins induce caspase-mediated apoptosis (i.e., programmed cell death) of cancer cells through interaction with sugar-binding receptors on the plasma membrane [22]. For instance, the following plant lectins have been used as caspase-mediated apoptosis for anticancer therapies: mistletoe lectin I for leukemia [23], Korean mistletoe for liver cancer [24] and squamous cell carcinoma [25], Dioclea violacea for glioma [26], and Polygonatum odoratum lectin for melanoma [27]. Con A is one of the representative anticancer lectins [28], which has been used for various anticancer treatments including breast cancer [29], melanoma [30], and hepatoma [28]. Activated caspase (a cysteine protease) could catalyze the cleavage of essential cellular proteins and trigger apoptosis [31]. In particular, since caspase-3 executed this cell death modality, we preferentially selected and tested caspase-3 as the main signaling molecule [32]. Accordingly, caspase-3 activity and subsequent apoptosis level by direct AAL treatment were determined in PANC-1 cells (Fig. 2). On the other hand, AAL was no toxic to normal pancreas cell H6C7 (Fig. 3). These results are closely related to the attraction of lectins to glycans on the cancer cell membranes. In our previous study, we demonstrated lectin-type dependent cellular surface affinities; (1) AAL showed a higher interaction with PANC-1 than Con A lectin, and (2) the affinity level of AAL to PANC-1 was also higher than in the other cell lines including H6C7, MDA-MB-231, HeLa, and human dermal fibroblasts [12]. In this regard, AAL is a potential candidate for a pancreatic cancer-specific therapeutic lectin with suitable safety for normal pancreas cells. To the best of our knowledge, this study was the first to investigate the anticancer properties of AAL lectin. Thus, sufficient follow-up studies on the in vivo safety and anticancer mechanism of AAL should be conducted to further confirm AAL as a successful anticancer agent and its mechanism of action.
Although direct treatment of AAL induced apoptosis in pancreatic cancer cells, administration of the protein without a delivery carrier could not achieve the expected therapeutic effect due to their short half-life [13]. Therefore, we synthesized mPEG-PEAD (Fig. 4) and fabricated mPEG-Coa as a AAL delivery carrier using polycation mPEG-PEAD and polyanion heparin.
Here, PEGylation to polycations provides steric stability to the coacervate complex in a harsh environment [33] and their colloidal stability in aqueous conditions could be improved [14,15]. The enhanced stability of mPEG-Coa by PEGylation and its usefulness as an excellent growth factor carrier were demonstrated in our previous study, which suggested that the optimal range of PEG chain length for cation modification (MW 350) should be considered in ionic environments. In addition, our mPEG-Coa showed excellent AAL loading efficiency of more than 99%. Similar high cargo loading efficiencies of over 96% were observed in our previous studies using mPEG-Coa for bone morphogenetic protein-2 [14] and vascular endothelial growth factor [15]. Thus, our mPEGylated PEAD-based Coa could be an effective AAL delivery platform with augmented colloidal stability. Moreover, the fabrication with 8:1 of mPEG-PEAD:heparin mass ratio (Fig. 5) was performed in subsequent studies, as the neutral surface charge of mPEG-Coa complex potentially prevents undesired non-specific adsorption of serum proteins, especially in a physiological environment, and further reduces the interference in diffusional release of cargo proteins in mPEG-Coa. The enhanced structural stability by mPEGylation was also demonstrated in our previous study. In this previous study, control Coa without PEG modification showed a randomly aggregated shape after 15 min of incubation in 0.3 M NaCl solution, whereas mPEG-Coa maintained its colloidal morphology for more than 30 min [14]. Similarly, our Coa also retained its spherical shape at neutral pH, whereas its structure immediately deformed and randomly aggregated in an acidic environment ( Fig. 6A and B). During the coacervation driven by electrostatic interactions between two polyelectrolytes (i.e., mPEG-PEAD cation and heparin), the change in the ionization degree of the components strongly affected the mutual affinity of the components, resulting in phase separation or transformation [34]. Therefore, mPEG-PEAD and heparin exposed to an acidic environment are protonated and the dissociation of mPEG-Coa complex commences. A similar phenomenon of the pH-dependent structural disruption was also observed in our previous study [35]. Here, cationic polymer poly(3-arginylamino propylene succinate-b-polypropylene glycol)-mediated Coa maintained its stable spherical shape for 24 h in a neutral environment, while dissociation of polyelectrolytes and subsequent deformation of Coa was observed within 1 h in an acidic environment (pH 5.0). It has been reported that the release of cargo proteins from PEAD-based Coa dominantly depends on (1) the dissociation constant (Kd) between heparin and cargo protein molecule and (2) hydrolysis of PEAD cation backbone [36]. Therefore, in acidic pH conditions, the modulated surrounding ionic strength and further protonation of mPEG-PEAD and heparin results in sequential dissociation of electrolytes, deformation of mPEG-Coa structure ( Fig. 6A and B), and accelerated release of cargo AAL (Fig. 6C). In particular, the pH-dependent cargo release profile indicated that our mPEG-Coa-mediated drug delivery system could burst and release the cargo AAL in the acidic tumor microenvironment (TME). In addition, the mPEG-Coa could not only regulate the sustained release profile of the cargo AAL, but also protect the cargo from the harsh external environment. After 24 h of trypsin exposure, 74.7% of the AAL remained when loaded on mPEG-Cao, whereas only 13.3% remained in the naked AAL (Fig. 6D). Cargo protection ability of Coa has also been demonstrated in previous studies [37,38]. For instance, when fibroblast growth factor-2 (FGF2) was loaded into Coa and exposed to trypsin, no degradation of cargo FGF2 was observed for 2 h, whereas naked FGF2 was completely degraded [37]. Moreover, after 10 h exposure of naked interleukin-12 (IL-12) to trypsin, only 2.1% of IL-12 was remained, whereas 70.1% of cargo IL-12 remained when loaded in Coa [38]. Furthermore, it has been also demonstrated that Coa-based delivery platform could not only prevent the degradation of cargo proteins from foreign enzymes, but also maintain the structure/ functionality of proteins [18,38]. In this study, since splenocytes secrete IFN-ϒ in response to IL-12, released IL-12 from Coa was collected and treated to mouse splenocyte. As a result, there was no significant difference in IFN secretion between the released IL-12-treated splenocyte group and the fresh IL-12-treated group, indicating that there was no effect on the functionality of released IL-12. In addition, released insulin-like growth factor-1 (IGF-1) from Coa, which embedded in 3D hydrogel was collected and treated with MCF-7 cells [18]. As a result, the growth of MCF-7 was promoted at the similar level as the group treated with fresh IGF-1. On the other hand, when bolus IGF-1 was directly loaded into the hydrogel (without coacervate-mediated protection), the promoted growth rate of MCF-7 cells by released bolus IFG-1 was not observed, as compared to the fresh IGF-1 treated group. These results demonstrated that our mPEG-Coa-based AAL delivery system could (1) protect incorporated cargo AALs from enzymatic degradation and sufficiently preserve their therapeutic activity and (2) induce pH-dependent AAL release, especially in acidic conditions (i.e., pH 6.5-6.8) of TME [39].
Finally, augmented anticancer efficacy of mPEG-Coamediated long-term AAL delivery, against pancreatic cancer cells, was investigated (Fig. 7). Here, empty Coa had no effect on PANC-1 cells for 5 days. Although cationic polymer-based delivery systems have been actively developed in recent decades, their toxicity hampers the clinical translation for tissue regeneration and disease treatment [36,40]. While the deleterious effects of polycations could damage cell membranes and induce apoptosis [41,42], suitable in vitro and in vivo biocompatibility of PEAD-based Coa has been demonstrated through a series of studies [14,15,36]. Interestingly, the administration of bolus AAL provided a more abundant dose of available AAL in the surrounding environment during the initial interaction with cancer cells, but facilitated cancer cell death was detected in mPEG-Coa-mediated AAL treatment. Moreover, viability of bolus AAL treated PANC-1 cells was gradually increased from days 1 (77.2% of viability) to 5 (93.6% of viability), indicating a recurrence proliferation of PANC-1 cells. However, the treatment using released AAL from mPEG-Coa effectively suppressed the proliferation of PANC-1 cells up to 32% on day 5. When the cargo protein is released from Coa by hydrolysis of PEAD backbone, it is most likely in the form of a cargo:heparin complex, where heparin could stabilize and protect the cargo protein and induce the binding of the protein to its receptor [38,43]. Therefore, the protein released from Coa could reveal better bioactivity than the freshly prepared bolus protein. For instance, interleukin-12 (IL-12) released from Coa exhibited improved bioactivity of up to 762.7% compared to the same amount of fresh IL-12 [38]. As compared to the inactivation of bolus AAL (i.e., without cargo protection) and subsequently reduced apoptosis-mediated PANC-1 cancer cells, released cargo AAL from mPEG-Coa (1) efficiently maintained its bioactivity via the protection efficacy of carriers and (2) effectively induced PANC-1 death, even with the less available therapeutic dose (i.e., 5.4, 5.6, and 5.7% cumulative release in pH 7.4 and 12.5, 24.2, and 27.1% in pH 6.5 on day 1, 3, and 5 in Fig. 6C) than directly applied bolus AALs. Therefore, rapid release of cargo AAL from mPEG-Coa by pH in TME could successfully induce early cancer cell inhibition. The late gradual release profile of AAL from mPEG-Coa also demonstrated the importance of a continuous supply of therapeutically available AAL doses above the threshold for anticancer treatment and prevention of proliferation recurrence.

Conclusion
In this study, we evaluated the anticancer efficacy of AAL, a novel protein drug candidate for the treatment of pancreatic cancer. AAL induced caspase-3 mediated apoptosis of PANC-1 cells with an efficacy that was higher than one of the representative anticancer Con A. In addition, AAL induced pancreatic cancer cell death in a concentration-dependent manner without affecting normal pancreas cells. Moreover, in this study, the modulated coacervate complex by mPEGylation into a polycation component (i.e., mPEG-Coa) could protect the incorporated cargo AALs from the surrounding environment and exhibited pH-responsive early burst and late gradual release kinetics, especially in acidic pH in TME. Subsequently, continuously supplied AALs above the therapeutic threshold from mPEG-Coa effectively suppressed the proliferation of pancreatic cancer cells. However, since this present study was the first trial of AAL-based cancer treatment, a follow-up study on the anticancer mechanism and the clinical safety of cargo AAL should be further performed. Nevertheless, it could be reasonably concluded that (1) AAL is a potentially effective treatment for pancreatic cancer and (2) continuous supply of this lectin-based anticancer agent through mPEG-Coa-mediated exogenous delivery could significantly inhibit cancer progress and proliferation recurrence. Furthermore, our previous results in enhanced cellular activities by delivering a variety of proteins/cytokines demonstrated the mPEG-Coa as an effective carrier that can improve therapeutic functionalities of not only AAL but also various conventional anticancer and protein drugs.