GPCRs in the round: SMA-like copolymers and SMALPs as a platform for investigating GPCRs.

G-protein-coupled receptors (GPCRs) are the largest family of membrane proteins, regulate a plethora of physiological responses and are the therapeutic target for 30-40% of clinically-prescribed drugs. They are integral membrane proteins deeply embedded in the plasma membrane where they activate intracellular signalling via coupling to G-proteins and ß-arrestin. GPCRs are in intimate association with the bilayer lipids and that lipid environment regulates the signalling functions of GPCRs. This complex lipid 'landscape' is both heterogeneous and dynamic. GPCR function is modulated by bulk membrane properties including membrane fluidity, microdomains, curvature, thickness and asymmetry but GPCRs are also regulated by specific lipid:GPCR binding, including cholesterol and anionic lipids. Understanding the molecular mechanisms whereby GPCR signalling is regulated by lipids is a very active area of research currently. A major advance in membrane protein research in recent years was the application of poly(styrene-co-maleic acid) (SMA) copolymers. These spontaneously generate SMA lipid particles (SMALPs) encapsulating membrane protein in a nano-scale disc of cell membrane, thereby removing the historical need for detergent and preserving lipid:GPCR interaction. The focus of this review is how GPCR-SMALPs are increasing our understanding of GPCR structure and function at the molecular level. Furthermore, an increasing number of 'second generation' SMA-like copolymers have been reported recently. These are reviewed from the context of increasing our understanding of GPCR molecular mechanisms. Moreover, their potential as a novel platform for downstream biophysical and structural analyses is assessed and looking ahead, the translational application of SMA-like copolymers to GPCR drug discovery programmes in the future is considered.

The Evolution of SNAP-Tag Labels.

The conjugation of proteins with synthetic molecules can be conducted in many different ways. In this Perspective, we focus on tag-based techniques and specifically on the SNAP-tag technology. The SNAP-tag technology makes use of a fusion protein between a protein of interest and an enzyme tag that enables the actual conjugation reaction. The SNAP-tag is based on the O6-alkylguanine-DNA alkyltransferase (AGT) enzyme and is optimized to react selectively with O6-benzylguanine (BG) substrates. BG-containing dye derivatives have frequently been used to introduce a fluorescent tag to a specific protein. We believe that the site-specific conjugation of polymers to proteins can significantly benefit from the SNAP-tag technology. Especially, polymers synthesized via reversible deactivation radical polymerization allow for the facile introduction of a BG end group to enable SNAP-tag conjugation.

Chemical Identity of Poly(N-vinylpyrrolidone) End Groups Impact Shape Evolution During the Synthesis of Ag Nanostructures.

Ag nanocubes (AgNCs) are predominantly synthesized by the polyol method, where the solvent (ethylene glycol) is considered the reducing agent and poly(N-vinylpyrrolidone) (PVP) the shape-directing agent. An experimental phase diagram for the formation of Ag nanocubes as a function of PVP monomer concentration (Cm) and molecular weight (Mw) demonstrated end groups of PVP impact the final Ag product. Measured rates of the initial Ag+ reduction at different PVP Cm and Mw confirmed the reducing effect originates from end-groups. PVP with well-defined aldehyde and hydroxyl end groups lead to the formation of Ag nanocubes and nanowires respectively, indicating the faster reducing agent formed kinetically preferred nanowires. We demonstrate PVP end-groups induce initial reduction of Ag+ to form seeds followed by autocatalytic reduction of Ag+ by ethylene glycol (and not solvent oxidation products) to form Ag nanostructures. The current study enabled a quantitative description of the role of PVP in nanoparticle shape-control and demonstrates a unique opportunity to design nanostructures by combining nanoparticle synthesis with polymer design to introduce specific physicochemical properties.

Influence of DIBMA Polymer Length on Lipid Nanodisc Formation and Membrane Protein Extraction.

Polymer-based lipid nanoparticles like styrene-maleic acid lipid particles have revolutionized the study of membrane proteins. More recently, alternative polymers such as poly(diisobutylene-alt-maleic acid) (DIBMA) have been used in this field. DIBMA is commonly synthesized via conventional radical copolymerization. In order to study the influence of its chain length on lipid nanodisc formation and membrane protein extraction, we synthesized DIBMA with molar masses varying from 1.2-12 kDa via RAFT-mediated polymerization. For molar masses in the range of 3-7 kDa, the rate of lipid nanodisc formation was the highest and similar to those of poly(styrene-co-maleic acid) (SMA) and commercially available DIBMA. ZipA solubilization efficiency was significantly higher than for commercially available DIBMA and similar to SMA (circa 75%). Furthermore, RAFT-made DIBMA with a molar mass of 1.2-3.9 kDa showed a much cleaner separation on SDS-PAGE, without the smearing that is typically seen for SMA and commercially available DIBMA.

Iterative RAFT-Mediated Copolymerization of Styrene and Maleic Anhydride toward Sequence- and Length-Controlled Copolymers and Their Applications for Solubilizing Lipid Membranes.

The use of poly(styrene-co-maleic acid) (SMA) for the solubilization of lipid membranes and membrane proteins is becoming more widespread, and with this, the need increases to better understand the chemical properties of the copolymer and how these translate into membrane solubilization properties. SMA comes in many different flavors that include the ratio of styrene to maleic acid, comonomer sequence distribution, average chain length, dispersity, and potential chemical modifications. In this work, the synthesis and membrane active properties are described for 2:1 (periodic) SMA copolymers with Mw varying from ∼1.4 to 6 kDa. The copolymers were obtained via an iterative RAFT-mediated radical polymerization. Characterization of these polymers showed that they represent a well-defined series in terms of chain length and overall composition (FMAnh ∼ 0.33), but that there is heterogeneity in comonomer sequence distribution (FMSS ∼ 0.50) and some dispersity in chain length (1.1 < D < 1.6), particularly for the larger copolymers. Investigation of the interaction of these polymers with phosphatidylcholine lipid self-assemblies showed that all copolymers inserted equally effectively into lipid monolayers, independent of the copolymer length. Nonetheless, smaller polymers were more effective at solubilizing lipid bilayers into nanodiscs, possibly because longer polymers are more prone to become intertwined with each other, thereby hampering their solubilization efficiency. Nanodisc sizes were independent of the copolymer length. However, nanodiscs formed with larger copolymers were found to undergo slower lipid exchange, indicating a higher stability. The results highlight the usefulness of having well-defined copolymers for systematic studies.

Poly(N-vinylpyrrolidone) Antimalaria Conjugates of Membrane-Disruptive Peptides.

The concepts of polymer-peptide conjugation and self-assembly were applied to antimicrobial peptides (AMPs) in the development of a targeted antimalaria drug delivery construct. This study describes the synthesis of α-acetal, ω-xanthate heterotelechelic poly(N-vinylpyrrolidone) (PVP) via reversible addition-fragmentation chain transfer (RAFT)-mediated polymerization, followed by postpolymerization deprotection to yield α-aldehyde, ω-thiol heterotelechelic PVP. A specific targeting peptide, GSRSKGT, for Plasmodium falciparum-infected erythrocytes was used to sparsely decorate the α-chain ends via reductive amination while cyclic decapeptides from the tyrocidine group were conjugated to the ω-chain end via thiol-ene Michael addition. The resultant constructs were self-assembled into micellar nanoaggregates whose sizes and morphologies were determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The in vitro activity and selectivity of the conjugates were evaluated against intraerythrocytic P. falciparum parasites.

Degradation of Proteins and Starch by Combined Immobilization of Protease, α-Amylase and ß-Galactosidase on a Single Electrospun Nanofibrous Membrane.

Two commercially available enzymes, Dextrozyme (α-amylase) and Esperase (protease), were covalently immobilized on non-woven electrospun poly(styrene-co-maleic anhydride) nanofiber mats with partial retention of their catalytic activity. Immobilization was achieved for the enzymes on their own as well as in different combinations with an additional enzyme, ß-galactosidase, on the same non-woven nanofiber mat. This experiment yielded a universal method for immobilizing different combinations of enzymes with nanofibrous mats containing maleic anhydride (MAnh) residues in the polymer backbone.

Synthesis and Cell Interaction of Statistical l-Arginine-Glycine-l-Aspartic Acid Terpolypeptides.

Copolymerizations and terpolymerizations of N-carboxyanhydrides (NCAs) of glycine (Gly), Nδ-carbobenzyloxy-l-ornithine (Z-Orn), and ß-benzyl-l-aspartate (Bz-Asp) were investigated. In situ 1H NMR spectroscopy was used to monitor individual comonomer consumptions during binary and ternary copolymerizations. The six relevant reactivity ratios were determined from copolymerizations of the NCAs of amino acids via nonlinear least-squares curve fitting. The reactivity ratios were subsequently used to maximize the occurrence of the Asp-Gly-Orn ( DGR') sequence in the terpolymers. Terpolymers with variable probability of occurrence of DGR' were prepared in the lab. Subsequently, the ornithine residues on the terpolymers were converted to l-arginine (R) residues via guanidination reaction after removal of the protecting groups. The resulting DGR terpolymers translate to traditional peptides and proteins with variable RGD content, due to the convention in nomenclature that peptides are depicted from N- to C-terminus, whereas the NCA ring-opening polymerization is conducted from C- to N-terminus. The l-arginine containing terpolymers were evaluated for cell interaction, where it was found that neuronal cells display enhanced adhesion and process formation when plated in the presence of statistical DGR terpolymers.

Influence of Poly(styrene- co-maleic acid) Copolymer Structure on the Properties and Self-Assembly of SMALP Nanodiscs.

Polymer stabilized nanodiscs are self-assembled structures composed of a polymer belt that wraps around a segment of lipid bilayer, and as such are capable of encapsulating membrane proteins directly from the cell membrane. To date, most studies on these nanodiscs have used poly(styrene- co-maleic acid) (SMA) with the term SMA-lipid particles (SMALPs) coined to describe them. In this study, we have determined the physical and thermodynamic properties of such nanodiscs made with two different SMA copolymers. These include a widely used and commercially available statistical poly(styrene- co-maleic acid) copolymer (coSMA) and a reversible addition-fragmentation chain transfer synthesized copolymer with narrow molecular weight distribution and alternating styrene and maleic acid groups with a polystyrene tail, (altSMA). We define phase diagrams for each polymer, and show that, regardless of polymer topological structure, self-assembly is driven by the free energy change associated with the polymers. We also show that nanodisc size is polymer dependent, but can be modified by varying polymer concentration. The thermal stability of each nanodisc type is similar, and both can effectively solubilize proteins from the E. coli membrane. These data show the potential for the development of different SMA polymers with controllable properties to produce nanodiscs that can be optimized for specific applications and will enable more optimized and widespread use of the SMA-based nanodiscs in membrane protein research.

Synthesis, Characterization, and Evaluation of Cytotoxicity of Poly(3-methylene-2-pyrrolidone).

The homo- and copolymerization of 3-methylene-2-pyrrolidone (3M2P) is introduced. 3M2P is readily polymerized via conventional free radical polymerization, and two reversible deactivation radical polymerization methods including reversible addition-fragmentation (chain) transfer and single-electron-transfer living radical polymerization. Poly(3M2P) has a high thermal stability and a very high glass transition temperature. Poly(3M2P) does not dissolve in most common organic solvents, but it has a high aqueous solubility. Cytotoxicity tests reveal that it is nontoxic to cells, even up to concentrations of 1 mg/mL. This adds poly(3M2P) to the family of water-soluble and biocompatible pyrrolidone-based vinyl polymers.

Deformation of the water/oil interface during the adsorption of sterically stabilized particles.

The adsorption of sterically stabilized colloids at water/oil (w/o) interfaces is studied experimentally by the formation of Pickering emulsions. Specifically, the effect of the steric stabilizer with respect to the rate of particle adsorption is investigated. Uniform, micrometer-sized poly(methyl methacrylate) (pMMA) particles, which are sterically stabilized with poly(isobutylene) (pIB), are used. The pIB concentration on the particle surface (ΓPIB) is controlled during the synthesis by adjusting the pIB/monomer ratio. Pickering emulsions are formed directly by the addition of water to the nonaqueous pMMA dispersions and subsequent emulsification. A strong dependence of the rate of particle adsorption on ΓPIB is found. The rate constant k for particle adsorption decays exponentially with ΓPIB, which suggest the use of a Boltzmann factor to model the experimentally found rate constants. The experimental results can be explained when the activation barrier for particle adsorption EA is of the same order as the average kinetic energy EK of a particle colliding with an emulsion droplet, which is equivalent to 10(5) kBT. Interestingly, this makes EA several orders of magnitude greater than the steric interaction with another particle. A possible mechanism that can lead to such a significant repulsive force is the inhibited drainage of solvent between the particle and o/w interface. Deformation of the o/w interface then occurs, when the solvent does not have time to drain, which results in a dramatic increase in the interfacial energy. This study identified the relevance of drainage in the formation of Pickering emulsions.

Templated hierarchical self-assembly of poly(p-aryltriazole) foldamers.

A biomimetic approach has been used for the templated self-assembly of a helical poly(para-aryltriazole) foldamer. The solvophobic folding process yields helices that further self-assemble into long nanotubes (see picture; scale bar: 100 nm). Constructs of controlled length and chirality can be generated by applying a poly(γ-benzyl-l-glutamate) scaffold at the appropriate assembly conditions, mimicking tobacco mosaic virus self-assembly.

Poly(N-vinylpyrrolidone)-block-poly(vinyl acetate) as a drug delivery vehicle for hydrophobic drugs.

Poly((N-vinylpyrrolidone)-block-poly(vinyl acetate)) (PVP-b-PVAc) block copolymers of varying molecular weight and hydrophobic block lengths were synthesized via controlled radical polymerization and investigated as carriers for the solubilization of highly hydrophobic riminophenazine compounds. These compounds have recently been shown to exhibit a strong activity against a variety of cancer types. PVP-b-PVAc self-assembles into polymer vesicles in aqueous media, and the dialysis method was used to load the water-insoluble drug (clofazimine) into these polymer vesicles. The polymer vesicles were characterized by 1H NMR spectroscopy to confirm vesicle formation and the incorporation of the anticancer drugs into the polymer vesicles. Dynamic light scattering was used to determine the particle size and particle size distribution of the drug-loaded vesicles as well as the stability of the vesicles under physiological conditions. The size of the polymer vesicles did not increase upon loading with clofazimine, and the particle size of 180-200 nm and the narrow particle size distribution were maintained. The morphology of the vesicles was examined by transmission electron microscopy. The polymer vesicles had a relatively high drug loading capacity of 20 wt %. In vitro cytotoxicity studies of PVP-b-PVAc and drug-loaded PVP-b-PVAc were performed against MDA-MB-231 multidrug-resistant breast epithelial cancer cells and MCF12A nontumorigenic breast epithelial cells. In vitro experiments demonstrated that the PVP-b-PVAc drug carrier showed no cytotoxicity, which confirms the biocompatibility of the PVP-b-PVAc drug carrier. The results indicate that the present PVP-b-PVAc block copolymer could be a potential candidate as a drug carrier for hydrophobic drugs.

Immobilized furanone derivatives as inhibitors for adhesion of bacteria on modified poly(styrene-co-maleic anhydride).

The ability of brominated furanones and other furanone compounds with 2(3H) and 2(5H) cores to inhibit bacterial adhesion of surfaces as well deactivate (destroy) them has been previously reported. The furanone derivatives 4-(2-(2-aminoethoxy)-2,5-dimethyl-3(2H)-furanone and 5-(2-(2-aminoethoxy)-ethoxy)methyl)-2(5H)-furanone were synthesized in our laboratory. These furanone derivatives were then covalently immobilized onto poly(styrene-co-maleic anhydride) (SMA) and electrospun to fabricate nonwoven nanofibrous mats with antimicrobial and cell-adhesion inhibition properties. The electrospun nanofibrous mats were tested for their ability to inhibit cell attachment by strains of bacteria commonly found in water ( Klebsiella pneumoniae Xen 39, Staphylococcus aureus Xen 36, Escherichia coli Xen 14, Pseudomonas aeruginosa Xen 5, and Salmonella tymphimurium Xen 26). Proton nuclear magnetic resonance spectroscopy ((1)H NMR), electrospray mass spectroscopy (ES-MS), and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) were used to confirm the structures of the synthesized furanones as well as their successful immobilization on SMA. To ascertain that the immobilized furanone compounds do not leach into filtered water, samples of water, filtered through the nanofibrous mats were analyzed using gas chromatography coupled with mass spectroscopy (GC-MS). The morphology of the electrospun nanofibers was characterized using scanning electron microscopy (SEM).

Investigation of the 3D Printability of Covalently Cross-Linked Polypeptide-Based Hydrogels.

The 3D printability of poly(l-lysine-ran-l-alanine) and four-arm poly(ethylene glycol) (P(KA)/4-PEG) hydrogels as 3D biomaterial inks was investigated using two approaches to develop P(KA)/4-PEG into 3D biomaterial inks. Only the "composite microgel" inks were 3D printable. In this approach, P(KA)/4-PEG hydrogels were processed into microparticles and incorporated into a polymer solution to produce a composite microgel paste. Polymer solutions composed of either 4-arm PEG-acrylate (4-PEG-Ac), chitosan (CS), or poly(vinyl alcohol) (PVA) were used as the matrix material for the composite paste. The three respective composite microgel inks displayed good 3D printability in terms of extrudability, layer-stacking ability, solidification mechanism, and 3D print fidelity. The biocompatibility of P(KA)/4-PEG hydrogels was retained in the 3D printed scaffolds, and the biofunctionality of bioinert 4-PEG and PVA hydrogels was enhanced. CS-P(KA)/4-PEG inks demonstrated excellent 3D printability and proved highly successful in printing scaffolds with a narrow strand diameter (∼200 µm) and narrow strand spacing (∼500 µm) while the integrity of the vertical and horizontal pores was maintained. Using different needle IDs and strand spacing, certain physical properties of the hydrogels could be tuned, while the 3D printed porosity was kept constant. This included the surface area to volume ratio, the macropore sizes, and the mechanical properties. The scaffolds demonstrated adequate adhesion and spreading of NIH 3T3 fibroblasts seeded on the scaffold surfaces for 4 days. Consequently, the scaffolds were considered suitable for potential applications in wound healing, as well as other soft tissue engineering applications. Apart from the contribution to new 3D biomaterial inks, this work also presented a new and facile method of processing covalently cross-linked hydrogels into 3D printed scaffolds. This could potentially "unlock" the 3D printability of biofunctional hydrogels, which are generally excluded from 3D printing applications.

Macrophage-Targeted Hydroxychloroquine Nanotherapeutics for Rheumatoid Arthritis Therapy.

Rheumatoid arthritis (RA) is an autoimmune disease with unclear pathogenesis. Hydroxychloroquine (HCQ), despite its moderate anti-RA efficacy, is among the few clinical drugs used for RA therapy. Macrophages reportedly play a vital role in RA. Here, we designed and explored macrophage-targeted HCQ nanotherapeutics based on mannose-functionalized polymersomes (MP-HCQ) for RA therapy. Notably, MP-HCQ exhibited favorable properties of less than 50 nm size, glutathione-accelerated HCQ release, and M1 phenotype macrophage (M1M) targetability, leading to repolarization of macrophages to anti-inflammatory M2 phenotype (M2M), reduced secretion of pro-inflammatory cytokines (IL-6), and upregulation of anti-inflammatory cytokines (IL-10). The therapeutic studies in the zymosan-induced RA (ZIA) mouse model showed marked accumulation of MP-HCQ in the inflammation sites, ameliorated symptoms of RA joints, significantly reduced IL-6, TNF-α, and IL-1ß, and increased IL-10 and TGF-ß compared with free HCQ. The analyses of RA joints disclosed greatly amplified M2M and declined mature DCs, CD4+ T cells, and CD8+ T cells. In accordance, MP-HCQ significantly reduced the damage of RA joints, cartilages, and bones compared to free HCQ and non-targeted controls. Macrophage-targeted HCQ nanotherapeutics therefore appears as a highly potent treatment for RA.

Pickering emulsions: wetting and colloidal stability of hairy particles--a self-consistent field theory.

The assembly of sterically stabilized colloids at liquid-liquid interfaces is studied with the self-consistent field (SCF) theory using the discretization scheme that was developed by Scheutjens, Fleer, and co-workers. The model is based on a poly(methyl methacrylate) (pMMA) particle with poly(isobutylene) (pIB) grafted to the surface. The stabilizing groups on the particle surface have a significant effect on the interfacial assembly and, therefore, also on the formation and properties of Pickering emulsions. The wetting behavior of the particle is altered by the presence of the stabilizing groups, which affects the equilibrium position of the particles at the interface. The stabilizing groups can also lead to an activation barrier before interfacial adsorption, analogous to the steric repulsion between two particles. These effects are numerically solved with the SCF theory. It is commonly known that flocculating conditions enhance the interfacial adsorption and yield stable Pickering emulsions, which is confirmed in this work. Additionally, it is concluded that those conditions are not an absolute requirement. There is a window of stabilizer concentrations Γ(pIB), 2.2-3.3 mg/m(2) pIB, that shows both partial wetting and colloidal stability. The activation barrier for interfacial assembly is 140-550 k(B)T and is an order of magnitude higher than the colloidal stability. The difference can be attributed to the unfavorable interaction of pIB with water and a difference in geometry (plate-sphere vs sphere-sphere). This study demonstrates the interplay and provides a quantitative comparison between the wetting behavior and the colloidal stability, and it gives a better understanding of the colloidal assembly at soft interfaces and formation of Pickering emulsions in general.

Release of bacteriocins from nanofibers prepared with combinations of poly(d,l-lactide) (PDLLA) and poly(ethylene oxide) (PEO).

Plantaricin 423, produced by Lactobacillus plantarum, and bacteriocin ST4SA produced by Enterococcus mundtii, were electrospun into nanofibers prepared from different combinations of poly(d,l-lactide) (PDLLA) and poly(ethylene oxide) (PEO) dissolved in N,N-dimethylformamide (DMF). Both peptides were released from the nanofibers with a high initial burst and retained 88% of their original antimicrobial activity at 37 °C. Nanofibers have the potential to serve as carrier matrix for bacteriocins and open a new field in developing controlled antimicrobial delivery systems for various applications.

Evaluation of Composition Effects on the Physicochemical and Biological Properties of Polypeptide-Based Hydrogels for Potential Application in Wound Healing.

In this study, the effect of crosslinking and concentration on the properties of a new library of low-concentration poly(Lys60-ran-Ala40)-based hydrogels for potential application in wound healing was investigated in order to correlate the hydrogel composition with the desired physicochemical and biofunctional properties to expand the assortment of poly-l-lysine (PLL)-based hydrogels suitable for wound healing. Controlled ring-opening polymerization (ROP) and precise hydrogel compositions were used to customize the physicochemical and biofunctional properties of a library of new hydrogels comprising poly(l-lysine-ran-l-alanine) and four-arm poly(ethylene glycol) (P(KA)/4-PEG). The chemical composition and degree of crosslinking via free amine quantification were analyzed for the P(KA)/4-PEG hydrogels. In addition, the rheological properties, pore morphology, swelling behavior and degradation time were characterized. Subsequently, in vitro cell studies for evaluation of the cytotoxicity and cell adhesion were performed. The 4 wt% 1:1 functional molar ratio hydrogel with P(KA) concentrations as low as 0.65 wt% demonstrated low cytotoxicity and desirable cell adhesion towards fibroblasts and thus displayed a desirable combination of properties for wound healing application.

Systemic administration of polymersomal oncolytic peptide LTX-315 combining with CpG adjuvant and anti-PD-1 antibody boosts immunotherapy of melanoma.

Oncolytic peptide LTX-315 while showing clinical promise in treating solid tumors is limited to intratumoral administration, which is not applicable for inaccessible or metastatic tumors. The cationic and amphipathic nature of oncolytic peptides engenders formidable challenges to developing systems for their systemic delivery. Here, we describe cRGD-functionalized chimaeric polymersomes (cRGD-CPs) as a robust systemic delivery vehicle for LTX-315, which in combination with CpG adjuvant and anti-PD-1 boost immunotherapy of malignant B16F10 melanoma in mice. cRGD-CPs containing 14.9 wt% LTX-315 (cRGD-CPs-L) exhibited a size of 53 nm, excellent serum stability, and strong and selective killing of B16F10 cells (versus L929 fibroblasts) in vitro, which provoked similar immunogenic effects to free LTX-315 as revealed by release of danger-associated molecular pattern molecules. The systemic administration of cRGD-CPs-L gave a notable tumor accumulation of 4.8% ID/g and significant retardation of tumor growth. More interestingly, the treatment of B16F10 tumor-bearing mice was further boosted by co-administration of polymersomal CpG and anti-PD-1 antibody, in which two out of seven mice were cured as a result of strong immune response and long-term immune memory protection. The immunotherapeutic effect was evidenced by secretion of IL-6, IFN-γ and TNF-α, tumor infiltration of CD8+ CTLs and Th, and induction of TEM and TCM in spleen. This study opens a new avenue to oncolytic peptides, which enables durable immunotherapy of tumors via systemic administration.