Cell-penetrating Ability Research Articles

Plasmin-Binding Tripeptide-Decorated Liposomes Loading Pyrazolo[3,4-d]pyrimidines for Targeting Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most fatal cancer types worldwide. HCC cells were proved to overexpress c-Src and Sgk1, a tyrosine and a serine-threonine kinase, respectively, whose role is crucial for the development and progression of the tumor. Pyrazolo[3,4-d]pyrimidine derivatives are a class of tyrosine kinase inhibitors that have shown good activity against HepG2. HCC cells were also proved to overexpress plasmin, which is localized on the cell surface bound to its receptors. In this study, a tripeptide with sequence d-Ala-Phe-Lys, which binds a specific reactive site of plasmin, was synthesized and characterized. This tripeptide was used to decorate liposomes encapsulating three selected pyrazolo[3,4-d]pyrimidines. Liposomes bearing tripeptide have been characterized, not showing remarkable differences with respect to the corresponding tripeptide-free liposomes. In vitro HepG2 cell uptake profiles and cytotoxicities showed that the presence of the tripeptide on the liposomal membrane surface improves the cell-penetrating ability of liposomes and increases the activity of two of the three tested compounds.

Streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular and tumor delivery of enzymes

Despite the extremely high substrate specificity and catalytically amplified activity of enzymes, the lack of efficient cellular internalization limits their application as therapeutics. To overcome this limitation and to harness enzymes as practical biologics for targeting intracellular functions, we developed the streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular delivery of various enzymes. The hybrid consists of streptavidin, which provides a stoichiometrically controlled loading site for the enzyme cargo and an L-DNA (mirror DNA) tetrahedron, which provides the intracellular delivery potential. Due to the cell-penetrating ability of the mirror DNA tetrahedron of this hybrid, enzymes loaded on streptavidin can be efficiently delivered into the cells, intracellularly expressing their activity. In addition, we demonstrate tumor delivery of enzymes in an animal model by utilizing the potential of the hybrid to accumulate in tumors. Strikingly, the hybrid is able to transfer the apoptotic enzyme specifically into tumor cells, leading to strong suppression of tumor growth without causing significant damage to other tissues. These results suggest that the hybrid may allow anti-proliferative enzymes and proteins to be utilized as anticancer drugs.

Cell-Penetrating Peptides Using Cyclic α,α-Disubstituted α-Amino Acids with Basic Functional Groups.

In the delivery of cell-impermeable molecules, cell-penetrating peptides (CPPs) have been attracting increasing attention as intracellular delivery tools. In the present study, we designed four types of cyclic α,α-disubstituted α-amino acids (dAAs) with basic functional groups on their five-membered rings and different chiralities at the α-position and introduced them into arginine (Arg)-rich peptides. The evaluation of cell-penetrating abilities indicated that these peptides exhibited better cell permeabilities than an Arg nonapeptide. Furthermore, peptides containing dAAs delivered plasmid DNA (pDNA) better than a commercially available transfection reagent with a longer incubation time. These results demonstrate that the introduction of cyclic dAAs with basic functional groups into Arg-rich peptides is an effective strategy for the design of CPPs as a pDNA delivery tool.

Novel Combination of Silver Nanoparticles and Carbon Nanotubes for Plasmonic Photo Thermal Therapy in Melanoma Cancer Model.

Purpose: Plasmonic photo thermal therapy (PPTT) is a therapeutic method in which the photon energy is rapidly transformed into heat via a series of radiative and non-radiative phenomena to ablate cancer. Plasmonic NPs, such as silver NPs (Ag NPs), have considerable properties in optical absorbance. Furthermore, good thermal conductivity and cell penetration ability of carbon nanotubes (CNTs) could improve the efficacy of Ag NPs for PPTT. Decoration of the multi-walled carbon nanotubes (MWCNTs) with silver has been developed to enhance thermal conductivity of the MWCNT particles.Methods: The Ag NPs were decorated on the CNTs and the ability of these particles (CNT/Ag NPs) in reduction of melanoma tumor size after PTT was evaluated experimentally. For comparison, the PTT of silver nanorods (Ag NRs) and CNTs were investigated. The melanoma tumor was induced by injection of B16/F10 cell line to the inbred mice. Different NPs were injected into the tumors and then irradiated via laser diode (λ=670 nm, P=500 mW, and I= 3.5 W/cm2) at scheduled time.Results: Monitoring of tumor sizes showed that integration of CNTs with silver could enhance the optical absorption of CNTs and improve tumor destruction in PPTT technique.Conclusion: The CNT/Ag NPs could act as a potent agent in PPTT method in curing solid tumors.

A simple in vitro tumor chemosensitivity assay based on cell penetrating peptide tagged luciferase.

The analysis of intracellular ATP can reveal the response of cells to different treatments and is important for individualized medicine. In the present study, we developed a cell penetrating peptides (CPPs) tagged luciferase (TAT-LUC) for tumor chemosensitivity assay. The activity of recombinant TAT-LUC was evaluated using ATP standard solution and tumor cells. This recombinant TAT-LUC was then used for the analysis of sensitivity index (SI) of four strains of tumor cells. The results showed that TAT-LUC could detect less than 10 nM extracellular ATP with a strong correlation between the luminescence intensity and the ATP content (R2 = 0.994). Without cell lysis, the detection limit for intracellular ATP analysis was 40 tumor cells. Furthermore, chemosensitivity of four strains of tumor cells (Skov-3/DDP, A549/DDP, MDA-MB-231, Huh-7) was determined by this assay successfully. The cell penetration ability of TAT-LUC enables the assay not only to reflect drug resistance of tumor cells real-timely but also to minimize the test time, which can be a valuable aid for personalized cancer chemotherapy.

Matrix Metalloproteinase Cleavable Nanoparticles for Tumor Microenvironment and Tumor Cell Dual-Targeting Drug Delivery.

Matrix metalloproteinases (MMPs), mostly abundant in the tumor extracellular matrix (ECM), tumor cells, and tumor vasculatures, are closely correlated with tumor progression and metastasis. In this case, making use of MMPs was supposed to achieve site-specific drug delivery and a satisfactory tumor treatment effect. Herein, we rationally developed a novel tumor microenvironment and tumor cell dual-targeting nanoparticle by integrating a chemotherapeutic-loaded drug-loaded carrier and a versatile polypeptide-LinTT1-PVGLIG-TAT (LPT) which is composed of a multitargeting peptide-LinTT1 and a cell-penetrating peptide-TAT. The functionalized nanoparticles exhibited a superior affinity to A549 lung-cancer cells and microenvironment including angiogenesis and tumor-associated macrophages (TAMs) in our study. In addition, cellular experiments demonstrated that the cell-penetrating ability of TAT was significantly shielded by the addition of LinTT1 to the fourth lysine of the TAT via an MMP cleavable linker PVGLIG and could be recovered under the catalysis of MMPs. This design was supposed to efficiently decrease the toxicological risk to normal tissues induced by the unselectivity of TAT. The finally treatment effect investigation showed that tumor-bearing mice treated with LPT-modified nanoparticles achieved an enhanced efficacy for inhibiting tumor growth and the longest survival time as compared to other groups. Collectively, this study provides a novel robust nanoplatform which could simultaneously target the tumor microenvironment and tumor cell drug delivery for increasing the efficacy of cancer therapy.

Multifunctional Gene Carriers with Enhanced Specific Penetration and Nucleus Accumulation to Promote Neovascularization of HUVECs in Vivo.

Recently, gene therapy has attracted much attention, especially for the treatment of vascular disease. However, it is still challenging to develop the gene carriers with high biocompatibility as well as highly efficient gene delivery to overcome multiple barriers. Herein, a frequently used cell-penetrating peptide PKKKRKV (TAT) was selected as a functional sequence of the gene carrier with distinctive cell-penetrating ability. REDV peptide with selectively targeting function for endothelial cells (ECs) and nuclear localization signals (NLS)were integrated with this TAT peptide to obtain a highly efficient gene delivery system with ECs specificity and nucleus accumulation capacity. Besides, the glycine sequences with different repeat numbers were inserted into the above integrated peptide. These glycine sequences acted as a flexible spacer arm to exert the targeting, cell-penetrating, and nucleus accumulation functions of each functional peptide. Three tandem peptides REDV-Gm-TAT-Gm-NLS (m = 0, 1, and 4) complexed with pZNF580 plasmid to form gene complexes. The results of hemocompatibility and cytocompatibility indicated that these peptides and gene complexes were nontoxic and biocompatible. The internalization efficiency and mechanism of these gene complexes were investigated. The internalization efficiency was improved as the introduction of targeting REDV and glycine sequence, and the REDV-G4-TAT-G4-NLS/pZNF580 (TP-G4/pZNF580) complexes showed the highest cellular uptake among the gene complexes. The TP-G4/pZNF580 complexes also presented significantly higher internalization efficiency (∼1.36 times) in human umbilical vein endothelial cells (HUVECs) than human umbilical artery smooth muscle cells. TP-G4/pZNF580 complexes substantially promoted the expression of pZNF580 by confocal live cell imaging, gene delivery efficiency, and HUVECs migration assay. The in vitro and in vivo revascularization ability of transfected HUVECs was further enhanced obviously. In conclusion, these multifunctional REDV-Gm-TAT-Gm-NLS peptides offer a promising and efficacious delivery option for neovascularization to treat vascular diseases.

In silico methods for design of biological therapeutics.

It has been twenty years since the first rationally designed small molecule drug was introduced into the market. Since then, we have progressed from designing small molecules to designing biotherapeutics. This class of therapeutics includes designed proteins, peptides and nucleic acids that could more effectively combat drug resistance and even act in cases where the disease is caused because of a molecular deficiency. Computational methods are crucial in this design exercise and this review discusses the various elements of designing biotherapeutic proteins and peptides. Many of the techniques discussed here, such as the deterministic and stochastic design methods, are generally used in protein design. We have devoted special attention to the design of antibodies and vaccines. In addition to the methods for designing these molecules, we have included a comprehensive list of all biotherapeutics approved for clinical use. Also included is an overview of methods that predict the binding affinity, cell penetration ability, half-life, solubility, immunogenicity and toxicity of the designed therapeutics. Biotherapeutics are only going to grow in clinical importance and are set to herald a new generation of disease management and cure.

Acid-activatable doxorubicin prodrug micelles with folate-targeted and ultra-high drug loading features for efficient antitumor drug delivery

Stimuli-responsive nanomedicine shows high therapeutic effects and low side effects to tumor cells and tissues, representing a preferable therapeutics for cancer therapy. Herein, we design an acid-stimuli-responsive doxorubicin polymeric prodrug (OM@DOX), and this amphiphilic prodrug has a unique chemical structure with prominent advantages, including high drug loading rate (as high as 61.5 wt%), pH-triggered drug release and targeted access to cells. This smart polymeric prodrug has a preferable size of ~40 nm and strong micellar stability in aqueous solution, which is benefited to the long blood circulation and efficient extravasation from tumor vessel. Moreover, the prodrug micelles showed a higher cytotoxicity against tumor cells (HeLa cells) than normal cells (L929 cells), likely suggesting the potential tumor-specific targeting ability. To render this prodrug micelles with targeting function, folic acid (FA) molecules conjugated prodrug (FA-OM@DOX) further showed selectively higher cytotoxicity to KB tumor cells (FA-receptor-positive) than A549 tumor cells (FA-receptor-negative). Considering the rapidly cell-penetrating ability and aforementioned features, we believe that the present prodrug strategy has the potential as a promising nanomedicine and providing inspired insights to design multifunctional drug delivery nanoplatforms.

Biophysical and biological properties of small linear peptides derived from crotamine, a cationic antimicrobial/antitumoral toxin with cell penetrating and cargo delivery abilities

Crotamine is a natural polypeptide from snake venom which delivers nucleic acid molecules into cells, besides having pronounced affinity for negatively charged membranes and antifungal activity. We previously demonstrated that crotamine derived short linear peptides were not very effective as antifungal, although the non-structured recombinant crotamine was overridingly more potent compared to the native structured crotamine. Aiming to identify the features necessary for the antifungal activity of crotamine, two linear short peptides, each comprising half of the total positively charged amino acid residues of the full-length crotamine were evaluated here to show that these linear peptides keep the ability to interact with lipid membrane model systems with different phospholipid compositions, even after forming complexes with DNA. Interestingly, the presence of cysteine residues in the structure of these linear peptides highly influenced the antifungal activity, which was not associated to the lipid membrane lytic activity. In addition to the importance of the positive charges, the crucial role of cysteine residues was noticed for these linear analogs of crotamine, although the tridimensional structure and lipid membrane lytic activity observed only for native crotamine was not essential for the antifungal activity. As these peptides still keep the ability to form complexes with DNA molecules with no prejudice to their ability to bind to lipid membranes, they may be potentially advantageous as membrane translocation vector, as they do not show lipid membrane lytic activity and may harbor or not antifungal activity, by keeping or not the semi-essential amino acid cysteine in their sequence.

Abstract 2167: Preclinical validation of an Omomyc cell-penetrating peptide as a viable anti-Myc therapy

Abstract Deregulation of the MYC oncoprotein promotes tumorigenesis in most, if not all, cancers and is often associated with poor prognosis. However, targeting MYC has long been considered impossible based on the assumption that it would cause catastrophic side effects in normal tissues. Despite this general preconceived notion, we showed that MYC inhibition exerts extraordinary therapeutic impact in various genetic mouse models of cancer, and causes only mild, well-tolerated and reversible side effects. For these studies we employed the systemic and conditional expression of a dominant negative of MYC, called Omomyc, which we designed and validated, and that can inhibit MYC transactivation function both in vitro and in vivo. To date, Omomyc has only been considered a proof of principle, with any potential clinical application limited to gene therapy. Here we actually show that the 11 kDa Omomyc polypeptide spontaneously transduces into cancer cells, demonstrating unexpected cell-penetrating ability. Once inside the nuclei, the polypeptide effectively blocks MYC binding to its target DNA sites, interfering with MYC transcriptional regulation and halting cell proliferation. Moreover, intranasal (i.n.) administration of the Omomyc polypeptide in mice results in its rapid and persistent distribution to lungs, as well as to other organs (i.e. intestine, liver, kidneys and brain). Importantly, i.n. treatment of mice bearing either Non-Small-Cell-Lung-Cancer (NSCLC) or glioblastoma (GBM) with the Omomyc cell-penetrating peptide (OmomycCPP) significantly reduces tumor burden compared to their control counterparts. Notably, tumor regression is accompanied by significant reprogramming of the tumor microenvironment and tumor immune response. In summary, our data indicate that this novel generation of polypeptides represents a new opportunity to potentially inhibit MYC pharmacologically in a variety of malignant diseases. Citation Format: Marie-eve Beaulieu, Toni Jauset, Daniel Massó-Vallés, Peter Rahl, Sandra Martinez-Martin, Loika Maltais, Mariano F. Zacarias-Fluck, Silvia Casacuberta, Erika Serrano del Pozo, Christopher Fiore, Laia Foradada, Matthew Guenther, Eduardo Romero Sanz, Marta Oteo Vives, Cynthia Tremblay, Martin Montagne, Miguel Ángel Morcillo Alonso, Jonathan R. Whitfield, Pierre Lavigne, Laura Soucek. Preclinical validation of an Omomyc cell-penetrating peptide as a viable anti-Myc therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2167. doi:10.1158/1538-7445.AM2017-2167

Dual Tumor-Targeting Nanocarrier System for siRNA Delivery Based on pRNA and Modified Chitosan.

Highly specific and efficient delivery of siRNA is still unsatisfactory. Herein, a dual tumor-targeting siRNA delivery system combining pRNA dimers with chitosan nanoparticles (CNPPs) was designed to improve the specificity and efficiency of siRNA delivery. In this dual delivery system, folate-conjugated and PEGylated chitosan nanoparticles encapsulating pRNA dimers were used as the first class of delivery system and would selectively deliver intact pRNA dimers near or into target cells. pRNA dimers simultaneously carrying siRNA and targeting aptamer, the second class of delivery system, would specifically deliver siRNA into the target cells via aptamer-mediated endocytosis or proper particle size. To certify the delivering efficiency of this dual system, CNPPs, pRNA dimers alone, chitosan nanoparticles containing siRNA with folate conjugation and PEGylation (CNPS), and chitosan nanoparticles containing pRNA dimers alone (CN) were first prepared. Then, we observed that treatment with CNPPs resulted in increased cellular uptake, higher cell apoptosis, stronger cell cytotoxicity, and more efficacious gene silencing compared to the other three formulations. Higher accumulation of siRNA in the tumor site, stronger tumor inhibition, and longer circulating time were also observed with CNPPs compared to other formulations. In conclusion, this dual nanocarrier system showed high targeting and favorable therapeutic efficacy both in vitro and in vivo. Thereby, a new approach is provided in this study for specific and efficient delivery of siRNA, which lays a foundation for the development of pRNA hexamers, which can simultaneously carry six different substances.

Enhanced and Prolonged Cell-Penetrating Abilities of Arginine-Rich Peptides by Introducing Cyclic α,α-Disubstituted α-Amino Acids with Stapling.

Cell-penetrating peptides are receiving increasing attention as drug delivery tools, and the search for peptides with high cell-penetrating ability and negligible cytotoxicity has become a critical research topic. Herein, cyclic α,α-disubstituted α-amino acids were introduced into arginine-rich peptides and an additional staple was provided in the side chain. The peptides designed in the present study showed more enhanced and prolonged cell-penetrating abilities than an arginine nonapeptide due to high resistance to protease and conformationally stable helical structures.

Design of new acid-activated cell-penetrating peptides for tumor drug delivery.

TH(AGYLLGHINLHHLAHL(Aib)HHIL-NH2), a histidine-rich, cell-penetrating peptide with acid-activated pH response, designed and synthesized by our group, can effectively target tumor tissues with an acidic extracellular environment. Since the protonating effect of histidine plays a critical role in the acid-activated, cell-penetrating ability of TH, we designed a series of new histidine substituents by introducing electron donating groups (Ethyl, Isopropyl, Butyl) to the C-2 position of histidine. This resulted in an enhanced pH-response and improved the application of TH in tumor-targeted delivery systems. The substituents were further utilized to form the corresponding TH analogs (Ethyl-TH, Isopropyl-TH and Butyl-TH), making them easier to protonate for positive charge in acidic tumor microenvironments. The pH-dependent cellular uptake efficiencies of new TH analogs were further evaluated using flow cytometry and confocal laser scanning microscopy, demonstrating that ethyl-TH and butyl-TH had an optimal pH-response in an acidic environment. Importantly, the new TH analogs exhibited relatively lower toxicity than TH. In addition, these new TH analogs were linked to the antitumor drug camptothecin (CPT), while butyl-TH modified conjugate presented a remarkably stronger pH-dependent cytotoxicity to cancer cells than TH and the other conjugates. In short, our work opens a new avenue for the development of improved acid-activated, cell-penetrating peptides as efficient anticancer drug delivery vectors.

Transbody against virus core protein potently inhibits hepadnavirus replication in vivo: evidence from a duck model of hepatitis B virus.

The therapeutic management of hepatitis B virus (HBV) infections remains challenging, and novel antiviral strategies are urgently required. The HBV transbody, a monoclonal antibody (MAb) against human HBcAg coupled with the trans-activator of transcription protein transduction domain (TAT PTD), was previously shown to possess cell-penetrating ability and potent antiviral activity in vitro. The purpose of the present study was to evaluate the antiviral activity of the HBcMAb-TAT PTD conjugate in vivo in a duck model of HBV. Female Peking ducks were injected i.p. with 0.03-0.3 mg·kg-1 ·day-1 of the DHBV transbody (DHBcMAb-TAT PTD conjugate) for 30 days. Serum DHBV DNA levels and liver DHBV DNA and covalently closed circular DNA (cccDNA) loads were determined at scheduled time points. Immunohistological examination of DHBcAg in the duck liver was also performed. The DHBV transbody significantly reduced the serum and liver DHBV DNA levels at doses of 0.1 and 0.3 mg·kg-1 ·day-1 and liver cccDNA levels at a dose of 0.3 mg·kg-1 ·day-1 after 30 days of treatment. The suppressive effects of the DHBV transbody at 0.3 mg·kg-1 ·day-1 on the serum and liver DHBV DNA and liver cccDNA levels remained significant, even at 15 days after treatment cessation. Similarly, immunohistochemistry of liver sections revealed decreased DHBcAg levels within hepatocytes 15 days after treatment termination. The DHBV transbody inhibits DHBV replication and possesses potent anti-DHBV activities in vivo. The cell-permeable antibody against the virus core antigen may be developed as a novel treatment for patients with hepadnavirus infections.

PH-Responsive, Lysine-Based, Hyperbranched Polymers Mimicking Endosomolytic Cell-Penetrating Peptides for Efficient Intracellular Delivery

The insufficient delivery of biomacromolecular therapeutic agents into the cytoplasm of mammalian cells remains a major barrier to their pharmaceutical applications. Cell-penetrating peptides (CPPs) are considered as potential carriers for cytoplasmic delivery of macromolecular drugs. However, due to the positive charge of most CPPs, strong nonspecific cell membrane bindings may lead to relatively high toxicity. In this study, we report a series of anionic, CPP-mimicking, lysine-based hyperbranched polymers, which caused complete membrane disruption at late endosomal pH while remaining nonlytic at physiological pH. The pH-responsive conformational alterations and the multivalency effect of the hyperbranched structures were demonstrated to effectively facilitate their interaction with cell membranes, thus leading to significantly enhanced membrane-lytic activity compared with their linear counterpart. The unique structures and pH-responsive cell-penetrating abilities make the novel hyperbranched polymers p...

Noninvasive delivery of oligonucleotide by penetratin-modified polyplexes to inhibit protein expression of intraocular tumor

Our present study aimed to develop an antisense oligonucleotide (ASO) delivery system to achieve gene silencing in intraocular tumor via topical instillation. ASO specific for luciferase was chosen as model drug, polyamidoamine (PG5) was employed to condense ASO, and penetratin (Pene) was used to enhance cellular uptake. Nanoscale PG5/ASO/Pene polyplex was stabilized via noncovalent bonding. In vitro evaluations indicated that PG5/ASO/Pene exhibited improved cell-penetrating and gene silencing ability compared with naked ASO and PG5/ASO. Subcutaneous and orthotopic tumor models expressing luciferase were established in nude mice. After treated by PG5/ASO/Pene, immunohistochemical results of subcutaneous tumors showed significant inhibition of luciferase expression via peritumoral injection, and bioluminescence from orthotopic tumor was obviously weakened via topical instillation. To date, few works were successful in noninvasive treatment of intraocular diseases using antisense strategy, this penetratin-modified polyplex could be a promising vector to inhibit protein expression by effectively delivering ASOs into the eye.

Cell-penetrating and cargo-delivery ability of a spider toxin-derived peptide in mammalian cells

Cell-penetrating peptides are short cationic peptides with inherent ability to cross the plasma membrane barrier as well as intracellularly deliver cargo molecules conjugated to them. Venoms from snakes, scorpions and spiders are rich in membrane-active peptides. Crotamine from snake venom as well as maurocalcine and imperatoxin isolated from scorpion venoms have been reported to possess cell-penetrating property in mammalian cells. Latarcins, a group of spider venom toxins, has also been reported to possess antimicrobial property. However, cell-penetrating ability of Latarcins is still not elucidated. This is the first report where cell-penetrating ability of a peptide derived from spider toxin, Latarcin 1 has been demonstrated. Interestingly, the structurally minimized sequence of Latarcin 1 (LDP – Latarcin-derived peptide) when conjugated with nuclear localization sequence from Simian Virus T40 antigen (LDP-NLS) translocates across cell membrane in HeLa cells. The chimeric LDP-NLS peptide also did not exhibit cytotoxicity towards mammalian cells in contrast to the LDP that showed lesser uptake and higher cytotoxicity. LDP-NLS also successfully delivered macromolecular protein cargo inside the cells.

Cyclotide Structure and Function: The Role of Membrane Binding and Permeation.

There is growing interest in the use of peptides as therapeutic drugs and, in particular, in the potential of cyclotides, a family of cyclic peptides with remarkable stability and amenability to sequence engineering, as scaffolds in drug design. As well as having an ultrastable structure, many natural cyclotides have intrinsic biological activities with potential pharmaceutical or agricultural applications. Some cyclotides also have the ability to cross membrane barriers and to enter into cells; in particular, cyclotides that belong to the Möbius and bracelet subfamilies have been found to harbor lipid-binding domains, which allow for the specific recognition of phosphatidylethanolamine phospholipids in biological membranes. This lipid selectivity is intimately correlated with the highly conserved three-dimensional structures of cyclotides and is important for their reported biological properties and cell penetration ability. The membrane binding features of Möbius and bracelet cyclotides contrast with the lack of membrane binding of trypsin inhibitor cyclotides, which have physicochemical properties and bioactivities different from those of the other two subfamilies of cyclotides but are also able to enter cells. This review discusses the structures of cyclotides with regard to their myriad of biological activities and describes the role of membrane binding in their functions and ability to enter cells.

Glioma Dual-Targeting Nanohybrid Protein Toxin Constructed by Intein-Mediated Site-Specific Ligation for Multistage Booster Delivery.

Malignant glioma is one of the most untreatable cancers because of the formidable blood-brain barrier (BBB), through which few therapeutics can penetrate and reach the tumors. Biologics have been booming in cancer therapy in the past two decades, but their application in brain tumor has long been ignored due to the impermeable nature of BBB against effective delivery of biologics. Indeed, it is a long unsolved problem for brain delivery of macromolecular drugs, which becomes the Holy Grail in medical and pharmaceutical sciences. Even assisting by targeting ligands, protein brain delivery still remains challenging because of the synthesis difficulties of ligand-modified proteins. Herein, we propose a rocket-like, multistage booster delivery system of a protein toxin, trichosanthin (TCS), for antiglioma treatment. TCS is a ribosome-inactivating protein with the potent activity against various solid tumors but lack of specific action and cell penetration ability. To overcome the challenge of its poor druggability and site-specific modification, intein-mediated ligation was applied, by which a gelatinase-cleavable peptide and cell-penetrating peptide (CPP)-fused recombinant TCS toxin can be site-specifically conjugated to lactoferrin (LF), thus constructing a BBB-penetrating, gelatinase-activatable cell-penetrating nanohybrid TCS toxin. This nanohybrid TCS system is featured by the multistage booster strategy for glioma dual-targeting delivery. First, LF can target to the BBB-overexpressing low-density lipoprotein receptor-related protein-1 (LRP-1), and assist with BBB penetration. Second, once reaching the tumor site, the gelatinase-cleavable peptide acts as a separator responsive to the glioma-associated matrix metalloproteinases (MMPs), thus releasing to the CPP-fused toxin. Third, CPP mediates intratumoral and intracellular penetration of TCS toxin, thereby enhancing its antitumor activity. The BBB penetration and MMP-2-activability of this delivery system were demonstrated. The antiglioma activity was evaluated in the subcutaneous and orthotopic animal models. Our work provides a useful protocol for improving the druggability of such class of protein toxins and promoting their in-vivo application for targeted cancer therapy.