pH-responsive poly(styrene-alt-maleic anhydride) alkylamide copolymers for intracellular drug delivery

Many macromolecular therapeutics such as peptides, proteins, antisense oligodeoxynucleotides (ASODN), and short interfering RNA (siRNA) are active only in the cytoplasm or nucleus of targeted cells. Endocytosis is the primary route for cellular uptake of these molecules, which results in their accumulation in the endosomal-lysosomal trafficking pathway and loss of therapeutic activity. In this article, we describe the synthesis and pH-dependent membrane-destabilizing activity of a new "smart" polymer family that can be utilized to enhance the intracellular delivery of therapeutic macromolecules through the endosomal membrane barrier into the cytoplasm of targeted cells. These polymers are propylamine, butylamine, and pentylamine derivatives of poly(styrene-alt-maleic anhydride) (PSMA) copolymers. The PSMA-alkylamide derivatives are hydrophilic and membrane-inactive at physiological pH; however, they become hydrophobic and membrane-disruptive in response to endosomal pH values as measured by their hemolytic activity. Results show that the pH-dependent membrane-destabilizing activity of PSMA derivatives can be controlled by varying the length of the alkylamine group, the degree of modification of the copolymer, and the molecular weight of the PSMA copolymer backbone. Butylamine and pentylamine derivatives of PSMA copolymers exhibited more than 80% hemolysis at endosomal pH values, which suggests their potential as a platform of "smart" polymeric carriers for enhanced cytoplasmic delivery of a variety of therapeutic macromolecules.     

细胞穿透肽:一种新型非病毒载体

细胞膜的选择通透性对维持细胞内环境的稳定起着非常重要的作用,但细胞膜的这种特性限制了一些生物大分子和药物进入细胞内,不利于对一些细胞内疾病的诊断和药物靶向治疗的应用。如何将一些具有诊断和治疗潜力的生物大分子、药物通过细胞膜进入细胞内一直是医学界研究的热点和难点。细胞穿透肽是一类能够携带多肽、蛋白质、核酸、纳米颗粒、病毒颗粒及药物等穿过细胞膜进入细胞,导致完整载物内化的短肽,为生物大分子和药物进入细胞内部提供了有力的运载工具,其作为载体具有的高转导效率和低毒性特点,已经得到了广泛关注和大量研究。目前,细胞穿透肽作为生物分子和药物细胞内化的运载体已经在荧光成像,肿瘤治疗,抗炎治疗及药物靶向治疗中发挥了潜在的诊断和治疗作用,显示出其诱人的应用前景。     

Cellular uptake of Aib-containing amphipathic helix peptide

Cell-penetrating peptides (CPPs) are useful tools for the delivery of hydrophilic bioactive molecules, such as peptides, proteins, and oligonucleotides, across the cell membrane. To realize the delivery of therapeutic macromolecules by CPPs, the CPPs are required to show resistance to protease and no cytotoxicity. In order to produce potent non-toxic and protease-resistant CPPs with high cellular uptake, we designed an amphipathic helix peptide using α-aminoisobutyric acid (Aib, U) and named it MAP(Aib). In the MAP(Aib) molecule, five Aib residues are aligned on the hydrophobic face of the helix and five lysine (K) residues are aligned on the hydrophilic face. MAP(Aib) showed potent resistance to trypsin and pronase compared with MAP, an amphipathic helix peptide formed by usual amino acids. Fluorescein-labeled MAP(Aib) efficiently traversed the A549 cell membrane, diffusing into the cytoplasm and slightly into the nucleus without exerting any cytotoxicity. In contrast, MAP was poorly taken up by the cell. These results indicate that the incorporation of Aib residues into CPPs markedly improves cellular uptake and MAP(Aib) may be a useful tool for the delivery of hydrophilic macromolecules.     

Cellular delivery of impermeable effector molecules in the form of conjugates with peptides capable of mediating membrane translocation.

Most molecules that are not actively imported by living cells are impermeable to cell membranes, including practically all macromolecules and even many small molecules whose physicochemical properties prevent passive membrane diffusion. The use of peptide vectors capable of transporting such molecules into cells in the form of covalent conjugates has become an increasingly attractive solution to this problem. Not only has this technology permitted the study of modulating intracellular target proteins, but it has also gained importance as an alternative to conventional cellular transfection with oligonucleotides. Peptide vectors derived from viral, bacterial, insect, and mammalian proteins endowed with membrane translocation properties have now been proposed as delivery vectors. These are discussed comprehensively and critically in terms of relative utility, applications to compound classes and specific molecules, and relevant conjugation chemistry. Although in most cases the mechanisms of membrane translocation are still unclear, physicochemical studies have been carried out with a number of peptide delivery vectors. Unifying and distinguishing mechanistic features of the various vectors are discussed. Until a few years ago speculations that it might be possible to deliver peptides, proteins, oligonucleotides, and impermeable small molecules with the aid of cellular delivery peptides not only to target cells in vitro, but in vivo, was received with scepticism. However, the first studies showing pharmacological applications of conjugates between macromolecules and peptide delivery vectors are now being reported, and therapies based on such conjugates are beginning to appear feasible.     

Different mechanisms for cellular internalization of the HIV-1 Tat-derived cell penetrating peptide and recombinant proteins fused to Tat.

Translocation through the plasma membrane is a major limiting step for the cellular delivery of macromolecules. A promising strategy to overcome this problem consists in the chemical conjugation (or fusion) to cell penetrating peptides (CPP) derived from proteins able to cross the plasma membrane. A large number of different cargo molecules such as oligonucleotides, peptides, peptide nucleic acids, proteins or even nanoparticles have been internalized in cells by this strategy. One of these translocating peptides was derived from the HIV-1 Tat protein. The mechanisms by which CPP enter cells remain unknown. Recently, convincing biochemical and genetic findings has established that the full-length Tat protein was internalized in cells via the ubiquitous heparan sulfate (HS) proteoglycans. We demonstrate here that the short Tat CPP is taken up by a route that does not involve the HS proteoglycans.     

Membrane permeability commonly shared among arginine-rich peptides

Delivery of proteins and other macromolecules using membrane-permeable carrier peptides is a recently developed novel technology, which enables us to modulate cellular functions for biological studies with therapeutic potential. One of the most often used carrier peptides is the arginine-rich basic peptide derived from HIV-1 Tat protein [HIV-1 Tat (48-60)]. Using this peptide, efficient intracellular delivery of molecules including proteins, oligonucleic acids and liposomes has been achieved. We have demonstrated that these features were commonly shared among many arginine-rich peptides such as HIV-1 Rev (34-50) and octaarginine. Not only the linear peptides but also branched-chain peptides showed efficient internalization with an optimum number of arginines (approximately eight residues). The structural and mechanistic features of the translocation of these membrane-permeable arginine-rich peptides are reviewed.     

First step of the cell-penetrating peptide mechanism involves Rac1 GTPase-dependent actin-network remodelling

BACKGROUND INFORMATION: Application of CPPs (cell-penetrating peptides) constitutes a promising strategy for the intracellular delivery of therapeutic molecules. The non-covalent approach based on the amphipathic peptide MPG has been successfully used to improve the delivery of biologically active macromolecules, both in cellulo and in vivo, through a mechanism independent of the endosomal pathway and mediated by the membrane potential. RESULTS: In the present study, we have investigated the first step of the cellular uptake mechanism of MPG and shown that both MPG and MPG-cargo complexes interact with the extracellular matrix through the negatively charged heparan sulfate proteoglycans. We demonstrated that initiation of cellular uptake constitutes a highly dynamic mechanism where the binding of MPG or the MPG-cargo to the extracellular matrix is rapidly followed by a remodelling of the actin network associated with the activation of the GTPase Rac1. We suggest that MPG-induced clustering of the glycosaminoglycan platform constitutes the 'onset' of the cellular uptake mechanism, thereby increasing membrane dynamics and membrane fusion processes. This process favours cell entry of MPG or MPG-DNA complexes, which is further controlled by the ability of MPG to induce a local membrane destabilization. CONCLUSIONS: Although CPPs are taken up through different pathways and mechanisms, the initial step involves electrostatic interactions with the glycosaminoglycan platform, and the dynamics of associated membrane microdomains can be generalized to most non-viral delivery systems.     

Heparan sulfate proteoglycan as a plasma membrane carrier

The plasma membrane defines the border of living cells and provides a barrier to extracellular components. Advances in molecular biology have resulted in the development of novel therapeutic strategies (e.g. gene therapy and cellular protein delivery) which rely on the entry of charged macromolecules into the intracellular compartment. Recent reports demonstrate an intriguing role for heparan sulfate proteoglycans in cellular internalization of viruses, basic peptides and polycation-nucleic-acid complexes and the possibility that they have important implications for gene transfer and protein delivery to mammalian cells. This review focuses on heparan sulfate proteoglycan as a plasma membrane carrier.     

细胞穿透肽在肿瘤治疗中的应用

随着人类对基因组信息解读的不断深入,越来越多的生物大分子作为候选药物进入生物治疗领域。但细胞表面的脂质双层膜具有选择通透性,这种天然屏障作用在保护细胞的同时也限制了绝大多数生物大分子进入细胞内部发挥治疗效应。目前流行的入胞转运方式如电穿孔、脂质体转染等均存在对细胞的额外毒副作用,且作用范围局限于体外实验。细胞穿透肽是一类以非受体依赖方式,非经典内吞方式直接穿过细胞膜进入细胞的多肽。它们可以与多种生物活性物质连接并携带其进入细胞,这一特性为它们成为理想的药物载体提供了可能。本文对使用细胞穿透肽作为载体转运具有抗癌作用的生物大分子进入细胞的抗癌实验予以介绍。     

Identification of a novel cell-penetrating peptide from human phosphatidate phosphatase LPIN3

Biomolecules such as proteins, DNA, and RNA are macromolecules and can not cross the cell membrane. However, cell-penetrating peptide (CPP) has been shown to deliver therapeutic biomolecules successfully into cells. The various and widely used CPPs including TAT, VP22, and Antp are mostly non-human originated CPPs, and are limited by their potential toxicity and immunogenicity. We report here on a newly identified novel cell-penetrating sequence (LPIN; RRKRRRRRK) from the nuclear localization sequence (NLS) of human nuclear phosphatase, LPIN3. LPIN-EGFP recombinant protein was concentration- and time-dependently delivered into cells and localized to the nucleus as well as the cytoplasm. It penetrated the cell membrane by lipid raft-mediated endocytosis by binding to heparan sulfate proteoglycan. LPIN-EGFP was successfully delivered into primary mouse splenocytes in vitro and it could be delivered into various tissues including liver, kidney, and intestine in mice after intra-peritoneal injection. This research suggests that LPIN-CPP could be used in a drug delivery system to deliver therapeutic biomolecules including peptides, proteins, DNA, and RNA and without the limitations of non-human originated CPPs such as TAT-CPP.     

Biological applications of protein transduction technology

The impermeable nature of the cell membrane to peptides, proteins, DNA and oligonucleotides limits the therapeutic potential of these biological agents. However, the recent discovery of short cationic peptides that cross the plasma membrane efficiently is opening up new possibilities for the intracellular delivery of such agents. These peptides are commonly referred to as protein transduction domains (PTDs) and are successfully used to transport heterologous proteins, peptides and other types of cargo into cells. Several recent reports have used the membrane transducing technology in vivo to deliver biologically active cargo into various tissues. This review discusses the structure of the most commonly used PTDs and how their ability to transduce membranes is used to regulate biological functions. It also considers future directions and the potential of this technology to move from the laboratory into the clinic.     

Cell-penetrating peptides: [corrected] from inception to application

Despite continuing advances in the development of macromolecules, including peptides, proteins, and oligonucleotides, for therapeutic purposes, the successful application of these hydrophilic molecules has so far been hampered by their inability to efficiently traverse the cellular plasma membrane. The discovery of a class of peptides (cell-penetrating peptides, CPPs) with the ability to mediate the non-invasive and efficient import of a whole host of cargoes, both in vitro and in vivo, has provided a new means by which the problem associated with cellular delivery can be circumvented. A complete understanding of the translocation mechanism(s) of CPPs has so far proven elusive. Initial studies indicated an ATP-independent, non-endocytotic mechanism, dependent on direct peptide-membrane interactions, making it an enticing challenge from a biophysical point of view. However, recent evidence cast doubt on many of the earlier results, and led to a re-evaluation of the translocation mechanism of CPPs. In this review a brief history of the field will be given, followed by an introduction to some of the better known and more widely used CPPs, including some of their current applications, and finally a discussion of the translocation mechanism(s) and the controversies surrounding it.     

COPA and SLC4A4 are Required for Cellular Entry of Arginine-Rich Peptides

Cell-penetrating peptides (CPPs) have gained attention as promising tools to enable the delivery of various molecules in a non-invasive manner. Among the CPPs, TAT and poly-arginine have been extensively utilized in numerous studies for the delivery of functional proteins, peptides, and macromolecules to analyze cellular signaling. However, the molecular mechanisms of cellular entry remain largely unknown. Here, we applied siRNA library screening to identify the regulatory genes for the cellular entry of poly-arginine peptide based on microscopic observation of the entry of fluorescent peptides in siRNA-treated cells. In this screening, we identified the cell membrane gene SLC4A4 and the trafficking regulator gene COPA, which also plays an important role in early endosome maturation. These results demonstrated that cellular entry of poly-arginine requires at least two different steps, probably binding on the cell surface and endosomal entry. The identification of genes for cellular entry of poly-arginine provides insights into its mechanisms and should further aid in the development of highly efficient cell-penetrating peptides.     

Intracellular traffic and fate of protein transduction domains HIV-1 TAT peptide and octaarginine. Implications for their utilization as drug delivery vectors

Transduction domains such as those derived from the HIV-TAT protein are candidate vectors for intracellular delivery of therapeutic macromolecules such as DNA and proteins. The mechanism by which they enter cells is controversial, and very little spatial information regarding the downstream fate of these peptides from the plasma membrane is available. We studied endocytic traffic of fluorescent conjugates of HIV-TAT peptide and octaarginine in human hematopoietic cell lines K562 (CD34-) and KG1a (CD34+) and substantiated our findings in epithelia cells. Both peptides were efficiently internalized to endocytic pathways of both hematopoietic cell lines; however, comparative analysis of the intracellular location of the peptides with endocytic probes revealed major differences in spatial organization of their endocytic organelles and their interaction with the peptides at low temperatures. Double labeling confocal microscopy demonstrates that prelabeled lysosomes of all the tested cells are accessible to internalized peptides within 60 min of endocytic uptake. Incubation of cells with nocodazole and cytochalasin D inhibited peptide traffic from early to late endosomal structures, demonstrating a cytoskeletal requirement for lysosomal delivery. Disruption of Golgi and endoplasmic reticulum dynamics was without effect on peptide localization, suggesting that endosomes and lysosomes rather than these organelles are the major acceptor compartments for these molecules.     

Simulation study of interaction mechanism between peptide and asymmetric membrane

Cell-penetrating peptides (CPPs) are widely used as drug carriers, owing to their superior ability to cross cell membrane both alone and with cargos, such as genes and other particles. Understanding the translocation mechanism of CPP is significant for many therapeutic purposes, including targeting drug and gene delivery. In this study, we performed a coarse-grained molecular dynamics simulation to investigate the interaction mechanism between polyarginine peptides and asymmetric membranes. Results showed that peptides can penetrate through the lipid bilayer by inducing a hydrophilic hole formation in the asymmetric membrane. Furthermore, the lengthy peptide chain length (R4–R16 peptides) and high membrane asymmetry positively affect peptide penetration. Our study provides insights into the molecular-level interactions between peptides and asymmetric membranes, as well as suggestions for targeted gene and drug delivery.     

Cell-penetrating peptides: from molecular mechanisms to therapeutics

The recent discovery of new potent therapeutic molecules which do not reach the clinic due to poor delivery and low bioavailability have made the delivery of molecules a keystone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including CPPs (cell-penetrating peptides), which represent a new and innovative concept to bypass the problem of bioavailability of drugs. CPPs constitute very promising tools and have been successfully applied for in vivo. Two CPP strategies have been described to date; the first one requires chemical linkage between the drug and the carrier for cellular drug internalization, and the second is based on the formation of stable complexes with drugs, depending on their chemical nature. The Pep and MPG families are short amphipathic peptides, which form stable nanoparticles with proteins and nucleic acids respectively. MPG- and Pep-based nanoparticles enter cells independently of the endosomal pathway and efficiently deliver cargoes, in a fully biologically active form, into a large variety of cell lines, as well as in animal models. This review focuses on the structure-function relationship of non-covalent MPG and Pep-1 strategies, and their requirement for cellular uptake of biomolecules and applications in cultured cells and animal models.     

TAT transduction: the molecular mechanism and therapeutic prospects

Research into the mechanism of protein transduction has undergone a renaissance in the past five years as many groups have sought to understand the behavior of transducing peptides to harness their enormous therapeutic and diagnostic potential. The field has benefited greatly from rigorous cell biological and biophysical studies of the mechanism used by cell penetrating peptides to enter cells and deliver their cargo. The recent identification of fluid phase endocytosis as the mode of cellular entry for TAT and other protein transduction domains has enhanced our understanding of how transduction facilitates intracellular delivery. Many outstanding questions and contradictions still remain to be resolved in the field. Nevertheless, the current body of work regarding the mechanism of uptake gives a much clearer picture of how these macromolecules enter cells and how we might enhance the bioavailability to take advantage of them clinically.     

Macromolecular therapeutics: emerging strategies for drug discovery in the postgenome era

The postgenome era offers a plethora of potential therapeutic targets. Many of these targets will be addressable using small organic molecules as drug candidates. However, certain aspects of cell function, particularly those that rely on protein-protein or protein-nucleic acid interactions, will be difficult to influence using small molecules. Thus, the possibility of using highly specific macromolecules as potential therapeutic agents is an intriguing concept. Recent developments in several areas of research have brought this possibility closer to fruition. Peptide and nucleic acid combinatorial libraries allow the generation of novel molecules having exquisite selectivity. Structural information and molecular modeling also contribute to the design of new macromolecules with therapeutic potential. Perhaps most importantly, approaches for delivering macromolecules into the cell interior have been developed and applied with considerable success. Thus, the therapeutic use of macromolecules, including oligonucleotides, peptides, and proteins, may be an idea whose time has come.     

Peptide-functionalized poly(ethylene glycol) star polymers: DNA delivery vehicles with multivalent molecular architecture

Exploring the development of nonviral nucleic acid delivery vectors with progressive, specific, and novel designs in molecular architecture is a fundamental way to investigate how aspects of chemical and physical structure impact the transfection process. In this study, macromolecules comprised of a four-arm star poly(ethylene glycol) and termini modified with one of five different heparin binding peptides have been investigated for their ability to bind, compact, and deliver DNA to mammalian cells in vitro. These new delivery vectors combine a PEG-derived stabilizing moiety with peptides that exhibit unique cell-surface binding ability in a molecular architecture that permits multivalent presentation of the cationic peptides. Five peptide sequences of varying heparin binding affinity were studied; each was found to sufficiently bind heparin for biological application. Additionally, the macromolecules were able to bind and compact DNA into particles of proper size for endocytosis. In biological studies, the PEG-star peptides displayed a range of toxicity and transfection efficiency dependent on the peptide identity. The vectors equipped with peptides of highest heparin binding affinity were found to bind DNA tightly, increase levels of cellular internalization, and display the most promising transfection qualities. Our results suggest heparin binding peptides with specific sequences hold more potential than nonspecific cationic polymers to optimize transfection efficiency while maintaining cell viability. Furthermore, the built-in multivalency of these macromolecules may allow simultaneous binding of both DNA at the core of the polyplex and heparan sulfate on the surface of the cell. This scheme may facilitate a bridging transport mechanism, tethering DNA to the surface of the cell and subsequently ushering therapeutic nucleic acids into the cell. This multivalent star shape is therefore a promising architectural feature that may be exploited in the design of future polycationic gene delivery vectors.