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Selective Uptake Into Drug Resistant Mammalian Cancer By Cell Penetrating Peptide-Mediated Delivery
Abstract
Research over the past decade has identified several of the key limiting features in multidrug resistance (MDR) cancer ther-apy applications, such as evolving glycoprotein receptors at the surface of the cell that limit therapeutic uptake, metabolic changes leading to protection from multidrug resistant mediators by enhanced degradation or efflux of therapeutics, and difficulty ensuring retention of intact and functional drugs once endocytosed. Nanoparticles have been demonstrated to be effective delivery vehicles for a plethora of therapeutic agents and, in the case of nucleic acid-based agents, they provide protective advantages. Functionalizing cell penetrating peptides (CPPs), also known as protein transduction domains, onto the surface of a fluorescent quantum dots creates a labelled delivery package to investigate the nuances and difficulties of drug transport in MDR cancer cells for potential future clinical applications of diverse nanoparticle therapeutic delivery strategies. In this study, eight distinct cell penetrating peptides were used (CAAKA, VP-22, HIV-TAT, Ku-70, and hCT(9-32), integrin-β3, HIV-gp41, and K-FGF) to examine the different cellular uptake profiles in cancer versus drug resistant melano-ma (A375 & A375-R), mesothelioma (MSTO & MSTO-R), and glioma (rat 9L & 9L-R, and human U87 & LN18), cell lines. The results of this study demonstrate that cell penetrating peptide uptake varies with the amount of drug resistance and cell type, likely due to changes in cell surface markers. This study provides insight to developing functional nanoplatform deliv-ery systems in drug resistant cancer models.
... CPPs could be advantageous for the intracellular delivery of therapeutically active molecules in various cancer types, which are challenging for the pharmaceutical sector [60]. According to research results, these peptides are an effective alternative against multidrug-resistant (MDR) cancer cells, allowing them to overcome difficulties of drug transport for potential future clinical applications [61]. ...
... Compared to cationic and amphipathic CPPs, the number of hydrophobic CPPs discovered is lower. To consider a CPP as hydrophobic, it must either contain a hydrophobic motif crucial for its internalization despite the rest of its sequence or be completely hydrophobic [67], such as Ku-70 (PMLKE) and K-FGF (AAVALLPAVLLAHLLAP) respectively, whose cellular internalization profile is comparable with other CPPs classes [61]. ...
... CPPs are a potentially powerful tool for overcoming difficulties in MDR cancer treatments. However, CPP uptake may vary according to drug resistance and cell type, probably due to differences in cell surface markers [61]. Therefore, the selection of CPPs for the develop CPP-based drug delivery systems must be further considered, and the selection of different CPPs could be a wise decision depending on the drug resistance in cancer cells. ...
The usage of Peptide-Based Drug-delivery systems (PBDs) is a strategy in which peptides carry a variety of compounds with pharmacological activity into cells. These innovative drug-delivery systems provide a promising perspective to fight cancer and other diseases, used for diagnosis and treatment. PBDs can be Cell-Penetrating Peptides that translocate across the cell membrane, allowing cargos’ intracellular delivery, and Tumor Homing Peptides which target biomarkers such as overexpressed receptors on tumor cells. PBDs bring groundbreaking advantages in cancer treatment: improved drug delivery and efficacy, decreases toxicity, and high selectivity resulting in lower accumulation on non-target tissues in the case of tumor homing peptides. These peptides are a hope to bring better drugs to fight cancer.
... Generally, the attachment of CPPs to the NP surface has been shown to be beneficial for the accessibility of CPPs, which potentially enhances recognition of the peptide with the cell surface [76]. Nevertheless, the chemical interaction between functional groups of the peptide and those of the NPs can have a significant influence on the formation of the secondary structure. ...
... Moreover, CPPs may be rationally modulated to target cancer cells [100]. Recently, Carnevale et al. compared the uptake profiles of several different CPP-NPs in cancer cells versus cancer-resistant cells [76]. In fact, the authors used eight different CPPs, which were attached via bidentate coordination using the thiol and amine of cysteine to the NP surface to generate multi-shelled QDs, and tested their ability to enter cancer cells. ...
Based on their tunable physicochemical properties and the possibility of producing cell-specific platforms through surface modification with functional biomolecules, nanoparticles (NPs) represent highly promising tools for biomedical applications. To improve their potential under physiological conditions and to enhance their cellular uptake, combinations with cell-penetrating peptides (CPPs) represent a valuable strategy. CPPs are often cationic peptide sequences that are able to translocate across biological membranes and to carry attached cargos inside cells and have thus been recognized as versatile tools for drug delivery. Nevertheless, the conjugation of CPP to NP surfaces is dependent on many properties from both individual components, and further insight into this complex interplay is needed to allow for the fabrication of highly stable but functional vectors. Since CPPs per se are nonselective and enter nearly all cells likewise, additional decoration of NPs with homing devices, such as tumor-homing peptides, enables the design of multifunctional platforms for the targeted delivery of chemotherapeutic drugs. In this review, we have updated the recent advances in the field of CPP-NPs, focusing on synthesis strategies, elucidating the influence of different physicochemical properties, as well as their application in cancer research.
... In a recent study, Carnevale et al. designed and tested CPP/NP conjugates against drug resistant cancer cell-lines. The first phase of this study demonstrated CPPs differences in cell selectivity attached to quantum dots (QD) in resistant cell lines versus naïve [95]. The authors observed that different CPPs exhibit selective uptake profiles for each cell-line. ...
... The authors observed that different CPPs exhibit selective uptake profiles for each cell-line. Although prominent uptake profiles were observed for the hCT (9-32), Ku-70 and HSV1-VP22 in resistant melanoma and rat glioblastoma cells [95]. ...
Cell-penetrating peptides (CPPs), also known as protein translocation domains, membrane translocating sequences or Trojan peptides, are small molecules of 6 to 30 amino acid residues capable of penetrating biological barriers and cellular membranes. Furthermore, CPP have become an alternative strategy to overcome some of the current drug limitations and combat resistant strains since CPPs are capable of delivering different therapeutic molecules against a wide range of diseases. In this review, we address the recent conjugation of CPPs with nanoparticles, which constitutes a new class of delivery vectors with high pharmaceutical potential in a variety of diseases.
... As underlined before, MDR of cancer cells is a major concern in all nanohybrid-based treatment strategies. Regarding the administration of functionalized QDs, the uptake of the fluorescent nanocarriers has been reported to differ depending on the drug-resistance profile of the cells [203]. Thus, the application of nanocarriers that are superior emitters for cell labeling and two-photon imaging may be considered, such as the recently described 2D cadmium chalcogenide nanoplatelets that display a 10 times greater two-photon absorption coefficient and better fluorescence response than QDs [204]. ...
Glioblastoma (GBM) is the most commonly diagnosed and most lethal primary malignant brain tumor in adults. Standard treatments are ineffective, and despite promising results obtained in early phases of experimental clinical trials, the prognosis of GBM remains unfavorable. Therefore, there is need for exploration and development of innovative methods that aim to establish new therapies or increase the effectiveness of existing therapies. One of the most exciting new strategies enabling combinatory treatment is the usage of nanocarriers loaded with chemotherapeutics and/or other anticancer compounds. Nanocarriers exhibit unique properties in antitumor therapy, as they allow highly efficient drug transport into cells and sustained intracellular accumulation of the delivered cargo. They can be infused into and are retained by GBM tumors, and potentially can bypass the blood–brain barrier. One of the most promising and extensively studied groups of nanostructured therapeutics are metal-based nanoparticles. These theranostic nanocarriers demonstrate relatively low toxicity, thus they might be applied for both diagnosis and therapy. In this article, we provide an update on metal-based nanostructured constructs in the treatment of GBM. We focus on the interaction of metal nanoparticles with various forms of electromagnetic radiation for use in photothermal, photodynamic, magnetic hyperthermia and ionizing radiation sensitization applications.
... Viral fusion peptides belong to the group of membranotropic CPPs [56]. The hydrophobic CPP family comprises K-FGF [71], C105Y [72] and gH625 [73][74][75][76][77]. ...
Biomedical research devotes a huge effort to the development of efficient non-viral nanovectors (NV) to improve the effectiveness of standard therapies. NVs should be stable, sustainable and biocompatible and enable controlled and targeted delivery of drugs. With the aim to foster the advancements of such devices, this review reports some recent results applicable to treat two types of pathologies, cancer and microbial infections, aiming to provide guidance in the overall design of personalized nanomedicines and highlight the key role played by peptides in this field. Additionally, future challenges and potential perspectives are illustrated, in the hope of accelerating the translational advances of nanomedicine
... 322 Cellular uptake of different NPs-CPP conjugates into drug resistant cancer cells showed that CPPs uptake is cell type-dependent. 323 CPPs in combination with NPs can also be used as imaging agents to follow the pharmacokinetics and pharmacodynamics of the therapeutics. Liu et al. demonstrated that arginine-rich CPPs significantly increase cellular uptake of QDs. ...
Nanoparticles (NPs) have attracted considerable attention in various fields, such as cosmetics, the food industry, material design, and nanomedicine. In particular, the fast-moving field of nanomedicine takes advantage of features of NPs for the detection and treatment of different types of cancer, fibrosis, inflammation, arthritis as well as neurodegenerative and gastrointestinal diseases. To this end, a detailed understanding of the NP uptake mechanisms by cells and intracellular localization is essential for safe and efficient therapeutic applications. In the first part of this review, we describe the several endocytic pathways involved in the internalization of NPs and we discuss the impact of the physicochemical properties of NPs on this process. In addition, the potential challenges of using various inhibitors, endocytic markers and genetic approaches to study endocytosis are addressed along with the principal (semi) quantification methods of NP uptake. The second part focuses on synthetic and bio-inspired substances, which can stimulate or decrease the cellular uptake of NPs. This approach could be interesting in nanomedicine where a high accumulation of drugs in the target cells is desirable and clearance by immune cells is to be avoided. This review contributes to an improved understanding of NP endocytic pathways and reveals potential substances, which can be used in nanomedicine to improve NP delivery.
... Carnevale et al. [78] studied the cellular uptake of eight CPPs [CAAKA, HSV1-VP22, HIV-TAT, HIV-gp41, Ku-70, hCT(9-32), integrin-β3, and K-FGF] incubated Recently, Lee et al. [79] conjugated, by adsorption, hydrophilic commercial QDs with a short CPP, and studied their transduction into A549 cells. The L6 CPP (RRWQWR) was derived from bovine lactoferricin and contained only three arginine residues. ...
Quantum dots (QDs) have attracted considerable attention as fluorescent probes for life sciences. The advantages of using QDs in fluorescence-based studies include high brilliance, a narrow emission band allowing multicolor labeling, a chemically active surface for conjugation, and especially, high photostability. Despite these advantageous features, the size of the QDs prevents their free transport across the plasma membrane, limiting their use for specific labeling of intracellular structures. Over the years, various methods have been evaluated to overcome this issue to explore the full potential of the QDs. Thus, in this review, we focused our attention on physical and biochemical QD delivery methods—electroporation, microinjection, cell-penetrating peptides, molecular coatings, and liposomes—discussing the benefits and drawbacks of each strategy, as well as presenting recent studies in the field. We hope that this review can be a useful reference source for researches that already work or intend to work in this area.
Graphic Abstract
Strategies for the intracellular delivery of quantum dots discussed in this review (electroporation, microinjection, cell-penetrating peptides, molecular coatings, and liposomes).
... There are relatively few numbers of hydrophobic CPPs, and their structure contains a large number of non-polar residues or only a few charged amino acids (less than 20% of the sequence). Natural hydrophobic CPPs found so far include C105Y (Rhee and Davis, 2006), Bip4 (Gomez et al., 2010), and K-FGF (Carnevale et al., 2018). Different from what is known for most amphiphilic cationic CPPs, the peptide sequence of hydrophobic CPPs does not significantly affect cell uptake (Gomez et al., 2010). ...
Cell-penetrating peptides (CPPs) are short peptides (fewer than 30 amino acids) that have been predominantly used in basic and preclinical research during the last 30 years. Since they are not only capable of translocating themselves into cells but also facilitate drug or CPP/cargo complexes to translocate across the plasma membrane, they have potential applications in the disease diagnosis and therapy, including cancer, inflammation, central nervous system disorders, otologic and ocular disorders, and diabetes. However, no CPPs or CPP/cargo complexes have been approved by the US Food and Drug Administration (FDA). Many issues should be addressed before translating CPPs into clinics. In this review, we summarize recent developments and innovations in preclinical studies and clinical trials based on using CPP for improved delivery, which have revealed that CPPs or CPP-based delivery systems present outstanding diagnostic therapeutic delivery potential.
... Carnevale et al. presented the results of a study in which fluorescent quantum dots (QDs) are incorporated into a conjugate in order to determine cellular uptake of cell penetrating peptides (CPPs), for potential use in a therapeutic delivery strategy for multidrug resistance (MDR) cancer cells [65]. Various CPPs such as hCT (9-32), Ku-70 and HSV1-VP22 were utilized in this study. ...
Proteins have received increasing attention as a novel platform for drug delivery and phototheranostic agents that may serve as new nanomedicines. In drug delivery system, proteins can act as powerful tools with self-assembled structures, triggering groups, receptors and agents to enhance cell penetration. Compared to other nanoparticle based theranostics, nano-platforms for theranostics by utilizing proteins have unique merits, such as higher loading capacities and high efficiency of photosensitizers in phototherapies. However, the full potential of protein-based theranostics has not been achieved thus far. In this review, recent advances made in this area were discussed following a format based on proteins in drug delivery systems, proteins-dye complexes for phototherapy of cancer, proteins nanoparticles for imaging-guided cancer therapy such as fluorescent imaging, positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound (US) Imaging, PA imaging and proteins-based nanoprobes and nanostructures. Importantly, this discussion also provides a view of future advances that will likely be made in this area and their potential clinical applications.
... 24 In a recent report, QDs were functionalized with different CPPs to understand cellular uptake mechanisms in multidrug resistance cancer cell lines. 25 Keeping in mind the need of novel and targeted bacterial internalization strategies with newer mechanisms of action, herein, the QD surface have been functionalized with CPP using the electrostatic approach that was studied thoroughly using fluorescence spectroscopy, gel electrophoresis, and ζpotential ( Figure 1i). The internalization of the QD−CPP conjugate into Escherichia coli (Gram negative) as well as Staphylococcus aureus (Gram positive) has been further investigated using confocal microscopy, which exhibited an effective internalizing signal into both strains ( Figure 1ii). ...
This report illustrates a strategy for designing a nanoconjugate derived vector which efficiently delivers antimicrobial drug directly into bacterial cells. The nanoconjugate comprises of negatively charged [email protected] quantum dot (QD) with its surface functionalized using catioinic BP-100 (KKLFKKILKYL-amide); a known cell penetrating peptide (CPP), via electrostatic approach. The interactions between QD and CPP in QDs functionalized CPPs (QD-CPP) have been well analysed using fluorescence spectroscopy, gel electrophoresis and zeta potential analysis. The QD-CPP conjugate was internalized into Gram-negative (E. coli) as well as Gram positive (S. aureus) bacterial strains with confocal studies exhibiting a strong signal in tested microorganisms. Further, to check the applicability of QD-CPP conjugate, as a delivery vector for generating an effective therapeutics, ampicillin molecules were conjugated on QD-CPP surface to generate QD-CPP-Amp conjugate. The CPP and drug molecules on the surface of QDs were well quantified using HPLC data. It was observed that the internalization and bacterial debilitation of QD-CPP-Amp conjugate is two to four fold effective as compared to bare ampicillin. The morphological changes to the bacterial cells upon the treatment with QD-CPP-Amp conjugates, were noted with no cytotoxic effect on tested mammalian cell lines. The results inferred that the proposed QD-CPP vector provides a targeted and proficient approach for cellular internalization of cargo (drug) in bacterial cells with effective tracking through florescent QDs.
Conspectus
For the delivery of drugs, different nanosized drug carriers (e.g., liposomes, lipid nanoparticles, and micelles) have been developed in order to treat diseases that afflict society. Frequently, these vehicles are formed by the self-assembly of small molecules to encapsulate the therapeutic cargo of interest. Over decades, nanoparticles have been optimized to make them more efficient and specific to fulfill tailor-made tasks, such as specific cell targeting or enhanced cellular uptake. In recent years, lipid-based nanoparticles in particular have taken center stage; however, off-targeting side effects and poor endosomal escape remain major challenges since therapies require high efficacy and acceptable toxicity.
To overcome these issues, many different approaches have been explored to make drug delivery more specific, resulting in reduced side effects, to achieve an optimal therapeutic effect and a lower required dose. The fate of nanoparticles is largely dependent on size, shape, and surface charge. A common approach to designing drug carriers with targeting capability is surface modification. Different approaches to functionalize nanoparticles have been investigated since the attachment of targeting moieties plays a significant role in whether they can later interact with surface-exposed receptors of cells. To this end, various strategies have been used involving different classes of biomolecules, such as small molecules, nucleic acids, antibodies, aptamers, and peptides.
Peptides in particular are often used since there are many receptors overexpressed in different specific cell types. Furthermore, peptides can be produced and modified at a low cost, enabling high therapeutic screening. Cell-penetrating peptides (CPPs) and cell-targeting peptides (CTPs) are frequently used for this purpose. Less studied in this context are fusogenic coiled-coil peptides. Lipid-based nanoparticles functionalized with these peptides are able to avoid the endolysosomal pathway; instead such particles can be taken up by membrane fusion, resulting in increased delivery of payload. Furthermore, they can be used for targeting cells/organs but are not directed at surface-exposed receptors. Instead, they recognize complementary peptide sequences, facilitating their uptake into cells.
In this Account, we will discuss peptides as moieties for enhanced cytosolic delivery, targeted uptake, and how they can be attached to lipid-based nanoparticles to alter their properties. We will discuss the properties imparted to the particles by peptides, surface modification approaches, and recent examples showing the power of peptides for in vitro and in vivo drug delivery. The main focus will be on the functionalization of lipid-based nanoparticles by fusogenic coiled-coil peptides, highlighting the relevance of this concept for the development of future therapeutics.
Growth factors play a pivotal role in orchestrating cellular growth and division by binding to specific cell surface receptors. Dysregulation of growth factor production or activity can contribute to the uncontrolled cell proliferation observed in cancer. Peptide-based nanoformulations (PNFs) have emerged as promising therapeutic strategies for growth factor-deficient cancers. PNFs offer multifaceted capabilities including targeted delivery, imaging modalities, combination therapies, resistance modulation, and personalized medicine approaches. Nevertheless, several challenges remain, including limited specificity, stability, pharmacokinetics, tissue penetration, toxicity, and immunogenicity. To address these challenges and optimize PNFs for clinical translation, in-depth investigations are warranted. Future research should focus on elucidating the intricate interplay between peptides and nanoparticles, developing robust spectroscopic and computational methodologies, and establishing a comprehensive understanding of the structure-activity relationship governing peptide-nanoparticle interactions. Bridging these knowledge gaps will propel the translation of peptide-nanoparticle therapies from bench to bedside. While a few peptide-nanoparticle drugs have obtained FDA approval for cancer treatment, the integration of nanostructured platforms with peptide-based medications holds tremendous potential to expedite the implementation of innovative anticancer interventions. Therefore, growth factor-deficient cancers present both challenges and opportunities for targeted therapeutic interventions, with peptide-based nanoformulations positioned as a promising avenue. Nonetheless, concerted research and development endeavors are essential to optimize the specificity, stability, and safety profiles of PNFs, thereby advancing the field of peptide-based nanotherapeutics in the realm of oncology research.
In this chapter, the delivery of biomolecules such as oligonucleotides, proteins, peptides and others by connection of the power of modern nanoparticles with the flexibility and efficacy of CPPs is summarized. This will increase the potential of therapeutic agents, requiring protection from degradation and release in a controlled manner.
Here, the brief summary of therapeutic developments and current situation with applications of CPPs is presented, hopefully leading to multiple CPP based drugs in near future.
Single-chain variable fragment antibody (scFv) is a small molecular weight antibody that can be used for both therapeutic and diagnostic purposes. To visualize the interaction with the target biomolecules, scFv must be labeled with fluorescent molecules. In this study, to achieve the efficient labeling of scFv, we developed scFv-fluorescent nanoparticle conjugates to utilize scFv as bioprobes. As fluorescent carriers, cadmium-free ZnS-AgInS2/ZnS core/shell nanoparticles were used, and scFv was immobilized onto the nanoparticles via the interaction of nickel ions on nitrilotriacetic acid and hexahistidine (His-tag) fused with scFv. UV-Vis, fluorescence spectra, NMR, and dynamic laser scattering were used to characterize the scFv immobilized fluorescent nanoparticles (scFv-FNPs). The amounts of scFv on FNPs were controlled by the concentration of scFv. The scFv-FNPs that were prepared were non-toxic and selectively bound to cancer cells. The scFv-FNPs could be used as bioanalytical tools, and the immobilization method described here is a promising method for labeling biomolecules with the His-tag.
Engineering of smart building molecules is key basis in designing intelligent drug delivery systems. As an emerging sophisticated delivery system strategy, the powerful functions of peptide drug conjugates (PDCs) are attributed to a smart linker and multifunctional peptide domain. Peptides exhibit a wide range of functions and properties, including easy chemical synthesis and versatile modification, tunable biocompatibility, diversified self-assembled nanostructures, specific recognition/binding, and deep penetration of the cell membrane/extracellular matrix. In addition, various types of linkers enable PDCs to release drugs responsively according to the disease microenvironment or treatment needs. Owing to these inherent advantages, PDCs have been widely explored for drug delivery. Herein, the latest developments in functional peptides and linkers commonly used to construct smart PDCs are reviewed. The purpose is to bring widespread attention to PDC design strategies and their contribution to fighting various diseases, as well as to provide guidance for research on intelligent PDC drug delivery systems.
Background
Efficient and controlled internalization of NPs into the cells depends on their physicochemical properties and dynamics of the plasma membrane. NPs-cell interaction is a complex process that decides the fate of NPs internalization through different endocytosis pathways.
Objective
The aim of this review is to highlight the physicochemical properties of synthesized nanoparticles (NPs) and their interaction with the cellular-dynamics and pathways like phagocytosis, pinocytosis, macropinocytosis, clathrin, and caveolae-mediated endocytosis and the involvement of effector proteins domain such as clathrin, AP2, caveolin, Arf6, Cdc42, dynamin and cell surface receptors during the endocytosis process of NPs.
Method
An electronic search was performed to explore the focused reviews and research articles on types of endocytosis and physicochemical properties of nanoparticles and their impact on cellular internalizations. The search was limited to peer-reviewed journals in the PubMed database.
Results
This article discusses in detail how different types of NPs and their physicochemical properties such as size, shape, aspect ratio, surface charge, hydrophobicity, elasticity, stiffness, corona formation, surface functionalization changes the pattern of endocytosis in the presence of different pharmacological blockers. Some external forces like a magnetic field, electric field, and ultrasound exploit the cell membrane dynamics to permeabilize them for efficient internalization with respect to fundamental principles of membrane bending and pore formation.
Conclusion
This review will be useful to attract and guide the audience to understand the endocytosis mechanism and their pattern with respect to physicochemical properties of NPs to improve their efficacy and targeting to achieve the impactful outcome in drug-delivery and theranostics applications.
Since the discovery of cell-penetrating peptides (CPP) in the 1980s, they have played a unique role in various fields owing to their excellent and unique cell membrane penetration function. In particular, in the treatment of tumours, CPPS have been used to deliver several types of 'cargos' to cancer cells. To address the insufficient targeting ability, non-selectivity, and blood instability, activatable cell-penetrating peptides, which can achieve targeted drug delivery in tumour treatment, enhance curative effects, and reduce toxicity have been developed. This study reviews the application of different cell-penetrating peptides in tumour-targeted delivery, overcoming multidrug resistance, organelle targeting, tumour imaging, and diagnosis, and summarises the different mechanisms of activatable cell-penetrating peptides in detail.
Synthetic or natural derived cell-penetrating peptides (CPPs) are vastly investigated as tools for the intracellular delivery of membrane-impermeable molecules. As viruses are intracellular obligate parasites, viral originated CPPs have been considered as suitable intracellular shuttling vectors for cargo transportation. A total of 310 CPPs were identified in the proteome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Screening the proteome of the cause of COVID-19 reveals that SARS-CoV-2 CPPs (SCV2-CPPs) span the regions involved in replication, protein-nucleotide and protein-protein interaction, protein-metal ion interaction, and stabilization of homo/hetero-oligomers. However, to find the most appropriate peptides as drug delivery vectors, one might face several hurdles. Computational analyses showed that 94.3% of the identified SCV2-CPPs are non-toxins, and 38% are neither antigenic nor allergenic. Interestingly, 36.70% of SCV2-CPPs were resistant to all four groups of protease families. Nearly 1/3 of SCV2-CPPs had sufficient inherent or induced helix and sheet conformation leading to increased uptake efficiency. Heliquest lipid-binding discrimination factor revealed that 44.30% of the helical SCV2-CPPs are lipid-binding helices. Although Cys-rich derived CPPs of helicase (NSP13) can potentially fold into a cyclic conformation in endosomes with a higher rate of endosomal release, the most optimal SCV2-CPP candidates as vectors for drug delivery were SCV2-CPP118, SCV2-CPP119, SCV2-CPP122, and SCV2-CPP129 of NSP12 (RdRp). Ten experimentally validated viral-derived CPPs were also used as the positive control to check the scalability and reliability of our protocol in SCV2-CPP retrieval. Some peptides with a cell-penetration ability known as bioactive peptides are adopted as biotherapeutics themselves. Therefore, 59.60%, 29.63%, and 32.32% of SCV2-CPPs were identified as potential antibacterial, antiviral, and antifungals, respectively. While 63.64% of SCV2-CPPs had immuno-modulatory properties, 21.89% were recognized as anti-cancers. Conclusively, the workflow of this study provides a platform for profound screening of viral proteomes as a rich source of biotherapeutics or drug delivery carriers.
Lipidated peptide amphiphiles exhibit high affinity for plasma cell membranes, good cell internalization and improved stability against degradation, which make them attractive as a drug delivery system. However, low drug loading capacity limits their application for drug delivery. Herein, we developed a gemini peptide amphiphile with enhanced membrane activity and endosomal escape, containing dodecanoic acid hydrophobic tails at the N-terminus, rhodamine on the pendant chain and an active targeting peptide sequence (GRGDS). The presence of rhodamine not only provides high drug loading capability due to the π-π stacking interaction between camptothecin (CPT) and rhodamine, but also promotes peptide amphiphiles to form compact spherical micelles, as well as allows for fluorescence imaging. Single-molecule total internal reflection fluorescence microscopy investigation reveals gemini peptide amphiphile has higher affinity for plasma cell membranes than single-tail peptide amphiphile. Meanwhile, better cell internalization of gemini peptide amphiphile was confirmed by flow cytometry and confocal laser scanning microscopy. Moreover, CPT-loaded gemini peptide amphiphile induced enhanced cytotoxicity in cancer cells when compared to free CPT, indicating that lipidated peptide amphiphile with π-conjugates on the pendant chain and two hydrophobic tails can serve as a highly efficient drug delivery vehicle.
Effective delivery of biologically active compounds including peptides, proteins, and nucleic acids into cells is an obstacle in the drug research. The intracellular delivery of biological agents generally relies on the endocytosis pathway, causing them to be trapped in endosomes and lysosomes. Cell‐penetrating peptides (CPPs) have previously been shown to be a useful transport tool for delivering many kinds of goods into cells through cell membranes. The latest discovery of cell penetrating poly(disulfide)s (CPDs) with excellent cytosolic delivery efficiency represents a major breakthrough in this field. In this work, we will focus on the latest advances in biological applications of the CPPs and new opportunities opened up by the CPDs.
The capability of cell-penetrating peptides (CPPs) to enable translocation of cargos across biological barriers carries promising pharmaceutical potential for the transport of drug molecules, as well as nanomaterials, into cells. Herein, we report on optimization of a CPP, namely sC18, in terms of its translocation efficiency, and on investigating those new CPPs regarding their interaction with silica nanoparticles (NPs). First, alanine scanning of sC18 yielded 16 cationic peptides from which two were selected for further studies. Whereas in the first case, a higher positive net charge and enhanced amphipathicity resulted in significantly higher internalization rates than sC18, the second one demonstrated reduced cellular uptake efficiencies, and served as a control. We then attached these CPPs to silica nanoparticles of different sizes (50, 150 and 300 nm) via electrostatic interactions and could demonstrate that the secondary alpha-helical structure of the peptides was preserved. Following, cellular uptake studies using HeLa cells showed that the tested CPP-NPs were successfully translocated into HeLa cells in a size-dependent manner. Moreover, dependent on the CPP used, we realized differences in translocation efficiency, which was similar to what we had observed for the free peptides. All in all, we highlight the high potential of sequential fine-tuning of CPPs and provide novel insights into their interplay with biologically inorganic benign nanoparticles. Given the high cellular permeability of CPPs and their ability to translocate into a wide spectrum of cell types, our studies may stimulate future research of CPPs with inorganic nanocarrier surfaces.
The selective infiltration of cell membranes and tissue barriers often blocks the entry of most active molecules. This natural defense mechanism prevents the invasion of exogenous substances and limits the therapeutic value of most available molecules. Therefore, it is particularly important to find appropriate ways of membrane translocation and therapeutic agent delivery to its target site. Cell penetrating peptides (CPPs) are a group of short peptides harnessed in this condition, possessing a significant capacity for membrane transduction and could be exploited to transfer various biologically active cargoes into the cells. Since their discovery, CPPs have been employed for delivery of a wide variety of therapeutic molecules to treat various disorders including cranial nerve involvement, ocular inflammation, myocardial ischemia, dermatosis and cancer. The promising results of CPPs-derived therapeutics in various tumor models demonstrated a potential and worthwhile scope of CPPs in chemotherapy. This review describes the detailed description of CPPs and CPPs-assisted molecular delivery against various tissues and organs disorders. An emphasis is focused on summarizing the novel insights and achievements of CPPs in surmounting the natural membrane barriers during the last 5 years.
Cell penetrating peptides as well as their applications have been extensively studied and multiple exciting reports about the topics emerge daily; this book hopefully is the proof for this statement. However, CPPs are not yet on the list of drugs for a patient therapy today and we can talk about the therapeutic potential of CPPs only. I believe that the CPP based drugs will be available in near future, however, several hurdles should be overcome before that. Below, the brief summary of therapeutic developments and current situation with applications of CPPs will be presented.
The membrane bilayer delimits the interior of individual cells and provides them with the ability to survive and function properly. However, the crossing of cellular membranes constitutes the principal impediment to gaining entry into cells, and the potential therapeutic application of many drugs is predominantly dependent on the development of delivery tools that should take the drug to target cells selectively and efficiently with only minimal toxicity. Cell-penetrating peptides are short and basic peptides are widely used due to their ability to deliver a cargo across the membrane both in vitro and in vivo. It is widely accepted that their uptake mechanism involves mainly the endocytic pathway, the drug is catched inside endosomes and lysosomes, and only a small quantity is able to reach the intracellular target. In this wide-ranging scenario, a fascinating novel hypothesis is that membranotropic peptides that efficiently cross biological membranes, promote lipid-membrane reorganizing processes and cause a local and temporary destabilization and reorganization of the membrane bilayer, may also be able to enter cells circumventing the endosomal entrapment; in particular, by either favoring the escape from the endosome or by direct translocation. This review summarizes current data on membranotropic peptides for drug delivery.
Kaposi's sarcoma (KS) is an angioproliferative disease frequently seen in patients with the acquired immunodeficiency syndrome (AIDS). Previous studies suggest that the HIV-1 protein Tat and Fibroblast Growth Factor 2 (FGF-2) have synergistic angiogenic effects in AIDS-KS tumors. However, the mechanisms by which FGF-2 is released and activated in KS tumors are not clearly defined. We carried out this study to determine whether an FGF-binding protein (FGFBP1 or BP1) that enhances the angiogenic activity of FGF-2 is expressed in AIDS-KS tumors, and to define whether BP1, FGF-2, and HIV-Tat protein-protein interactions could play a potential clinically role in the pathogenesis of AIDS-KS.
BP1 was localized in AIDS-KS lesions by immunohistochemistry and in situ hybridization studies. The binding of radiolabeled FGF-2 to His-tagged BP1 or the FGF-receptor 1 was assessed in the presence and absence of HIV-Tat and other viral proteins. Mice carrying tetracycline-regulated BP1 transgene mice were used to determine whether activation of BP1 during wound healing induces KS-like lesions.
BP1 expression was detected in AIDS-KS tumor keratinocytes, spindle cells, and infiltrating mononuclear cells. In addition, HIV-Tat competed for the binding of FGF-2 to immobilized BP1, but does not affect the interactions of FGF-2 with its high affinity receptor (FGFR-1). In contrast, two other HIV-proteins, Nef and gp120, did not affect the binding of FGF-2 to BP1 or to FGFR-1. Finally, up-regulation of BP1 expression in tetracycline-regulated -conditional BP1 transgenic mice subjected to skin wounds, induced KS-like skin lesions.
Taking into consideration the results of previous studies showing that both HIV-Tat and BP1 enhance the mitogenic and angiogenic activity of locally-stored FGF-2, both in vitro and in vivo, our findings suggest a novel mechanism by which the release and activity of FGFs can be modulated in AIDS-KS tumors by HIV-Tat as well as BP1.
Mesenchymal stem cells (MSC) have been identified as having great potential as autologous cell therapeutics to treat traumatic brain injury, spinal injury, as well as neuronal and cardiac ischemic events. All future clinical applications of MSC cell therapies must allow the MSC to be harvested, transfected, and induced to express a desired protein or selection of proteins to have medical benefit. For the full potential of MSC cell therapy to be realized, it is desirable to systematically alter the protein expression of therapeutically beneficial biomolecules in harvested MSC cells with high fidelity in a single transfection event. We have developed a delivery platform based on the use of a solid gold nanoparticle that has been surface modified to produce a fusion containing a zwitterionic, pentapeptide designed from Bax-inhibiting peptide (Ku70) to enhance cellular uptake and a linearized expression vector to induce enhanced expression of brain derived neurotrophic factor (BDNF) in rat derived MSCs. Ku70 is observed to effect >80% transfection following a single treatment of femur bone marrow isolated rat MSCs with efficiencies for the delivery of a 6.6kbp gene on either a AuNP or CdSe/ZnS QD. Gene expression is observed within 4d by optical measurements and secretion observed within 10d by Western Blot analysis. The combination of being able to selectively engineer the NP, co-localize biological agents, and enhance the stability of those agents have provided the strong impetus to utilize this novel class of materials to engineer primary MSCs.
Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.
Förster resonance energy transfer (FRET) may be regarded as a "smart" technology in the design of fluorescence probes for biological sensing and imaging. Recently, a variety of nanoparticles that include quantum dots, gold nanoparticles, polymer, mesoporous silica nanoparticles and upconversion nanoparticles have been employed to modulate FRET. Researchers have developed a number of "visible" and "activatable" FRET probes sensitive to specific changes in the biological environment that are especially attractive from the biomedical point of view. This article reviews recent progress in bringing these nanoparticle-modulated energy transfer schemes to fruition for applications in biosensing, molecular imaging and drug delivery.
Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
Cell-penetrating peptides (CPPs) have been previously shown to be powerful transport vector tools for the intracellular delivery of a large variety of cargoes through the cell membrane. Intracellular delivery of plasmid DNA (pDNA), oligonucleotides, small interfering RNAs (siRNAs), proteins and peptides, contrast agents, drugs, as well as various nanoparticulate pharmaceutical carriers (e.g., liposomes, micelles) has been demonstrated both in vitro and in vivo. This review focuses on the peptide-based strategy for intracellular delivery of CPP-modified nanocarriers to deliver small molecule drugs or DNA. In addition, we discuss the rationales for the design of 'smart' pharmaceutical nanocarriers in which the cell-penetrating properties are hidden until triggered by exposure to appropriate environmental conditions (e.g., a particular pH, temperature, or enzyme level), applied local microwave, ultrasound, or radiofrequency radiation.
We found that Ku70, a known DNA repair factor, has a novel function to bind and inhibit Bax (Bcl-2-associated X protein), a key mediator of apoptosis. Pentapeptides derived from the Bax-binding domain of Ku70 were cell-permeable and protected cells from Bax-mediated apoptosis. These pentapeptides were called BIPs (Bax-inhibiting peptides). BIPs may become a useful therapeutic tool to reduce cellular damage. We also generated BIP mutant pentapeptides that do not inhibit Bax, but retain their cell-penetrating activity. Since both BIPs and BIP mutants are cell-permeable, these peptides were designated CPP5s (cell-penetrating pentapeptides). Among the CPP5s discovered, VPTLK (BIP) and KLPVM (BIP mutant) were confirmed to possess protein transduction activity by examination of the delivery of GFP (green fluorescent protein) into cells by these peptides. The mechanism of cell penetration by CPP5s is not known. CPP5s enter the cell at 0 and 4 degrees C. In preliminary studies, various inhibitors of endocytosis and pinocytosis did not show any significant suppression of CPP5 cell entry. CPP5s have very low toxicity in vitro and in vivo and so may be useful tools in order to develop non-toxic drug-delivery technologies.
Short regulatory oligonucleotides (ONs) have a great therapeutic potential for the modulation of gene expression due to their high specificity and low toxicity. The major obstacles for in vivo clinical applications of ONs are the poor permeability of plasma membrane to nucleic acids and the sensitivity of ONs to enzymatic degradation. Hence, various delivery vehicles have been developed to ensure the transduction of ONs into cells. Among these, the cell-penetrating peptides (CPPs) have gained quickly broadening popularity as promising nonviral transmembrane delivery vectors. For coupling of nucleic acids to CPPs, two distinct strategies may be applied-covalent and noncovalent. The majority of earlier studies have used covalent coupling of CPPs to ONs. However, the number of studies demonstrating very high therapeutic potential of noncovalent complexes of ONs with novel CPP-based delivery vehicles is explosively increasing. In this review, the recent developments in the application of CPP-mediated oligonucleotide delivery by noncovalent strategy will be discussed.
Lentiviruses, including HIV-1, have transmembrane envelope (Env) glycoproteins with cytoplasmic tails that are quite long compared with those of other retroviruses. However, mainly because of the lack of biochemical studies performed in cell types that are targets for HIV-1 infection, no clear consensus exists regarding the function of the long lentiviral Env cytoplasmic tail in virus replication. In this report, we characterize the biological and biochemical properties of an HIV-1 mutant lacking the gp41 cytoplasmic tail. We find that the gp41 cytoplasmic tail is necessary for the efficient establishment of a productive, spreading infection in the majority of T cell lines tested, peripheral blood mononuclear cells, and monocyte-derived macrophages. Biochemical studies using a high-level, transient HIV-1 expression system based on pseudotyping with the vesicular stomatitis virus glycoprotein demonstrate that in HeLa and MT-4 cells, mutant Env incorporation into virions is reduced only 3-fold relative to wild type. In contrast, gp120 levels in virions produced from a number of other T cell lines and primary macrophages are reduced more than 10-fold by the gp41 truncation. The Env incorporation defect imposed by the cytoplasmic tail truncation is not the result of increased shedding of gp120 from virions or reduced cell-surface Env expression. These results demonstrate that in the majority of T cell lines, and in primary cell types that serve as natural targets for HIV-1 infection in vivo, the gp41 cytoplasmic tail is essential for efficient Env incorporation into virions.
Quantum dots (QDs) are luminescent semiconductor nanocrystals that are widely used as fluorescent probes in biomedical applications, including cellular imaging and tumor tracking. Cell-penetrating peptides (CPPs), also called protein transduction domains (PTDs), are short basic peptides that permeate cell membranes and are able to deliver a variety of macromolecule cargoes, such as DNAs, RNAs, proteins, and nanomaterials. Here we review strategies to couple QDs to CPPs, by either covalent linkages or noncovalent interactions, to provide a tool to study intracellular delivery. This facilitated transport of QDs by CPPs into cells is both simple and efficient. Accordingly, CPP-QD nanoparticles are likely to be of broad utility in biological research and advance the development of medical and pharmaceutical therapeutics.
A major challenge of drug delivery using colloids via the airway is to understand the mechanism implied in their interactions with epithelial cells. The purpose of this work was to characterize the process of endocytosis and exocytosis of cationic nanoparticles (NPs) made of maltodextrin which were developed as a delivery system for antigens in vaccine applications. Confocal microscopy demonstrated that these NP are rapidly endocytosed after as little as 3 min incubation, and that the endocytosis was also faster than NP binding since most of the NPs were found in the middle of the cells around the nuclei. A saturation limit was observed after a 40 min incubation, probably due to an equilibrium becoming established between endocytosis and exocytosis. Endocytosis was dramatically reduced at 4 degrees C compared with 37 degrees C, or by NaN(3) treatment, both results suggesting an energy dependent process. Protamine pretreatment of the cells inhibited NPs uptake and we found that clathrin pathway is implied in their endocytosis. Cholesterol depletion increased NP uptake by 300% and this phenomenon was explained by the fact that cholesterol depletion totally blocked NP exocytosis. These results suggest that these cationic NPs interact with anionic sites, are quickly endocytosed via the clathrin pathway and that their exocytosis is cholesterol dependent, and are similar to those obtained in other studies with viruses such as influenza.
The benefit of cytoreductive surgery for glioblastoma multiforme (GBM) is unclear, and selection bias in past series has been observed. The 5-aminolevulinic acid (ALA) study investigated the influence of fluorescence-guided resections on outcome and generated an extensive database of GBM patients with optimized resections. We evaluated whether the Radiation Therapy Oncology Group recursive partitioning analysis (RTOG-RPA) would predict survival of these patients and whether there was any benefit from extensive resections depending on RPA class. A total of 243 per-protocol patients with newly diagnosed GBM were operated on with or without ALA and treated by radiotherapy. Postoperative MRI was obtained in all patients. Patients were allocated into RTOG-RPA classes III-V based on age, KPS, neurological condition, and mental status (as derived from the NIH Stroke Scale). Median overall survival among RPA classes III, IV, and V was 17.8, 14.7, and 10.7 months, respectively, with 2-year survival rates of 26%, 12%, and 7% (p = 0.0007). Stratified for degree of resection, survival of patients with complete resections was clearly longer in RPA classes IV and V (17.7 months vs. 12.9 months, p = 0.0015, and 13.7 months vs. 10.4 months, p = 0.0398; 2-year rates: 21.0% vs. 4.4% and 11.1% vs. 2.6%, respectively), but was not in the small subgroup of RPA class III patients (19.3 vs. 16.3 months, p = 0.14). Survival of patients from the ALA study is correctly predicted by the RTOG-RPA classes. Differences in survival depending on resection status, especially in RPA classes IV and V, support a causal influence of resection on survival.
Integrins are major two-way signaling receptors responsible for the attachment of cells to the extracellular matrix and for cell-cell interactions that underlie immune responses, tumor metastasis, and progression of atherosclerosis and thrombosis. We report the structure-function analysis of the cytoplasmic tail of integrin beta 3 (glycoprotein IIla) based on the cellular import of synthetic peptide analogs of this region. Among the four overlapping cell-permeable peptides, only the peptide carrying residues 747-762 of the carboxyl-terminal segment of integrin beta 3 inhibited adhesion of human erythroleukemia (HEL) cells and of human endothelial cells (ECV) 304 to immobilized fibrinogen mediated by integrin beta 3 heterodimers, alpha IIb beta 3, and alpha v beta 3, respectively. Inhibition of adhesion was integrin-specific because the cell-permeable beta 3 peptide (residues 747-762) did not inhibit adhesion of human fibroblasts mediated by integrin beta 1 heterodimers. Conversely, a cell-permeable peptide representing homologous portion of the integrin beta 1 cytoplasmic tail (residues 788-803) inhibited adhesion of human fibroblasts, whereas it was without effect on adhesion of HEL or ECV 304 cells. The cell-permeable integrin beta 3 peptide (residues 747-762) carrying a known loss-of-function mutation (Ser752Pro) responsible for the genetic disorder Glanzmann thrombasthenia Paris I did not inhibit cell adhesion of HEL or ECV 304 cells, whereas the beta 3 peptide carrying a Ser752Ala mutation was inhibitory. Although Ser752 is not essential, Tyr747 and Tyr759 form a functionally active tandem because conservative mutations Tyr747Phe or Tyr759Phe resulted in a nonfunctional cell permeable integrin beta 3 peptide. We propose that the carboxyl-terminal segment of the integrin beta 3 cytoplasmic tail spanning residues 747-762 constitutes a major intracellular cell adhesion regulatory domain (CARD) that modulates the interaction of integrin beta 3-expressing cells with immobilized fibrinogen. Import of cell-permeable peptides carrying this domain results in inhibition "from within" of the adhesive function of these integrins.
Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an alpha-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the alpha-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nM. In contrast, a peptide with a full alpha-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4 degrees C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest.
The development of antisense and gene therapy has focused mainly on improving methods for oligonucleotide and gene delivery into cells. In the present work, we describe a potent new strategy for oligonucleotide delivery based on the use of a short peptide vector, termed MPG (27 residues), which contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen. The formation of peptide vector/oligonucleotide complexes was investigated by measuring changes in intrinsic tryptophan fluorescence of peptide and of mansyl-labelled oligonucleotides. MPG exhibits relatively high affinity for both single- and double-stranded DNA in a nanomolar range. Based on both intrinsic and extrinsic fluorescence titrations, it appears that the main binding between MPG and oligonucleotides occurs through electrostatic interactions, which involve the basic-residues of the peptide vector. Further peptide/peptide interactions also occur, leading to a higher MPG/oligonucleotide ratio (in the region of 20/1), which suggests that oligonucleotides are most likely coated with several molecules of MPG. Premixed complexes of peptide vector with single or double stranded oligonucleotides are delivered into cultured mammalian cells in less than 1 h with relatively high efficiency (90%). This new strategy of oligonucleotide delivery into cultured cells based on a peptide vector offers several advantages compared to other commonly used approaches of delivery including efficiency, stability and absence of cytotoxicity. The interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and crossing of the plasma membrane. The mechanism of cell delivery of oligonucleotides by MPG does not follow the endosomal pathway, which explains the rapid and efficient delivery of oligonucleotides in the nucleus. As such, we propose this peptide vector as a powerful tool for potential development in gene and antisense therapy.
VP22, a structural protein of herpes simplex virus, exhibits unusual trafficking properties which we proposed might be exploited
in gene and protein delivery applications. To pursue the use of the protein itself for cargo delivery into cells, we developed
an expression system for the C-terminal half of VP22, residues 159–301 (VP22.C1), and purified the protein in high yields.
Addition of short oligonucleotides (ODNs) induced the assembly of novel particles, which were regular spheres with a size
range of 0.3 to 1.0 μm in diameter, incorporating both protein and ODN. Following the particles in living cells using fluorescently
tagged ODNs, we show that they enter efficiently within 2–4 h, and reside stably in the cell cytoplasm for up to several days.
Remarkably, however, light activation induced particle disruption and release of the protein and ODN to the nucleus and cytoplasm
within seconds, a process that we have captured by time lapse microscopy. In addition to delivering antisense ODNs, ribozymes,
and RNA/DNA hybrids, the VP22.C1 protein could also be modified to include peptides or proteins. These particles have the
potential for delivery of a wide range of therapeutic agents in gene therapy and vaccine development.
Multidrug resistance (MDR) represents a major hindrance to the efficacy of cancer chemotherapeutics. While surgical resection, radiation, and chemotherapy can be used to reduce tumor size, the subsequent appearance of drug resistant cells is a frequent problem. One of the main contributors to the development of MDR is increased expression of multi-drug resistant protein 1 (MDR1), also known as P-glycoprotein (P-gp). P-gp is a membrane-associated efflux pump that can efficiently remove internalized taxane-base chemotherapeutics thus preventing drug accumulation and maintaining cellular viability. Consequently, investigation into the molecular mechanisms responsible for regulation of P-gp expression is necessary to facilitate treatment of MDR tumors. Using molecular and biochemical approaches, we identified that the micro-RNA, miRNA149, contributes to the development of MDR within malignant mesothelioma cells by regulating the expression of MDR1.
Gold nanoparticles provide an excellent platform for biological and material applications due to their unique physical and chemical properties. However, decreased colloidal stability and formation of irreversible aggregates while freeze-drying nanomaterials, limits their use in real world applications. Here, we report the use of engineered surface ligands that provide non-fouling characteristics, exceptional colloidal stability, and the ability to lyophilize and re-disperse multiple times. The design of the surface ligand enables efficient conjugation; the use of avidin provides a universal toolkit for further functionalization that maintains all the favorable attributes of the precursor particles.
Each year, the American Cancer Society estimates the numbers of new cancer cases and deaths that will occur in the United States in the current year and compiles the most recent data on cancer incidence, mortality, and survival. Incidence data were collected by the National Cancer Institute (Surveillance, Epidemiology, and End Results [SEER] Program), the Centers for Disease Control and Prevention (National Program of Cancer Registries), and the North American Association of Central Cancer Registries. Mortality data were collected by the National Center for Health Statistics. In 2016, 1,685,210 new cancer cases and 595,690 cancer deaths are projected to occur in the United States. Overall cancer incidence trends (13 oldest SEER registries) are stable in women, but declining by 3.1% per year in men (from 2009-2012), much of which is because of recent rapid declines in prostate cancer diagnoses. The cancer death rate has dropped by 23% since 1991, translating to more than 1.7 million deaths averted through 2012. Despite this progress, death rates are increasing for cancers of the liver, pancreas, and uterine corpus, and cancer is now the leading cause of death in 21 states, primarily due to exceptionally large reductions in death from heart disease. Among children and adolescents (aged birth-19 years), brain cancer has surpassed leukemia as the leading cause of cancer death because of the dramatic therapeutic advances against leukemia. Accelerating progress against cancer requires both increased national investment in cancer research and the application of existing cancer control knowledge across all segments of the population. CA Cancer J Clin 2016. © 2016 American Cancer Society.
Peptides and mini proteins have many biological and biomedical implications, which motivates the development of accurate methods, suitable for large-scale experiments, to predict their experimental or native conformations solely from sequences. In this study, we report PEP-FOLD2, an improved coarse grained approach for peptide de novo structure prediction and compare it with PEP-FOLD1 and the state-of-the-art Rosetta program. Using a benchmark of 56 structurally diverse peptides with 25–52 amino acids and a total of 600 simulations for each system, PEP-FOLD2 generates higher quality models than PEP-FOLD1, and PEP-FOLD2 and Rosetta generate near-native or native models for 95% and 88% of the targets, respectively. In the situation where we do not have any experimental structures at hand, PEP-FOLD2 and Rosetta return a near-native or native conformation among the top five best scored models for 80% and 75% of the targets, respectively. While the PEP-FOLD2 prediction rate is better than the ROSETTA prediction rate by 5%, this improvement is non-negligible because PEP-FOLD2 explores a larger conformational space than ROSETTA and consists of a single coarse-grained phase. Our results indicate that if the coarse-grained PEP-FOLD2 method is approaching maturity, we are not at the end of the game of mini-protein structure prediction, but this opens new perspectives for large-scale in silico experiments.
The ability to efficiently access cytosolic proteins is desired in both biological research and medicine. However, targeting intracellular proteins is often challenging, because to reach the cytosol, exogenous molecules must first traverse the cell membrane. This review provides a broad overview of how certain molecules are thought to cross this barrier, and what kinds of approaches are being made to enhance the intracellular delivery of those that are impermeable. We first discuss rules that govern the passive permeability of small molecules across the lipid membrane, and mechanisms of membrane transport that have evolved in nature for certain metabolites, peptides, and proteins. Then, we introduce design strategies that have emerged in the development of small molecules and peptides with improved permeability. Finally, intracellular delivery systems that have been engineered for protein payloads are surveyed. Viewpoints from varying disciplines have been brought together to provide a cohesive overview of how the membrane barrier is being overcome.
Recent advances in cell transfection have suggested that delivery of a gene on a gold nanoparticle (AuNP) can enhance transfection efficiency. The mechanism of transfection is poorly understood, particularly when the gene is appended to an AuNP, as expression of the desired exogenous protein is dependent not only on the efficiency of the gene being taken into the cell, but also on efficient endosomal escape and cellular processing of the nucleic acid. Design of a multicolor surface energy transfer (McSET) molecular beacon by independently dye labeling a linearized plasmid and short duplex DNA (sdDNA) appended to an AuNP, allows spatiotemporal profiling the transfection events providing insight into package uptake, disassembly, and final plasmid expression. Delivery of the AuNP construct encapsulated in Lipofectamine2000® is monitored in Chinese hamster ovary cells using live-cell confocal microscopy. The McSET beacon signals the location and timing of the AuNP release and endosomal escape events for the plasmid and the sdDNA discretely, which are correlated with plasmid transcription by fluorescent protein expression within the cell. It is observed that delivery of the construct leads to endosomal release of the plasmid and sdDNA from the AuNP surface at different rates, prior to endosomal escape. Slow cytosolic diffusion of the nucleic acids is believed to be the limiting step for transfection, impacting the time dependent expression of protein. The overall protein expression yield is enhanced when delivered on an AuNP, possibly due to better endosomal escape or lower degradation prior to endosomal escape.
Cell-penetrating peptides (CPP) are able to efficiently transport cargos across cell membranes without being cytotoxic to cells, thus present a great potential in drug delivery and diagnosis. While the role of cationic residues in CPPs has been well studied, that of Trp is still not clear. Herein 7 peptide analogs of RW9 (RRWWRRWRR, an efficient CPP) were synthesized in which Trp were systematically replaced by Phe residues. Quantification of cellular uptake reveals that substitution of Trp by Phe strongly reduces the internalization of all peptides despite the fact that they strongly accumulate in the cell membrane. Cellular internalization and biophysical studies show that not only the number of Trp residues but also their positioning in the helix and the size of the hydrophobic face they form are important for their internalization efficacy, the highest uptake occurring for the analog with 3 Trp residues. Using CD and ATR-FTIR spectroscopy we observe that all peptides became structured in contact with lipids, mainly in α-helix. Intrinsic tryptophan fluorescence studies indicate that all peptides partition in the membrane in about the same manner (Kp~10(5)) and that they are located just below the lipid headgroups (~10Å) with slightly different insertion depths for the different analogs. Plasmon Waveguide Resonance studies reveal a direct correlation between the number of Trp residues and the reversibility of the interaction following membrane washing. Thus a more interfacial location of the CPP renders the interaction with the membrane more adjustable and transitory enhancing its internalization ability.
Copyright © 2014 Elsevier B.V. All rights reserved.
The blood-brain barrier (BBB) remains a formidable obstacle in medicine, preventing efficient penetration of chemotherapeutic and diagnostic agents to malignant gliomas. Here, a transactivator of transcription (TAT) peptide-modified gold nanoparticle platform (TAT-Au NP) with a 5 nm core size is demonstrated to be capable of crossing the BBB efficiently and delivering cargoes such as the anticancer drug doxorubicin (Dox) and Gd3+ contrast agents to brain tumor tissues. Treatment of mice bearing intracranial glioma xenografts with pH-sensitive Dox-conjugated TAT-Au NPs via a single intravenous administration leads to significant survival benefit when compared to the free Dox. Furthermore, it is demonstrated that TAT-Au NPs are capable of delivering Gd3+ chelates for enhanced brain tumor imaging with a prolonged retention time of Gd3+ when compared to the free Gd3+ chelates. Collectively, these results show promising applications of the TAT-Au NPs for enhanced malignant brain tumor therapy and non-invasive imaging.
Significant quantities of synthetic nanoparticles circulating in the body are cleared and retained long in the resident macrophages of the mononuclear phagocytic system (MPS), increasing the likelihood of nanoparticle-mediated chronic toxicity. To date, there has been limited effort to understand how these nanoparticles leave the macrophages. Here, we demonstrate that the native surface chemistries of gold nanoparticles (GNPs) and their subsequent opsonization by serum proteins play critical roles in the exocytosis patterns in macrophages. The cationic GNPs were retained in the cells for a relatively long time, likely due to their intracellular agglomeration. Contrarily, the PEGylated GNPs migrated in the cytoplasm in the form of individual particles and exited the cells rapidly because the PEG coating mitigated interactions between GNPs and intracellular proteins. Additionally, their exocytosis pattern was not significantly governed by the size, particularly in the range from 10 to 40 nm. These results suggest that systemic excretion and toxicity of nanoparticles cleared in the MPS could be modulated by engineering their surface chemistry.
The intrinsic property of cell-penetrating peptides (CPPs) to deliver therapeutic molecules (nucleic acids, drugs, imaging agents) to cells and tissues in a non-toxic manner, has pointed them as potential components of future drugs and disease diagnostic agents. These versatile peptides are simple to synthesize, functionalize, and characterize yet are able to deliver covalently or non-covalently conjugated bioactive cargos (from small chemical drugs to large plasmid DNA) inside cells, primarily via endocytosis, in order to obtain high levels of gene expression, gene silencing or tumor targeting. Tipically CPPs are often passive and non-selective, yet must must be functionalized or chemically modified to create effective delivery vectors that succeed in targeting specific cells or tissues. Furthermore, the design of clinically effective systemic delivery systems requires the same amount of attention to detail in both design of the delivered cargo and the cell penetrating peptide used to deliver it.
Cancer cell resistance to chemotherapy is still a heavy burden that impairs treatment of cancer patients. Both intrinsic and acquired resistance results from the numerous genetic and epigenetic changes occurring in cancer cells. Most of the hallmarks of cancer cells provide general mechanisms to sustain stresses such as the ones induced by chemotherapeutic drugs. Moreover, specific changes in the target bring resistance to specific drugs like modification in nucleotide synthesis enzymes upon anti-metabolite exposure, in microtubule composition upon spindle poison treatment, in topoisomerase activity upon topoisomerase inhibitor incubation or in intracellular signaling pathways when targeting tyrosine kinase receptors. Finally, the stemness properties of a few cancer cells as well as components of the tumor stroma, like fibroblasts and tumor-associated macrophages but also hypoxia, also help tumor to resist to anticancer agents. These processes provide an additional level of complexity to the understanding of the tumor resistance phenomenon. This review aims to describe the different general mechanisms as well as some examples of specific on target modifications inducing cancer cell resistance to chemotherapy at the molecular level. Perspectives to develop more efficient treatment, using genomic signature or more specific biomarkers to characterize putative resistance mechanisms in patients before choosing the more appropriate treatment, will also be discussed.
Cell-penetrating peptides (CPPs) are short cationic peptides that have been extensively studied as drug delivery vehicles for proteins, nucleic acids and nanoparticles. However, the formulation of CPP-based therapeutics into different pharmaceutical formulations and their stability in relevant biological environments have not been given the same attention. Here, we show that a newly developed CPP, PepFect 14 (PF14), forms non-covalent nanocomplexes with short interfering RNA (siRNA), which are able to elicit efficient RNA-interference (RNAi) response in different cell-lines. RNAi effect is obtained at low siRNA doses with a unique kinetic profile. Furthermore, the solid dispersion technique is utilized to formulate PF14/siRNA nanocomplexes into solid formulations that are as active as the freshly prepared nanocomplexes in solution. Importantly, the nanocomplexes are stable and active in mediating RNAi response after incubation with simulated gastric fluid (SGF) that is highly acidic. These results demonstrate the activity of PF14 in delivering and protecting siRNA in different pharmaceutical forms and biological environments.
With more than ten new FDA approvals since 2001, peptides are emerging as an important therapeutic alternative to small molecules. However, unlike small molecules, peptides on the market today are limited to extracellular targets. By contrast, cell-penetrating peptides (CPPs) can target intracellular proteins and also carry other cargoes (e.g. other peptides, small molecules or proteins) into the cell, thus offering great potential as future therapeutics. In this review I present a classification scheme for CPPs based on their physical-chemical properties and origin, and I provide a general framework for understanding and discovering new CPPs.
Aqueous dispersions of solid lipid nanoparticles (SLN™) are basically stable for up to 3 years, however some systems show particle growth followed by gelation. To assess the destabilizing factors, a poloxamer 188 stabilized Compritol SLN formulation was prepared. Its stability was investigated as a function of storage temperature, light exposure and packing material (untreated and siliconized vials of glass quality I). In general, introduction of energy to the system (temperature, light) led to particle growth and subsequent gelation. This process was accompanied by a decrease in zeta potential from approximately −25 mV to −15 mV. The effect of the packing material was less pronounced. However, siliconization of the vials almost eliminated particle growth. By optimization of the storage conditions (8°C, in the dark, siliconized vials), a stability of the less stable aqueous Compritol SLN over 3 years was achieved.
We recently reported the cloning of a rearranged human oncogene following transfection of DNA from Kaposi's sarcoma into NIH 3T3 cells. To identify the protein(s) encoded in two novel mRNAs of 3.5 and 1.2 kb expressed in NIH 3T3 transformants, we constructed a cDNA library. One of the cDNA clones isolated (KS3) corresponded to the 1.2 kb mRNA and transformed NIH 3T3 cell when inserted into a mammalian expression vector. The 1152 nucleotide KS3 cDNA encodes a protein of 206 amino acids with significant homology to the growth factors basic FGF and acidic FGF. Expression of the KS3 product as a bacterial fusion protein or in COS cells allowed us to determine that both proteins had significant growth-promoting activity and that the COS cell protein was glycosylated. Thus one of the mRNAs transcribed from the KS oncogene encodes a growth factor that could transform cells by an autocrine mechanism and appears to represent a new member of the FGF family.
Nanoparticles generated by complex coacervation of plasmid DNA (pDNA) and modified chitosans namely chitosan-thioglycolic acid (TGA) conjugate and chitosan-HIV-1 Tat peptide conjugate were evaluated as gene delivery systems. In order to optimize transfection efficiency, chitosan-HIV-1 Tat peptide conjugate was combined with chitosan-TGA before its complexation with pDNA. Particle size and zeta potential measurements were performed to characterize the generated nanoparticles. The nanoparticles transfection efficiencies were assessed by exploitation of the green fluorescent protein (GFP) reporter gene. HEK293 cells were incubated for 24 h with the nanoparticles and the GFP positive cells were observed by fluorescence microscopy. The nanoparticles in the size range of 200-300 nm could transfect HEK293 cells as a model cell line with different transfection efficiencies. Unlike chitosan-TGA, chitosan-HIV-1 Tat peptide led to increased zeta potential of nanoparticles as compared to unmodified chitosan. The transfection efficiency of the nanoparticles generated by combination of chitosan-HIV-1 Tat peptide with chitosan-TGA was comparatively higher than that of the nanoparticles generated by either chitosan-TGA or the combination of chitosan-HIV-1 Tat peptide with unmodified chitosan. After 72 h of incubation, the combination of chitosan-HIV-1 Tat peptide with chitosan-TGA was found to be 7.12- and 67.37 times more efficient than unmodified chitosan and pDNA alone, respectively and showed a synergistic effect in transfection of pDNA into the cells. Moreover, none of the nanoparticles showed any severe cytotoxicity. Accordingly, this strategy might result in a potent carrier for gene delivery.
Combinatorial peptide chemistry and orthogonal high-throughput screening were used to select peptides that spontaneously translocate across synthetic lipid bilayer membranes without permeabilization. A conserved sequence motif was identified that contains several cationic residues in conserved positions in an otherwise hydrophobic sequence. This 9-residue motif rapidly translocates across synthetic multibilayer vesicles and into cells while carrying a large polar dye as a "cargo" moiety. The extraordinary ability of this family of peptides to spontaneously translocate across bilayers without an energy source of any kind is distinctly different from the behavior of the well-known, highly cationic cell-penetrating peptides, such as the HIV tat peptide, which do not translocate across synthetic bilayers, and enter cells mostly by active endocytosis. Peptides that translocate spontaneously across membranes have the potential to transform the field of drug design by enabling the delivery of otherwise membrane-impermeant polar drugs into cells and tissues. Here we describe the chemical tools needed to rapidly identify spontaneous membrane translocating peptides.
Advances in cancer research are enabling fast-paced discovery and translation of results into potential clinical tools. Here we consider some of the most influential findings of the past two years, selected by experts in the cancer field.
Uptake and intracellular transport of D-penicillamine coated quantum dots (DPA-QDs) of 4 nm radius by live HeLa cells have been investigated systematically by spinning disk and 4Pi confocal microscopies. Unlike larger nanoparticles, these small DPA-QDs were observed to accumulate at the plasma membrane prior to internalization, and the uptake efficiency scaled nonlinearly with the nanoparticle concentration. Both observations indicate that a critical threshold density has to be exceeded for triggering the internalization process. By using specific inhibitors, we showed that DPA-QDs were predominantly internalized by clathrin-mediated endocytosis and to a smaller extent by macropinocytosis. Clusters of DPA-QDs were found in endosomes, which were actively transported along microtubules toward the perinuclear region. Later on, a significant fraction of endocytosed DPA-QDs were found in lysosomes, while others were actively transported to the cell periphery and exocytosed with a half-life of 21 min.
In this study, a cell-penetrating peptide, the transactivating transcriptional factor (TAT) domain from HIV, was linked to a chitosan/doxorubicin (chitosan/DOX) conjugate to form a chitosan/DOX/TAT hybrid. The synthesized chitosan/DOX/TAT conjugate showed a different intracellular distribution pattern from a conjugate without TAT. Unlike both free DOX and the conjugate without TAT, the chitosan/DOX/TAT conjugate was capable of efficient cell entry. The chitosan/DOX/TAT conjugate was found to be highly cytotoxic, with an IC(50) value of approximately 480 nM, 2 times less than that of chitosan/DOX (980 nM). The chitosan/DOX/TAT provided decreases in tumor volume of 77.4 and 57.5% compared to free DOX and chitosan/DOX, respectively, in tumor-bearing mice. Therefore, this study suggests that TAT-mediated chitosan/DOX conjugate delivery is effective in slowing tumor growth.
Novel classes and applications of cell-penetrating peptides (CPPs) are being constantly discovered since they were first identified 2 decades ago. These short cationic peptides (nanomolecules) either by covalent binding or by noncovalent binding can traverse cell membranes and deliver a variety of molecules that are unable to overcome the permeability barrier in their own capacity. The ability of the CPPs to deliver variety of macromolecules, such as oligonucleotides, therapeutic drugs, proteins, and medical imaging agents, by forming nanoparticulate carriers in a range of cells has led them to emerge as a potential tool for both macromolecule delivery application and to gain insight into the fundamentals of mechanism of cellular uptake across the plasma membrane. This review explores the recent advances, challenges, and future prospects in the field of CPP-mediated cargo delivery in mammalian and plant cells. Studies have been conducted into the peptide chemistry and stability of CPP-macromolecular complexes. Most of the CPPs have been shown to be nontoxic and do not interfere with the functionality of the macromolecules delivered across the cell membrane. The mechanism of uptake of CPP-cargo complexes and the uptake of CPPs alone across the plasma membrane remains unresolved. As the world of CPPs is rapidly advancing in both mammalian and plant system, there is a promising future for the various applications of transduction and transfection into intact cells.
To study the clinical characteristics, treatment and prognosis of patients with glioblastoma multiforme (GBM) in China, we retrospectively analyzed 205 Chinese patients with histologically proven GBM. A univariate analysis of prognosis factors for survival time was performed and significant factors were tested in a multivariate analysis using the Cox regression method. Median overall survival time was 12.0 months (95% confidence interval [CI] 11.0-13.1 months). Survival rates after diagnosis were 82% at 6 months, 52% at 12 months, 27% at 18 months and 17% at 24 months. Age, preoperative Karnofsky's performance status score and tumour location were independent preoperative predictors of prognosis and among the treatment methods of GBM, radiotherapy was the strongest predictor of prognosis followed by radical surgery and chemotherapy. The median survival time post diagnosis for Chinese patients is comparable to the 11.0-15.9 month range observed in western patients. The data suggest a lack of ethnic differences in GBM prognosis of Chinese and western patients.
Applications of water-dispersible near-infrared (NIR)-emitting quantum dots (QDs) have been hampered by their instability and low photoluminescence (PL) efficiencies. In this paper, water-soluble highly luminescent NIR-emitting QDs were developed through constructing CdTe/CdSe/ZnS core/shell/shell nanostructure. The CdTe/CdSe type-II structure yields the QDs with NIR emission. By varying the size of CdTe cores and the thickness of the CdSe shell, the emission wavelength of the obtained nanostructure can span from 540 to 825 nm. In addition, the passivation of the ZnS shell with a substantially wide bandgap confines the excitons within the CdTe/CdSe interface and isolates them from the solution environment and consequently improves the stability of the nanostructure, especially in aqueous media. An effective shell-coating route was developed for the preparation of CdTe/CdSe core/shell nanostructures by selecting capping reagents with a strong coordinating capacity and adopting a low temperature for shell deposition. An additional ZnS shell was deposited around the outer layer of CdTe/CdSe QDs to form the core/shell/shell nanostructure through the decomposition of single molecular precursor zinc diethyldithiocarbamate in the crude CdTe/CdSe reaction solution. The water solubilization of the initially oil-soluble CdTe/CdSe/ZnS QDs was achieved through ligand replacement by 3-mercaptopropionic acid. The as-prepared water-soluble CdTe/CdSe/ZnS QDs possess PL quantum yields as high as 84% in aqueous media, which is one of the best results for the luminescent semiconductor nanocrystals.
The selective permeability of the plasma membrane prohibits most exogenous agents from gaining cellular access. Since many therapeutics and reporter molecules must be internalized for activity, crossing the plasma membrane is essential. A very effective class of transporters harnessed for this purpose are cell penetrating peptides (CPPs), a group of short cationic sequences with a remarkable capacity for membrane translocation. Since their discovery in 1988, CPPs have been employed for the delivery of a wide variety of cargo including small molecules, nucleic acids, antibodies and nanoparticles. This review describes recent advances in the use of CPPs for biological and therapeutic applications. In particular, an emphasis is placed on novel systems and insights acquired since 2006. Basic research on CPPs has recently yielded techniques that provide further information on the controversial mechanism of CPP uptake and has also resulted in the development of new model membrane systems to evaluate these mechanisms. In addition, recent use of CPPs for the development of new cellular imaging tools, biosensors, or biomolecular delivery systems have been highlighted. Lastly, novel peptide delivery vectors, designed to tackle some of the drawbacks of CPPs and enhance their versatility, will be described. This review will illustrate the diverse applications for which CPPs have been harnessed and also demonstrate the remarkable advancements these peptides have facilitated in cell biology.
Cell-penetrating peptides (CPPs) are short peptides able to penetrate cell membranes and translocate different cargoes into cells. Although recently the topic of many research articles, to our best knowledge no single systematic study of CPPs has been carried out as yet, meaning information can only by gathered piece by piece from different sources. We therefore decided to start analytical screening of CPP specificity in cell lines. We used 22 different CPPs, which have all been published before, and present the first analytical screen in 4 selected cell lines (MDCK, HEK293, HeLa, and Cos-7). Furthermore, we examined the influence of different conditions, such as protease inhibitors, incubation conditions, endocytosis inhibitors, temperature, and cytotoxicity. We clearly demonstrate that the 22 CPPs can be classified into 3 groups based on their internalization properties, even after trypsinization. Moreover, we show that additional agents, which should increase cellular uptake or dissolve endosomal/lysosomal entrapped CPPs, only have low effects. Our intensive screening under standardized conditions provides the opportunity to compare cellular uptake of CPPs, an important step for the use of CPPs as peptidic vectors in the medical field.
Conjugation of the cell-penetrating peptide derived from the human immunodeficiency virus-1 transactivator protein (TAT) to semiconductor quantum dots (QDs) is an effective way to enhance transmembrane delivery of QDs for intracellular and molecular imaging. In this work, the internalization pathway of TAT-QDs was studied systematically in living cells. Cellular uptake of TAT-QDs, under different endocytosis-inhibiting conditions, was compared by fluorescence imaging and flow cytometry. The results suggest TAT-QDs internalize through lipid-raft-dependent macropinocytosis, which is different from that of FITC-labeled TAT. They also provide new information for better understanding of the TAT-mediated cell uptake mechanism.
Epidemiologists in the Brain Tumor Epidemiology Consortium (BTEC) have prioritized areas for further research. Although many risk factors have been examined over the past several decades, there are few consistent findings, possibly because of small sample sizes in individual studies and differences between studies in patients, tumor types, and methods of classification. Individual studies generally have lacked samples of sufficient size to examine interactions. A major priority based on available evidence and technologies includes expanding research in genetics and molecular epidemiology of brain tumors. BTEC has taken an active role in promoting understudied groups, such as pediatric brain tumors; the etiology of rare glioma subtypes, such as oligodendroglioma; and meningioma, which, although it is not uncommon, has only recently been registered systematically in the United States. There also is a pressing need for more researchers, especially junior investigators, to study brain tumor epidemiology. However, relatively poor funding for brain tumor research has made it difficult to encourage careers in this area. In this report, BTEC epidemiologists reviewed the group's consensus on the current state of scientific findings, and they present a consensus on research priorities to identify which important areas the science should move to address.
While developing an assay to measure the activity of the tat protein from human immunodeficiency virus 1 (HIV-1), we discovered that the purified protein could be taken up by cells growing in tissue culture and subsequently trans-activate the viral promoter. Trans-activation is dramatically increased by a variety of lysosomotrophic agents. For example, trans-activation can be detected at tat concentrations as low as 1 nM in the presence of chloroquine. Experiments using radioactive protein show that tat becomes localized to the nucleus after uptake and suggest that chloroquine protects tat from proteolytic degradation. These results raise the possibility that, under some conditions, tat might act as a viral growth factor to stimulate viral replication in latently infected cells or alter expression of cellular genes.
HIV-1 encodes a potent trans-activator protein, tat, which is essential for viral gene expression. To study tat domains that function in trans-activation, we chemically synthesized the 86 amino acid tat protein (tat-86) and tat mutant peptides. Remarkably, tat-86 is rapidly taken up by cells, and produces a massive and specific stimulation of HIV-LTR-driven RNA synthesis. Mutant peptides of 21 to 41 amino acids exhibit significant activity. Only two regions are essential for trans-activation; we suggest that one represents an activation region and the other, a nucleic acid binding or nuclear targeting region. Amino acid substitutions within these regions greatly reduce trans-activation, demonstrating the functional significance of these domains. The N-terminal 37 amino acids and exon 2 are not essential. Thus, tat is similar to regulatory proteins of Ad E1A and BPV1 E5 oncogenes, requiring only small domains for autonomous function.
We show that the HSV-1 structural protein VP22 has the remarkable property of intercellular transport, which is so efficient that following expression in a subpopulation the protein spreads to every cell in a monolayer, where it concentrates in the nucleus and binds chromatin. VP22 movement was observed both after delivery of DNA by transfection or microinjection and during virus infection. Moreover, we demonstrate that VP22 trafficking occurs via a nonclassical Golgi-independent mechanism. Sensitivity to cytochalasin D treatment suggests that VP22 utilizes a novel trafficking pathway that involves the actin cytoskeleton. In addition, we demonstrate intercellular transport of a VP22 fusion protein after endogenous synthesis or exogenous application, indicating that VP22 may have potential in the field of protein delivery.
The intercellular transport property of VP22 chimeric proteins offers the opportunity for the improvement of gene therapy delivery systems. Since enhanced therapeutic effects of transduced genes already have been exemplified for chimeric proteins VP22-p53 and VP22-tk, we were interested in examining whether spread of VP22 chimeric proteins is a general biological phenomenon not restricted to distinct tissues or species.
To study intercellular spread of VP22-GFP fusion proteins, 15 different mammalian cell lines were transfected with 200-2000 ng of VP22-GFP or GFP expression plasmids. Expression of VP22-GFP or GFP was monitored by fluorescence microscopy of live GFP fluorescence and direct FACS analysis. For selected cell lines, antibody detection of VP22-GFP spread was analysed by confocal microscopy as a control.
Spread of VP22-GFP fusion proteins was detected in all 15 cell lines tested, and quantified by FACS analysis. Experimental conditions were found to be critical in the investigation of VP22-mediated intercellular spread.
Results of our study indicate that spread of VP22 chimeric proteins is a general biological phenomenon not restricted to distinct tissues or species. Therefore, further evidence is provided that VP22-enhanced gene therapeutic effects may be obtained irrespective of the target organ/tissue to be addressed.
The carcinogenic process involves a complex series of genetic and biochemical changes that enables transformed cells to proliferate, migrate to secondary sites and, in some cases, acquire mechanisms that make cancer cells resistant to chemotherapy. This phenomenon in its most common form is known as multidrug resistance (MDR). It is usually mediated by overexpression of P-glycoprotein (P-gp) or other plasma membrane ATPases that export cytotoxic drugs used in chemotherapy, thereby reducing their efficacy. However, additional adaptive changes are likely to be required in order to confer a full MDR phenotype. Recent studies have shown that acquisition of MDR is accompanied by upregulation of lipids and proteins that constitute lipid rafts and caveolar membranes, notably glucosylceramide and caveolin. These changes may be related to the fact that in MDR cells a significant fraction of cellular P-gp is associated with caveolin-rich membrane domains, they may be involved in drug transport and they could have an impact on drug-induced apoptosis and on the phenotypic transformation of MDR cancer cells.
The Ku heterodimer (Ku70 and Ku80 subunits) contributes to genomic integrity through its ability to bind DNA double-strand breaks and facilitate repair by the non-homologous end-joining pathway. The crystal structure of the human Ku heterodimer was determined both alone and bound to a 55-nucleotide DNA element at 2.7 and 2.5 A resolution, respectively. Ku70 and Ku80 share a common topology and form a dyad-symmetrical molecule with a preformed ring that encircles duplex DNA. The binding site can cradle two full turns of DNA while encircling only the central 3-4 base pairs (bp). Ku makes no contacts with DNA bases and few with the sugar-phosphate backbone, but it fits sterically to major and minor groove contours so as to position the DNA helix in a defined path through the protein ring. These features seem well designed to structurally support broken DNA ends and to bring the DNA helix into phase across the junction during end processing and ligation.