pH-dependent reversibly activatable cell-penetrating peptides improve the antitumor effect of artemisinin-loaded liposomes.

Artemisinin (ART) is well known as an antimalarial drug, and it can also be used to treat inflammation as well as cancer. Although many researchers have reported the antitumor activity of ART, most of these studies were investigated in vitro. In addition, ART is sparingly soluble in water, limiting its clinical relevance in drug development. Based on the data from our preliminary study, ART is not cytotoxic at low micromolar concentrations. Thus, we hypothesized that smart nanocarriers are beneficial for not only increasing the solubility of ART but also elevating the concentration of the drug at the target, thereby inducing the ideal antitumor effect. In this article, a reversibly activatable cell-penetrating peptide ((HE)10-G5-R6 or HE-R6) was introduced to modify artemisinin (ART)-loaded liposomes (ART-Lip-HE-R6) against tumors, and in vitro and in vivo performance were investigated. ART-Lip-HE-R6 exhibited sustained release under different pH conditions. The internalization and cytotoxicity of liposomes were enhanced at low pH, i.e., 6.5, after modification with HE-R6 versus nonmodified liposomes. Moreover, a longer retention time in tumors could be observed in the ART-Lip-HE-R6 group, followed by higher efficiency of tumor suppression. In conclusion, Lip-HE-R6 might be a promising delivery system for ART in cancer therapy.

Enhanced insulin receptor interaction by a bifunctional insulin-transferrin fusion protein: an approach to overcome insulin resistance.

Bifunctional fusion protein design has been widely utilized as a strategy to increase the efficacy of protein therapeutics. Previously, we proposed a novel application of the bifunctional fusion protein design through the introduction of proinsulin-transferrin (ProINS-Tf) fusion protein as a liver-specific protein prodrug to achieve a glucose-lowering effect in type 1 diabetic mice. In this report, we studied the binding characteristics of this activated fusion protein to the insulin receptor to elucidate its mechanism in eliciting insulin receptor-mediated signaling. We found that, with the assistance of the transferrin moiety binding to the transferrin receptor, the activated ProINS-Tf exhibited significantly higher binding affinity to the insulin receptor compared with the native insulin, resulting in a prolonged and stronger Akt phosphorylation. This enhanced induction by activated ProINS-Tf overcame insulin resistance in palmitate-treated HepG2 cells. ProINS-Tf also demonstrated a better glucose-lowering effect than native insulin, even with a much lower dose and less frequent injections, in non-obese diabetic mice with insulin resistance symptoms. The activated ProINS-Tf, serving as a bivalent protein molecule, could be a new insulin analog to overcome insulin resistance, which is associated with several diseases, including type 2 diabetes and non-alcoholic fatty liver disease.

Acid-sensitive hybrid polymeric micelles containing a reversibly activatable cell-penetrating peptide for tumor-specific cytoplasm targeting.

Cell-penetrating peptides (CPPs) have become a novel drug delivery system due to their distinct advantages, including high cell transmembrane potency and ability to carry cargo molecules inside cells. However, owing to their cationic charge and non-specificity characteristics, the clinical application of CPPs is limited. In the current study, we engineered a reversibly activatable cell-penetrating peptide (RACPP), containing oligoarginine fused to a pH-sensitive masking sequence via a polyglycine linker ((HE)10G5R6 or HE-CPP) with ultra-pH-sensitivity. The HE-CPP sequence was coupled to the surface of polyethyleneglycol-polylactic acid (PEG-PLA) polymer micelles (PMs-HE-CPP) to realize improve specificity and targeted delivery of encapsulated paclitaxel (PTX). PTX/PMs-HE-CPP showed the satisfactory encapsulated efficiency, loading capacity, size distribution as well as reversible charge-conversion in response to the surrounding pH. The zeta potential of PMs-HE-CPP was negative at pH 7.5, moderately positive at pH 6.5, and even more positive at a lower pH. Coumarin 6-loaded PMs-HE-CPP (C6/PMs-HE-CPP) showed enhanced tumor cellular uptake at a mildly acidic tumor microenvironment (pH 6.5) via energy-dependent and clathrin-mediated endocytosis. Furthermore, PTX/PMs-HE-CPP had significantly higher cytotoxicity toward mice breast cancer (4T1) cells at pH 6.5 versus at pH 7.4. In vivo imaging studies in 4T1-BALB/c tumor xenograft models confirmed the tumor-targeting characteristic of PMs-HE-CPP. PTX/PMs-HE-CPP also exhibited improved anti-tumor efficacy against unmodified polymer micelles and Taxol® in this tumor model. Accordingly, not only do RACPPs show the great potential to endow CPPs with specificity and reversible net-charge converting characteristic, they are also able to improve the targeting effect of nanoparticles.

Characterization and Oral Delivery of Proinsulin-Transferrin Fusion Protein Expressed Using ExpressTec.

Proinsulin-transferrin fusion protein (ProINS-Tf) has been designed and successfully expressed from the mammalian HEK293 cells (HEK-ProINS-Tf). It was found that HEK-ProINS-Tf could be converted into an activated form in the liver. Furthermore, HEK-ProINS-Tf was demonstrated as an extra-long acting insulin analogue with liver-specific insulin action in streptozotocin (STZ)-induced type 1 diabetic mice. However, due to the low production yield from transfected HEK293 cells, there are other interesting features, including the oral bioavailability, which have not been fully explored and characterized. To improve the protein production yield, an alternative protein expression system, ExpressTec using transgenic rice (Oryza sativa L.), was used. The intact and active rice-derived ProINS-Tf (ExpressTec-ProINS-Tf) was successfully expressed from the transgenic rice expression system. Our results suggested that, although the insulin-like bioactivity of ExpressTec-ProINS-Tf was slightly lower in vitro, its potency of in vivo blood glucose control was considerably stronger than that of HEK-ProINS-Tf. The oral delivery studies in type 1 diabetic mice demonstrated a prolonged control of blood glucose to near-normal levels after oral administration of ExpressTec-ProINS-Tf. Results in this report suggest that ExpressTec-ProINS-Tf is a promising insulin analog with advantages including low cost, prolonged and liver targeting effects, and most importantly, oral bioactivity.

Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery.

Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc)2), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc)2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc)2-mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc)2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics.

Tissue barriers and novel approaches to achieve hepatoselectivity of subcutaneously-injected insulin therapeutics.

Current subcutaneously (s.c.)-injected insulin (INS) products result in a hyperinsulin exposure to peripheral tissues (skeletal muscle and adipose) while INS hardly accesses to liver after injection. This unphysiological distribution raises risks of hypoglycemia episode and causes weight gain after long term treatment. An ideal INS replacement therapy requires the distribution or action of exogenous INS to more closely mimic physiological INS in terms of its preferential hepatic action. However, there are 2 factors that limit the ability of s.c. injected INS to restore the liver: peripheral gradient in INS deficient diabetes patients: (1) the transport of INS in capillary endothelium and peripheral tissues from the injection site; and (2) peripheral INS receptor (IR) mediated INS degradation. In this review, the tissue barriers against efficient liver targeting of s.c. injected INS are discussed and current advances in developing hepatoselective insulin therapeutics are introduced.

Proinsulin-Transferrin Fusion Protein Exhibits a Prolonged and Selective Effect on the Control of Hepatic Glucose Production in an Experimental Model of Type 1 Diabetes.

An ideal basal insulin (INS) replacement therapy requires the distribution or action of exogenous INS to more closely mimic physiological INS in terms of its preferential hepatic action. In this paper, we introduce a novel strategy to exert liver-specific INS action by hepatic activation of INS's precursor, proinsulin (ProINS). We demonstrated the conversion of human ProINS-transferrin (Tf) fusion protein, ProINS-Tf, into an active and immuno-reactive form of INS-Tf in the liver via the slow Tf receptor mediated recycling pathway. ProINS-Tf displayed prolonged basal blood glucose lowering effects for up to 40 h in streptozotocin-induced type 1 diabetic mice following a single subcutaneous injection. The effect of ProINS-Tf on blood glucose levels was observed predominantly under fasting conditions, with little effect under free-feeding conditions. In addition, both the pyruvate tolerance assay in normal mice and the Akt-phosphorylation assay in H-4-II-E hepatoma cells indicated that the hepatic-activated ProINS-Tf possessed a much longer effect on the control of hepatic glucose production than INS. These results indicated that ProINS-Tf may serve as an effective and safe hepatoselective INS analog to reduce the frequency of INS injections as well as avert severe hypoglycemia episodes and other side effects frequently encountered with long-acting INS therapeutics due to their peripheral action.

Selective Intracellular Delivery of Recombinant Arginine Deiminase (ADI) Using pH-Sensitive Cell Penetrating Peptides To Overcome ADI Resistance in Hypoxic Breast Cancer Cells.

Arginine depletion strategies, such as pegylated recombinant arginine deiminase (ADI-PEG20), offer a promising anticancer treatment. Many tumor cells have suppressed expression of a key enzyme, argininosuccinate synthetase 1 (ASS1), which converts citrulline to arginine. These tumor cells become arginine auxotrophic, as they can no longer synthesize endogenous arginine intracellularly from citrulline, and are therefore sensitive to arginine depletion therapy. However, since ADI-PEG20 only depletes extracellular arginine due to low internalization, ASS1-expressing cells are not susceptible to treatment since they can synthesize arginine intracellularly. Recent studies have found that several factors influence ASS1 expression. In this study, we evaluated the effect of hypoxia, frequently encountered in many solid tumors, on ASS1 expression and its relationship to ADI-resistance in human MDA-MB-231 breast cancer cells. It was found that MDA-MB-231 cells developed ADI resistance in hypoxic conditions with increased ASS1 expression. To restore ADI sensitivity as well as achieve tumor-selective delivery under hypoxia, we constructed a pH-sensitive cell penetrating peptide (CPP)-based delivery system to carry ADI inside cells to deplete both intra- and extracellular arginine. The delivery system was designed to activate the CPP-mediated internalization only at the mildly acidic pH (6.5-7) associated with the microenvironment of hypoxic tumors, thus achieving better selectivity toward tumor cells. The pH sensitivity of the CPP HBHAc was controlled by recombinant fusion to a histidine-glutamine (HE) oligopeptide, generating HBHAc-HE-ADI. The tumor distribution of HBHAc-HE-ADI was comparable to ADI-PEG20 in a mouse xenograft model of human breast cancer cells in vivo. In addition, HBHAc-HE-ADI showed increased in vitro cellular uptake in cells incubated in a mildly acidic pH (hypoxic conditions) compared to normal pH (normoxic conditions), which correlated with pH-sensitive in vitro cytotoxicity in hypoxic MDA-MB-231 and human prostate cancer PC3 cells. Together, we conclude that the HBHAc-HE-based peptide delivery offers a useful means to overcome hypoxia-induced resistance to ADI in breast cancer cells, and to target the mildly acidic tumor microenvironment.

Lipid-based drug carriers for prodrugs to enhance drug delivery.

The combination of lipid drug delivery systems with prodrugs offers several advantages including improved pharmacokinetics, increased absorption, and facilitated targeting. Lipidization and use of lipid carriers can increase the pharmacological half-life of the drug, thus improving pharmacokinetics and allowing less frequent dosing. Lipids also offer advantages such as increased absorption through the intestines for oral drug absorption and to the CNS for brain delivery. Furthermore, the use of lipid delivery systems can enhance drug targeting. Endogenous proteins bind lipids in the blood and carry them to the liver to enable targeting of this organ. Drugs with significant side effects in the stomach can be specifically delivered to enterocytes by exploiting lipases for prodrug activation. Finally, lipids can be used to target the lymphatic system, thus bypassing the liver and avoiding first-pass metabolism. Lymphatic targeting is also important for antiviral drugs in the protection of B and T lymphocytes. In this review, both lipid-drug conjugates and lipid-based carriers will be discussed. An overview, including the chemistry and assembly of the systems, as well as examples from the clinic and in development, will be provided.

Interaction between cell-penetrating peptides and acid-sensitive anionic oligopeptides as a model for the design of targeted drug carriers.

Overcoming the nonspecific cellular uptake of cell-penetrating peptides (CPPs) is a major hurdle in their clinical application. Using pH as the activation switch, histidine-glutamic acid (HE) dipeptide repeats were fused to CPPs to trigger the membrane-penetrating activity at mildly acidic pH environments (i.e., pH 6.5 or below) while masking the internalization at neutral pH (i.e., pH 7.0 or above). In this study, a series of recombinant GST-fusion proteins containing an HE oligopeptide sequence (i.e., (HE)n with n = 8, 10, or 12) and a cationic CPP (i.e., YG(RG)6, YGR6G6, or Tat) were engineered for a pH-sensitive study comparing their cellular uptake and surface binding in cultured HeLa cells. Circular dichroism (CD) spectroscopy was performed to correlate differences between CPPs in secondary structure with the pH sensitivity. YGR6G6 with clustered arginine residues exhibited greater pH sensitivity in cellular uptake than YG(RG)6 with separated arginine residues. Increasing the stretch of HE repeats decreased cellular uptake and surface binding for both YG(RG)6 and YGR6G6. The ratio of cellular internalization at pH 7.5 vs 6.0 was not changed by the presence of serum. CD spectral data revealed that both (HE)10-Tat and (HE)10-YGR6G6 exhibited an unordered secondary structure, whereas (HE)10-YG(RG)6 adopted an antiparallel ß-sheet conformation. This ß-sheet conformation presumably stabilized the association of (HE)10 with YG(RG)6, leading to weakened pH sensitivity of (HE)10-YG(RG)6. On the other hand, the random-coiled structures, that is, (HE)10-YGR6G6 and (HE)10-Tat, both showed higher pH sensitivity as determined in cell experiments. The data presented in this study provide a basis for the future design of pH-sensitive HE-CPP carrier for targeted drug delivery.

Tumor targeting of a cell penetrating peptide by fusing with a pH-sensitive histidine-glutamate co-oligopeptide.

Cell penetrating peptides (CPPs) have been well established as potential carriers for intracellular delivery of protein/peptide therapeutics. However, their lack of selectivity impedes their application in vivo. In order to increase their specificity, a highly pH-sensitive histidine-glutamate (HE) co-oligopeptide was fused with a CPP, i.e. model amphipathic peptide (MAP), and was expressed as a fusion protein with glutathione S-transferase (GST) acting as a cargo protein. Compared with two other fusion proteins containing either HE or MAP, only the fused peptide (HE-MAP) could effectively deliver the cargo GST protein to cells at pH 6.5 or below, while maintaining low delivery to cells at pH 7.0 and above. Using a xenograft mouse model of human breast cancer, fluorescent imaging showed that only HE-MAP could effectively target GST to the tumor site, while reducing non-specific association of MAP in other organs. The data presented in this report demonstrate the diagnostic and/or therapeutic potential of the fused peptide, HE-MAP, for targeting the acidic tumor microenvironment. The concise design for this pH-sensitive peptide offers a simple way to overcome CPP's lack of selectivity, which could lead to increased application of CPPs and macromolecular therapeutics.

Proinsulin-transferrin fusion protein as a novel long-acting insulin analog for the inhibition of hepatic glucose production.

Proinsulin-transferrin (ProINS-Tf) fusion protein was evaluated for its in vivo pharmacokinetics, efficacy, and mechanism. Our previous studies have shown that ProINS-Tf was converted to active insulin-transferrin (INS-Tf) via the transferrin (Tf)-receptor-mediated pathway in hepatoma cells. We hypothesized that this fusion protein can be administered as a prodrug and be converted to a biologically active protein with specificity for the liver versus other insulin (INS)-sensitive tissues (muscle and adipose). Administration as an inactive prodrug with liver-specific action compared with other INS-sensitive tissues conceivably reduces negative side effects seen with other INS analogs. In this report, the data show that ProINS-Tf exhibited a slow, but sustained, in vivo hypoglycemic efficacy and long plasma half-life. The fusion protein showed activity in the liver, as evidenced by decreased expression of two key hepatic glucose production (HGP) enzymes, PEPCK and glucose-6-phosphatase, and increased glycogen levels under feeding conditions. Furthermore, the INS receptor (IR) phosphorylation (activation) in liver and muscle tissues was compared with postinjection of INS or ProINS-Tf. While INS activated IR in both the liver and muscle, ProINS-Tf only showed activation in the liver. Thus, ProINS-Tf fusion protein can potentially be administered as a prodrug with sustained Tf-mediated activation and selectivity in inhibiting HGP.

Fusion protein linkers: property, design and functionality.

As an indispensable component of recombinant fusion proteins, linkers have shown increasing importance in the construction of stable, bioactive fusion proteins. This review covers the current knowledge of fusion protein linkers and summarizes examples for their design and application. The general properties of linkers derived from naturally-occurring multi-domain proteins can be considered as the foundation in linker design. Empirical linkers designed by researchers are generally classified into 3 categories according to their structures: flexible linkers, rigid linkers, and in vivo cleavable linkers. Besides the basic role in linking the functional domains together (as in flexible and rigid linkers) or releasing the free functional domain in vivo (as in in vivo cleavable linkers), linkers may offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles.

Fatty acids as therapeutic auxiliaries for oral and parenteral formulations.

Many drugs have decreased therapeutic activity due to issues with absorption, distribution, metabolism and excretion. The co-formulation or covalent attachment of drugs with fatty acids has demonstrated some capacity to overcome these issues by improving intestinal permeability, slowing clearance and binding serum proteins for selective tissue uptake and metabolism. For orally administered drugs, albeit at low level of availability, the presence of fatty acids and triglycerides in the intestinal lumen may promote intestinal uptake of small hydrophilic molecules. Small lipophilic drugs or acylated hydrophilic drugs also show increased lymphatic uptake and enhanced passive diffusional uptake. Fatty acid conjugation of small and large proteins or peptides has exhibited protracted plasma half-lives, site-specific delivery and sustained release upon parenteral administration. These improvements are most likely due to associations with lipid-binding serum proteins, namely albumin, LDL and HDL. These molecular interactions, although not fully characterized, could provide the ability of using the endogenous carrier systems for improving therapeutic outcomes.

Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.).

BACKGROUND: Transferrin (TF) plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR)-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF) is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF), and evaluate its suitability for biopharmaceutical applications. RESULT: Through a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2) and Caco-2 human colon carcinoma cells (HTB-37), we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240) and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72), for supporting their proliferation, differentiation, and physiological function of antibody production. CONCLUSION: The functional similarity between rice derived rhTF and native hTF in their cellular iron delivery, TFR binding, and TFR-mediated endocytosis and intracellular processing support that rice-derived rhTF can be used as a safe and animal-free alternative to serum hTF for bioprocessing and biopharmaceutical applications.

Vesicle-to-cytosol transport of disulfide-linked cargo mediated by an amphipathic cell-penetrating peptide.

The effect of linker stability on the intracellular localization and apoptotic activity of cytochrome c (Cyt c) conjugated to an amphipathic cell-penetrating peptide (CPP), model amphipathic peptide (MAP), was tested in HeLa cells. While conjugates linked with a stable thioether cross-linkage were only found in the vesicular compartment, a portion of the conjugate linked with a reducible disulfide bond was also detected in the cytosol. The apoptotic function of the reducible and non-reducible Cyt c-MAP conjugates was also evaluated quantitatively using the caspase 3 and annexin V/propidium iodide detection assays in the presence of a proteasome inhibitor used to inhibit cytosolic Cyt c degradation. Analysis for early phase apoptosis revealed that linker stability was important for biological activity. Only the reducible disulfide-linked Cyt c-MAP conjugate, and not free Cyt c or thioether-linked Cyt c-MAP, initiated apoptosis in proteasome-inhibited cells which correlated with the cytosolic localization profiles of the proteins. The co-treatment of disulfide-linked Cyt c-MAP with a disulfide reduction inhibitor decreased the amount of Cyt c delivered to the cytosol, which correlated with a lack of apoptotic activity. These findings indicated the presence of a vesicle-to-cytosolic delivery process for disulfide-linked MAP conjugates, which can be used to improve CPP-based drug delivery systems transporting cargo to cytosolic sites.

Pharmacokinetics of recombinant bifunctional fusion proteins.

INTRODUCTION: The development of biotechnology has enabled the creation of various recombinant fusion proteins as a new class of biotherapeutics. The uniqueness of fusion proteins lies in their ability to fuse two or more protein domains, providing vast opportunities to generate novel combinations of functions. Pharmacokinetic (PK) studies, which are critical components in preclinical and clinical drug development, have not been fully explored for fusion proteins. The lack of general PK models and study guidelines has become a bottleneck for translation of fusion proteins from basic research to the clinic. AREAS COVERED: This article reviews the current status of PK studies for fusion proteins, covering the processes that affect PK. According to their PK properties, a classification of fusion proteins is suggested along with examples from the clinic or under development. Current limitations and future perspectives for PK of fusion proteins are also discussed. EXPERT OPINION: A PK model for bifunctional fusion proteins is presented to highlight the importance of mechanistic studies for a thorough understanding of the PK properties of fusion proteins. The model suggests investigating the receptor binding and subsequent intracellular disposition of individual domains, which can have dramatic impact on the PK of fusion proteins.

Recombinant peptide constructs for targeted cell penetrating peptide-mediated delivery.

A recently designed nanoconstruct was engineered using recombinant technology to contain a cell-penetrating peptide (CPP), i.e. Model Amphipathic Peptide (MAP), attached to a pH-sensitive masking peptide sequence. CPPs such as MAP exhibit unique internalization properties which enable them to deliver attached bioactive molecules, including proteins and peptides, into the cytosolic or nuclear compartment of cells. However, their application in drug delivery is limited due to lack of specificity, as they are widely distributed in most tissues following in vivo administration. In order to overcome this hurdle, a highly pH-sensitive histidine-glutamic acid (HE) copolymer sequence was linked to MAP to prevent non-specific internalization of the construct in non-target cells. Our results show that this nanoconstruct is highly pH-sensitive in a mildly acidic pH, exhibiting high binding and internalization at pH 6.8 and below, but low binding and internalization at pH>7. This unique sensitivity in the mildly acidic pH range can be applied to targeting the activation of membrane permeable properties in mildly acidic pH environments, such as the surface of tumor cells or in the early endosomes of target cells. Therefore, the design could lead to a significant advancement in the application of CPPs and in the therapeutic potential of the biotechnology field.

MAP-mediated nuclear delivery of a cargo protein.

Radiolabeled cytochrome c (Cyt c), either as a free protein or as cell penetrating peptide (CPP)-conjugates, was tested for cellular uptake and nuclear transport in Human embryonic kidney 293 (HEK293) cells and HeLa cells. Conjugation of Cyt c with either the amphipathic peptide model amphipathic peptide (MAP) or the cationic peptide oligoarginine via a disulfide linkage significantly increased the total internalization and nuclear localization of Cyt c in both cell lines, though to a greater extent following conjugation with MAP. The nuclear localization was also evaluated qualitatively by confocal laser scanning microscopy (CLSM). CLSM images depicted high amounts of colocalization of fluorescently labeled Cyt c-MAP and the nucleus in HEK293 cells. In addition, prevention of disulfide reduction at the cell surface, or comparison of reducible disulfide or non-reducible thioether MAP-conjugates, showed that maintenance of an intact conjugate using a stable linkage enhanced MAP-mediated nuclear delivery. Furthermore, nuclear transport of the MAP-cargo conjugate appears to be dependent on vesicle fusion events following internalization via endocytosis. The findings presented in this report demonstrate the MAP-mediated transport of a small protein such as Cyt c into the nuclear compartment, which can be used to improve current CPP-cargo delivery of macromolecules with nuclear biological functions.

Comparison of cationic and amphipathic cell penetrating peptides for siRNA delivery and efficacy.

Cell penetrating peptides (CPPs) are short strands of arginine- and/or lysine-rich peptides (<30 amino acids) that use their cationic nature for efficient intracellular accumulation. CPPs have been used for small interfering RNA (siRNA) delivery by direct complexation with the siRNA anionic phosphate backbone. During this process, however, part of the CPP cationic charges are neutralized, and the resultant loss of free positive charges may substantially compromise CPP's internalization capabilities and eventually reduce siRNA delivery efficiency. The purpose of this study was to design a novel type of polyplex for siRNA delivery to overcome the CPP neutralization issue. This novel polyplex consists of three components: siRNA, 21mer oligolysine (K21) chemically modified to incorporate CPP conjugation sites (K21-PDP), and CPP delivery moiety. The siRNA was first neutralized by cationic charges of K21-PDP to form a polyplex. Then a cationic (hexaarginine, R6) or an amphipathic (model amphipathic peptide, MAP) CPP was conjugated to the polyplex. Agarose gel shift assays indicated that the siRNA could be released from the polyplex after K21-PDP degradation or polyplex dilution. Furthermore, the total intracellular internalization of these two CPP-polyplexes was studied. Compared with R6-polyplex, MAP-polyplex exhibited 170- and 600-fold greater uptake of fluorescently labeled siRNA at 1 and 6 h post-transfection, respectively. MAP-polyplex also exhibited comparable GFP silencing effects as Lipofectamine 2000 complex in Huh7.5 cells stably transfected to express GFP-light chain 3 protein, whereas R6-polyplex did not demonstrate significant silencing activity. Further studies indicated that the K21-PDP-siRNA polyplex formation and conjugation of MAP to the polyplex were essential for siRNA polyplex uptake and gene silencing. MAP-polyplex was also shown to be unaffected by the presence of 10% FBS during transfection. In addition, MAP-polyplex uptake was dependent on vesicle formation and fusion due to 70 and 54% loss of uptake at 4 and 16 °C, respectively, compared to incubation at 37 °C. Therefore, the amphipathic CPP is a more suitable carrier moiety for delivery of siRNA polyplex.