Cheol Moon

Nontoxic membrane translocation peptide from protamine, low molecular weight protamine (LMWP), for enhanced intracellular protein delivery: in vitro and in vivo study

Naturally derived, nontoxic peptides from protamine by the authors, termed low molecular weight protamines (LMWPs), possess high arginine content and carry significant sequence similarity to that of TAT, by far the most potent protein transduction domain peptide. Therefore, it was hypothesized that these LMWPs would also inherit the similar translocation activity across the cell membrane, which enables any impermeable species to be transduced into the cells. LMWPs were prepared by enzymatic digestion of protamine, examined their capability of transducing an impermeable protein toxin into the tumor cells by chemical conjugation, and determined cytotoxicity of transduced protein toxin (e.g., gelonin) against cancer cell lines and a tumor‐bearing mouse. In vitro results showed that LMWPs could indeed translocate themselves into several mammalian cell lines as efficiently as TAT, thereby transducing impermeable gelonin into the cells by chemical conjugation. In vivo studies further confirmed that LMWP could carry an impermeable gelonin across the tumor mass and subsequently inhibit the tumor growth. In conclusion, the presence of equivalent cell translocation potency, absence of toxicity of peptide itself, and the suitability for low‐cost production by simple enzymatic digestion could expand the range of clinical applications of LMWPs, including medical imaging and gene/protein therapies.

L-Asparaginase encapsulated intact erythrocytes for treatment of acute lymphoblastic leukemia (ALL)

As a primary drug for the treatment of acute lymphoblastic leukemia (ALL), encapsulation of L-asparaginase (ASNase) into red blood cells (RBC) has been popular to circumvent immunogenicity from the exogenous protein. Unlike existing methods that perturbs RBC membranes, we introduce a novel method of RBC-incorporation of proteins using the membrane-translocating low molecular weight protamine (LMWP). Confocal study of fluorescence-labeled LMWP-ovalbumin, as a model protein conjugate, has shown significant fluorescence inside RBCs. Surface morphology by scanning electron microscopy of the RBCs loaded with LMWP-ASNase was indistinguishable with normal RBCs. These drug loaded RBCs also closely resembled the profile of the native erythrocytes in terms of osmotic fragility, oxygen dissociation and hematological parameters. The in vivo half-life of enzyme activity after administering 8 units of RBC/LMWP-ASNase in DBA/2 mice was prolonged to 4.5+/-0.5 days whereas that of RBCs loaded with ASNase via a hypotonic method was 2.4+/-0.7 days. Furthermore, the mean survival time of DBA/2 mice bearing mouse lymphoma cell L5178Y was improved by approximately 44% compared to the saline control group after treatment with the RBC loaded enzymes. From these data, an innovative, novel method for encapsulating proteins into intact and fully functional erythrocytes was established for potential treatment of ALL.

Synthetic Skin-Permeable Proteins Enabling Needleless Immunization†

Protein drugs, due to their large size and hydrophilic nature, are normally precluded from effective delivery such as cell entry or tissue diffusion. Among the transport barriers, the skin poses as a formidable challenge to proteins due to the impermeable stratum corneum. The existing techniques for percutaneous protein delivery must rely on sophisticated delivery systems, such as the use of complicated nanocarriers or mechanical devices, to overcome the skin barrier for noninvasive delivery. A challenge in manufacturing of such systems is their complicated processes and potential negative impact on protein drug stability. Moreover, the high manufacturing cost of these advanced systems often offsets their remarkable advantages. To circumvent these problems that confront the current methods, we hypothesized the concept of “skin-permeable proteins” which would possess skin penetrating ability, and thereby eliminate a need for a transport vehicle. However, naturally occurring proteins with skin penetrating ability rarely exist. Herein, we present a novel strategy for chemically constructing artificial skin-permeable proteins, featured by a simple conjugation of a protein to a cell-penetrating peptide (CPP), which would display a penetration effect on the stratum corneum barrier, and transport the attached proteins into the skin. Furthermore, the feasibility of application in transcutaneous immunization is demonstrated. CPPs are known for their versatility in carrying macro- or supra-molecules through the cell membrane barriers that challenge the conventional drug delivery approaches.[1] The CPPs are capable of transporting their cargos, often linked by a covalent bond, into almost all cell types.[2] Among such CPPs, the low molecular weight protamine (LMWP) peptide (VSRRRRRRGGRRRRR), developed in our laboratory by enzymatic digestion of protamine (an FDA approved drug), offers distinct advantages. First, LMWP is as potent as the virus-derived TAT peptide, the most-studied CPP to date, in mediating cellular translocation of the attached cargos.[3] Secondly, unlike other CPPs, the toxicity profile of LMWP has already been thoroughly established. LMWP was shown to be non-immunogenic,[4] and its use in dogs did not elicit acute toxic responses.[5] Lastly, while other CPPs must be chemically synthesized, LMWP can be produced in mass quantities direct from native protamine with limited processing time and cost.[6] In this investigation, the artificial skin-permeable protein was synthesized by conjugating LMWP to ovalbumin (OVA), a representative antigenic protein, via a cleavable disulfide bond (Scheme 1). The LMWP-OVA conjugates were purified by heparin affinity chromatography, and the final product, generally possessing a 1:1 molar ratio of LMWP:OVA, was verified by MALDI-TOF-MS. Scheme 1 Chemical conjugation of LMWP to OVA As noted, skin keratinocytes are a physical barrier that provides the front line of defense against infection and also poses a challenge to protein delivery. On the other hand, keratinocytes execute a “part-time” antigen-presenting function by secreting immune mediators and transferring antigens to local antigen-presenting cells.[7] LMWP was shown to exhibit an ability to translocate the linked cargos of varying sizes into keratinocytes (Figure 1), demonstrating the potential for percutaneous protein delivery. Figure 1 Uptake by human keratinocyte cells of a) rhodamine B; b) OVA; and c) BSA; compared with those of d) LMWP-rhodamine B; e) LMWP-OVA; and f) LMWP-BSA conjugates. Protein cargos were labeled with FITC. (Scale bar = 100 μm) The plausibility of percutaneous delivery in vivo was examined by topical application of LMWP-linked lysozyme, OVA, or bovine serum albumin (BSA), to represent a broad range of protein sizes. All the LMWP-linked proteins successfully penetrated the stratum corneum and accumulated primarily in the epidermis (Figure 2), whereas the control proteins without LMWP linkage remained on the surface of skin. Figure 2 In vivo transcutaneous delivery mediated by LMWP. a), b), and c) represented unmodified free lysozyme, OVA, and BSA, whereas d), e), and f) represented LMWP-linked lysozyme, OVA, and BSA, respectively. Arrows represented the direction of skin penetration. ... The skin penetration mechanism of CPPs is still under debate. However, the interaction between CPP and lipid bilayer is believed to play a major role in the cell penetration process.[8] The skin permeability is governed by the physical state and structural organization of the extracellular lipids.[9] Hence, the skin penetration function of LMWP could account for its interaction with the skin extracellular lipid matrices. Such interaction would lead to disruption of the ordered lipid orientation, thereby creating channels for transducing protein cargos through the stratum corneum. As a typical example of protein percutaneous delivery, the immunological milieu of the skin is an ideal site for noninvasive vaccine delivery. The epidermis is rich in mature Langerhans cells (LCs), which represent a network of immune cells that underlie 25% of the total surface area in human skin,[10] and thus the epidermis is the target skin layer for transcutaneous immunization (TI). TI can be achieved by topically applying antigens, which, with the aid of a transdermal delivery system, penetrate into skin and subsequently elicit the desired immunity. The network of LCs acts as an immunological line of defense and initiates immune responses by conveying the captured antigens to other cells of the immune system, e.g. lymphocytes, melanocytes and Mercel cells.[11] Therefore, the unique epidermal accumulation of the LMWP-linked proteins offers an ideal situation to alert such antigen-presenting cells. The constructed skin-permeable antigen of LMWP-OVA was tested for the feasibility of TI on Balb/c mice. Humoral IgG is the primary protection induced by preventive vaccines to neutralize and eliminate of pathogens. Figure 3a revealed that a significant elevation of anti-OVA IgG concentration in the blood was observed following topical application of LMWP-OVA with cholera toxin as adjuvant. The IgG levels in TI groups treated with the high- (TI-H) and medium-dose (TI-M) of antigen displayed no statistical differences (p > 0.05) from those in animals given OVA through the standard intramuscular immunization method (IM group). The control group, receiving topical native OVA, exhibited markedly lower levels of IgG, due to poor percutaneous absorption of unmodified OVA. These findings indicated that the epidermis-accumulated LMWP-OVA was captured by LCs that subsequently migrated to lymphoid tissues and presented the antigens, effectively eliciting robust humoral immune responses. Furthermore, disulfide linkage could be cleaved by the elevated level of glutathione and reductase activity in the cytosol,[12] allowing release of OVA from LMWP, thus retaining a full intrinsic immunogenicity. As evidence, LMWP-OVA in TI method triggered OVA-specific IgG responses comparable to the IM injection of OVA. Since the conjugation of LMWP to OVA might affect its intrinsic antigenic determinants, a cleavable linkage could ease such concern. Figure 3 Transcutaneous immunization study. Mice were topically immunized with high- (500 μg; TI-H), medium- (250 μg; TI-M), and low-dose (100 μg; TI-L) of LMWP-OVA. a) High levels of anti-OVA IgG were observed in all TI groups, with no ... TI shows advantages over conventional injection vaccination by offering the opportunity to elicit specific immune responses, such as targeted immunity to the female reproductive tract[13] and cytotoxic T lymphocytes (CTL) effect.[14] Secretory IgA (sIgA) is the predominant humoral defense mechanism at mucosal surface, and it therefore protects the host from initial infections. As shown in Figure 3b, the anti-OVA sIgA levels measured in vaginal secretions were significantly higher in TI-H and TI-M groups than those in the IM group, confirming the promise of TI in achieving local protective immunity against female genital infection. Furthermore, interferon-γ(IFN-γ), the representative cytokine known to enhance the CD8+ CTL-mediated cytotoxicity against infected cells, was also present at a level significantly higher in the TI groups than in the IM group (Figure 3c). Notably, local immune response in skin could also benefit from production of high levels of IFN-γ, due to its effect on promoting CTL recognition of antigen molecules in keratinocytes[15] and subsequently their expedited lysis.[16] In addition, a primer-booster vaccination conducted by combining the IM injection of OVA with transcutaneous boosters of LMWP-OVA showed the immunity induction comparable to the multi-shot IM standard method (Figure S 1). The self-administrable boosters would eliminate follow-up visits to clinics for a multi-dose protocol. Hence this immunization strategy could improve not only patient compliance but also vaccination coverage in underserved areas with limited medical settings. In conclusion, this methodology for constructing artificial skin-permeable antigens may offer simple and needle-free vaccination modalities without the need for sophisticated drug carriers or expensive medical devices. Such a method could be beneficial especially to developing countries that struggle to fulfill effective vaccination coverage.

Combination of antibody targeting and PTD-mediated intracellular toxin delivery for colorectal cancer therapy.

The bottlenecks of current chemotherapy in the treatment of colorectal cancer lie in the ineffectiveness of the existing anti-cancer small molecule drugs as well as the dose-limiting toxicity caused by the nonselective action on normal tissues by such drugs. To address these problems, we introduce a novel therapeutic strategy based on tumor targeting using a non-internalizing anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) and intracellular delivery of the extremely potent yet cell-impermeable protein toxin gelonin via the aid of a cell-penetrating peptide (also termed as protein transduction domain; PTD). A chimeric TAT-gelonin fusion protein was genetically engineered, and it displayed remarkably enhanced anti-cancer activity against human colorectal cancer cells, with IC50 values being several orders of magnitude lower than the unmodified gelonin. On the other hand, a chemically synthesized conjugate of heparin and a murine anti-CEA mAb, T84.66 (termed T84.66-Hep) was found able to bind highly specifically to CEA over-expressing LS174T colorectal cancer cells. When mixing together, TAT-gelonin and T84.66-Hep could associate tightly and automatically through an electrostatic interaction between the cationic TAT and anionic heparin. In preliminary in vivo studies using LS174T s.c. xenograft tumor bearing mouse, selective and significantly augmented (58-fold) delivery of TAT-gelonin to the tumor target was observed, when compared with administration of TAT-gelonin alone. More importantly, efficacy studies also revealed that only the TAT-gelonin/T84.66-Hep complex yielded a significant inhibition of tumor growth (46%) without causing gelonin-induced systemic toxicity. Overall, this study suggested a generic strategy to effectively yet safely deliver potent PTD-modified protein toxins to the tumor.

Influence of formulation variables in transdermal drug delivery system containing zolmitriptan

The effects of different formulation variables including pressure sensitive adhesive (PSA), thickness of the matrix, solvent system, inclusion of crystallization inhibitor, loading amount of drug and enhancers on the transdermal absorption of zolmitriptan were investigated. Acrylic adhesive with hydroxyl functional group provided good adhesion force and high flux of zolmitriptan. Pseudopolymorphs of zolmitriptan were found to possess different solid-state properties that affected the permeation rate. Polyoxyethylene alkyl ethers significantly increased the permeation of zolmitriptan through hairless mouse skin. However, these enhancers induced crystallization of zolmitriptan. Kollidon(®) 30 delayed the crystallization without altering the permeation profile of zolmitriptan. Stability studies suggested that terpenes did not induce crystallization of zolmitriptan in the patch and stable formulations could be produced by using cineole and limonene, or their combination.

Development of Solid Self-Emulsifying Formulation for Improving the Oral Bioavailability of Erlotinib

To improve the solubility and oral bioavailability of erlotinib, a poorly water-soluble anticancer drug, solid self-emulsifying drug delivery system (SEDDS) was developed using solid inert carriers such as dextran 40 and Aerosil® 200 (colloidal silica). The preliminary solubility of erlotinib in various oils, surfactants, and co-surfactants was determined. Labrafil M2125CS, Labrasol, and Transcutol HP were chosen as the oil, surfactant, and co-surfactant, respectively, for preparation of the SEDDS formulations. The ternary phase diagram was evaluated to show the self-emulsifying area. The formulations were optimized using the droplet size and polydispersity index (PDI) of the resultant emulsions. Then, the optimized formulation containing 5% Labrafil M2125CS, 65% Labrasol, and 30% Transcutol was spray dried with dextran or Aerosil® and characterized for surface morphology, crystallinity, and pharmacokinetics in rats. Powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC) exhibited the amorphous form or molecular dispersion of erlotinib in the formulations. The pharmacokinetic parameters of the optimized formulations showed that the maximum concentration (Cmax) and area under the curve (AUC) of erlotinib were significantly increased, compared to erlotinib powder (p < 0.05). Thus, this SEDDS could be a promising method for enhancing the oral bioavailability of erlotinib.

Lichen Secondary Metabolites in Flavocetraria cucullata Exhibit Anti-Cancer Effects on Human Cancer Cells through the Induction of Apoptosis and Suppression of Tumorigenic Potentials

Lichens are symbiotic organisms which produce distinct secondary metabolic products. In the present study, we tested the cytotoxic activity of 17 lichen species against several human cancer cells and further investigated the molecular mechanisms underlying their anti-cancer activity. We found that among 17 lichens species, F. cucullata exhibited the most potent cytotoxicity in several human cancer cells. High performance liquid chromatography analysis revealed that the acetone extract of F. cucullata contains usnic acid, salazinic acid, Squamatic acid, Baeomycesic acid, d-protolichesterinic acid, and lichesterinic acid as subcomponents. MTT assay showed that cancer cell lines were more vulnerable to the cytotoxic effects of the extract than non-cancer cell lines. Furthermore, among the identified subcomponents, usnic acid treatment had a similar cytotoxic effect on cancer cell lines but with lower potency than the extract. At a lethal dose, treatment with the extract or with usnic acid greatly increased the apoptotic cell population and specifically activated the apoptotic signaling pathway; however, using sub-lethal doses, extract and usnic acid treatment decreased cancer cell motility and inhibited in vitro and in vivo tumorigenic potentials. In these cells, we observed significantly reduced levels of epithelial-mesenchymal transition (EMT) markers and phosphor-Akt, while phosphor-c-Jun and phosphor-ERK1/2 levels were only marginally affected. Overall, the anti-cancer activity of the extract is more potent than that of usnic acid alone. Taken together, F. cucullata and its subcomponent, usnic acid together with additional component, exert anti-cancer effects on human cancer cells through the induction of apoptosis and the inhibition of EMT.

Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides.

Brain delivery of macromolecular therapeutics (e.g., proteins) remains an unsolved problem because of the formidable blood–brain barrier (BBB). Although a direct pathway of nose-to-brain transfer provides an answer to circumventing the BBB and has already been intensively investigated for brain delivery of small drugs, new challenges arise for intranasal delivery of proteins because of their larger size and hydrophilicity. In order to overcome the barriers and take advantage of available pathways (e.g., epithelial tight junctions, uptake by olfactory neurons, transport into brain tissues, and intra-brain diffusion), a low molecular weight protamine (LMWP) cell-penetrating peptide was utilized to facilitate nose-to-brain transport. Cell-penetrating peptides (CPP) have been widely used to mediate macromolecular delivery through many kinds of biobarriers. Our results show that conjugates of LMWP–proteins are able to effectively penetrate into the brain after intranasal administration. The CPP-based intranasal method highlights a promising solution for protein therapy of brain diseases.

Lichen Secondary Metabolite, Physciosporin, Inhibits Lung Cancer Cell Motility.

Lichens produce various unique chemicals that can be used for pharmaceutical purposes. To screen for novel lichen secondary metabolites showing inhibitory activity against lung cancer cell motility, we tested acetone extracts of 13 lichen samples collected in Chile. Physciosporin, isolated from Pseudocyphellaria coriacea (Hook f. & Taylor) D.J. Galloway & P. James, was identified as an effective compound and showed significant inhibitory activity in migration and invasion assays against human lung cancer cells. Physciosporin treatment reduced both protein and mRNA levels of N-cadherin with concomitant decreases in the levels of epithelial-mesenchymal transition markers such as snail and twist. Physciosporin also suppressed KITENIN (KAI1 C-terminal interacting tetraspanin)-mediated AP-1 activity in both the absence and presence of epidermal growth factor stimulation. Quantitative real-time PCR analysis showed that the expression of the metastasis suppressor gene, KAI1, was increased while that of the metastasis enhancer gene, KITENIN, was dramatically decreased by physciosporin. Particularly, the activity of 3’-untranslated region of KITENIN was decreased by physciosporin. Moreover, Cdc42 and Rac1 activities were decreased by physciosporin. These results demonstrated that the lichen secondary metabolite, physciosporin, inhibits lung cancer cell motility through novel mechanisms of action.

Immobilized thermolysin for highly efficient production of low-molecular-weight protamine--an attractive cell-penetrating peptide for macromolecular drug delivery applications.

Macromolecules present a remarkable potential as future therapeutics. However, their translation into clinical practice has been hampered by an inherently low bioavailability. Cell-penetrating peptides (CPP) have been recently shown to significantly improve on the bioavailability of macromolecules. Yet, the high cost associated with development and production of these peptides is a major factor hindering their rapid deployment beyond the laboratory. Here, we describe a facile and robust methodology for efficient and large-scale production of low-molecular-weight protamine-a potent CPP of great clinical potential. Our methodology is based on the immobilization of thermolysin, an enzyme catalyzing digestion of native protamine, on chemically surface-modified gels produced by silica sol-gel chemistry. Thermolysin was immobilized at extremely high matrix loading of 733 mg/g matrix and exhibited good thermal and pH stability, indicating robustness with respect to processing conditions. The mechanical properties of the silica matrix further allowed utilization of the immobilized thermolysin in both batch and packed-bed reactor systems to produce the LMWP peptide in high yields. Results presented here are of high significance as this efficient and cost-effective production of high purity LMWP could enable clinical translation of many potential macromolecular drugs.