Low molecular weight protamine (LMWP): A nontoxic protamine substitute and an effective cell-penetrating peptide

COREL-07229; No of Pages 14 Journal of Controlled Release xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

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Huining He a,b,1, Junxiao Ye b,1, Ergang Liu b, Qiuling Liang a, Quan Liu b, Victor C. Yang a,b,c,d,⁎

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Low molecular weight protamine (LMWP): A nontoxic protamine substitute and an effective cell-penetrating peptide

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Article history: Received 4 April 2014 Accepted 27 May 2014 Available online xxxx

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Keywords: Low molecular weight protamine Protein transduction domain (PTD) Cell-penetrating peptide (CPP) Heparin antagonist Protamine

Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300072, PR China State Key Laboratory for Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA d Department of Molecular Medicine and Biopharmaceutical Sciences, College of Medicine & College of Pharmacy, Seoul National University, Seoul, South Korea b

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Low molecular weight protamine (LMWP) is a peptide fragment produced in our laboratory from enzymatic digestion of native protamine. More than 30 papers studying the properties and applications of LMWP have been published by our group in various journals since its initial discovery in 1999. Results have shown that LMWP could completely neutralize the anticoagulant functions of both heparin and low molecular weight heparin (LMWH), with reduced antigenicity and cross-reactivity toward the mice-derived anti-protamine antibodies. Aside from its potential as a heparin/LMWH antagonist, LMWP also shows the ability to retard insulin adsorption by the formation of an insoluble complex, making it a less toxic long-lasting insulin product than the conventional neutral protamine Hagedorn (NPH) insulin for diabetic control. Importantly, LMWP (Sequence: VSRRRRRRGGRRRR), with 10 arginine residues in its structure, could function as a cell-penetrating peptide (CPP), also termed protein transduction domain (PTD), to achieve effective intracellular protein or gene delivery in clinical practice. In this paper, we present a thorough review of our work related to LMWP, with the aim of providing readers an insight into its potential to be a clinical protamine substitute as well as a non-toxic cell penetrating peptide applicable to achieve intracellular protein and gene delivery. © 2014 Published by Elsevier B.V.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Preparation and characterization of low molecular weight protamine . . . . . . . . . . . . . . . . . . . . . . . . Toxicity evaluation of LMWP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. In vitro antigenicity study of LMWP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. In vivo immunogenicity evaluation of LMWP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. In vivo toxicity evaluation for LMWP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vitro/in vivo evaluation of the efficacy and toxicity of LMWP in neutralizing heparin and low molecular weight heparin (LMWH) 3.1. In vitro neutralization of heparin by LMWP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. In vitro neutralization of LMWH by LMWP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. In vivo efficacy of LMWP in heparin neutralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LMWP functions as an effective protein transduction domain (PTD) peptide for protein therapeutics . . . . . . . . . . . . . . 4.1. Chemically and biologically synthesized protein drugs for PTD mediated protein delivery . . . . . . . . . . . . . . . 4.1.1. Chemically and biologically synthesized PTD-modified gelonin for enhanced anti-tumor activity . . . . . . . . 4.1.2. LMWP enhanced intestinal absorption of the insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. Cell permeable PTD-cocaine esterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4. Cell-penetrating peptide mediated encapsulation of protein therapeutics into intact red blood cells . . . . . . . 4.2. Magnetic targeted prodrug strategy based on electrostatic interaction . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Antibody targeted, protamine triggered, electrically modified prodrug-type strategy . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA. Tel.: +1 734 764 4273; fax: +1 734 763 9772. E-mail address: [email protected] (V.C. Yang). 1 These authors contributed equally.

http://dx.doi.org/10.1016/j.jconrel.2014.05.056 0168-3659/© 2014 Published by Elsevier B.V.

Please cite this article as: H. He, et al., Low molecular weight protamine (LMWP): A nontoxic protamine substitute and an effective cellpenetrating peptide, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.05.056

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LMWP functions as an efficient and nontoxic gene carrier . . . . . . . . . . . . . 5.1. Non-covalent LMWP/nucleic acid complex formed by electrostatic interactions 5.2. Covalent LMWP–siRNA conjugate . . . . . . . . . . . . . . . . . . . . . 6. LMWP mediated cell visualization using magnetic nanoparticles . . . . . . . . . . 7. Discussion and prospectives . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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The native protamine, obtained from fish, has an average molecular weight of about 4500 Da. It consists of a group of heterogeneous polycationic peptides, with nearly 67% of its amino acid composition being arginine [1]. The protamines are a diverse family of small arginine-rich proteins that are synthesized to replace histones late in the haploid phase of spermatogenesis. Protamine binds to DNA, which is believed essential for condensing the spermatid genome and DNA stabilization. Protamine would yield a denser packaging of DNA in spermatozoon compared to that of histones, but the combination of the protamine and DNA must be decompressed before protein synthesis [2]. The polycationic protamine can electrostatically bind with the polyanionic heparin to form a stable complex that is devoid of anticoagulant activity. Therefore, protamine is routinely used after cardiac or vascular surgeries to reverse the anticoagulant function of heparin, thereby preventing post-operative bleeding [3–5]. Aside from its use as a heparin antagonist, protamine also has other major pharmacological applications. It is reported that protamines have the ability to combine with insulin in formulating protamine zinc insulin (PZI) and neutral protamine Hagedorn (NPH) insulin, thereby prolonging the adsorption of insulin [6]. These formulations allow insulin-dependent diabetic patients to achieve euglycemia with less frequent insulin injections. Despite its common use in clinical practice, protamine nevertheless is still a toxic drug. Intravenous administration of protamine is often associated with adverse respiratory (e.g. wheezing, bronchospasm, pulmonary edema, hypertension), and cardiovascular reactions (e.g. hypotension, preserved cardiac contractility, systemic vasodilatation, bradycardia, ventricular fibrillation) [7–16]. Protamine toxicity ranges from mild hypotension [17–19] to severe vascular collapse requiring prompt intervention [20–23] or even idiosyncratic fatal cardiac arrest [24–27]. Recent research also suggested that protamine could be a potential cause of fulminating noncardiogenic pulmonary edema after cardiopulmonary bypass [28]. In addition, unbound protamine was claimed to affect myocyte contractile processes causing myocardial depressant reactions [29,30], as well as altering calcium overload in the myocardium [31]. Moreover, protamine was known to possess its own anticoagulant functions [32,33], and its use in heparin reversal at times was accompanied by the phenomenon termed as heparin rebound [34,35]. Furthermore, several cases of protamine reactions masquerading as insulin allergy in diabetics have recently been reported [36–38]. To overcome this clinical dilemma, we proposed in 1999 a novel strategy of developing a short-chained low molecular weight protamine (LMWP, Sequence: VSRRRRRRGGRRRR) fragment [39], containing a compact arginine sequence and an average molecular weight of approximately 1880 Da via enzymatic digestion of native protamine using thermolysin [39–42]. In vitro studies delivered a great success that such LMWP fragments could completely neutralize the anticoagulant functions of both heparin and LMWH, based on the anti-Xa chromogenic and aPTT clotting time assays [42]. In vivo results revealed that although intravenous injection of protamine to mice led to obvious production of anti-protamine antibodies similar, injection of LMWP did not elicit any detectable immunogenic responses. In addition, these LMWP fragments displayed

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a markedly reduced antigenicity and cross-reactivity toward the mouse-derived anti-protamine antibodies. Furthermore, while protamine was known to retard insulin adsorption by the formation of an insoluble complex (PZI or NPH) at the physiological pH, our results also clearly demonstrated that LMWP could retain the same, if not superior, ability as protamine in retarding insulin adsorption [43]. Aside from its potential in neutralizing the anticoagulant functions of both heparin and LMWH and enhancing insulin adsorption, LMWP also carried significant structural similarity to that of the universally used protein transduction domain (PTD) peptide — TAT. Indeed, in our preliminary studies, we found that LMWP was almost equally potent as TAT in mediating cell transduction of its attached cargos. Based on its significantly reduced toxicity, LMWP could be applied to gene therapy to overcome the current toxicity-related limitation of the gene carriers. In contrast to TAT, however, the toxicity profiles of LMWP have already been fully established. Animal studies conducted in our laboratory demonstrated that LMWP was not toxic, immunogenic or antigenic. An additional advantage of using LMWP as the gene carrier is that, unlike TAT which must be synthesized chemically, LMWP can be readily produced, in mass quantities from native protamine, thereby significantly reducing the costs and the production period [44]. In this article, we fully present a thorough review of our previous work related to LMWP.

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1.1. Preparation and characterization of low molecular weight protamine 143 The enzymatic digestion method employed for the preparation of LMWP was published in 1999 [39] and employed frequently in our previous publications [40,41,44–50]. To perform the enzymatic digestion of protamine sulfate, thermolysin was mixed with protamine using a mole ratio 1:100 in the PBS solution containing 20 mM CaCl2, and then incubated for 30 min at room temperature. After that EDTA (50 mM) was added into the reaction mixture to quench the proteolytic activity of thermolysin [39]. Under such a situation, a number of fragments, composed of highly cationic peptides, with molecular weights ranging from 700 to 1900 Da, were produced. The lyophilized product thus prepared contained a mixture of highly cationic peptide impurities, and therefore peptides were further fractionated using a heparin affinity chromatography. A step NaCl gradient, produced by mixing solutions of PBS and 2 M NaCl, was used to separate the peptide fractions (Fig. 2). The precise molecular weight and amino acid sequence of each isolated fragment were further identified using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Among all the fragments, the TDSP-5 (sequence: VSRRRRRRGGRRRR) fragment, also termed LMWP, was utilized in the subsequent studies. (See Fig. 1.)

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In general, the use of protamine at times is associated with adverse reactions. Nevertheless, the chain-shortened arginine-rich LMWP peptide containing the heparin-neutralizing domain was expected to possess significantly reduced crosslinking and antigenic/immunogenic properties of protamine [51].

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Please cite this article as: H. He, et al., Low molecular weight protamine (LMWP): A nontoxic protamine substitute and an effective cellpenetrating peptide, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.05.056

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Fig. 1. Scheme illustrates the development and applications of low molecular weight protamine.

As known, commercial protamine is a non-human protein drug, and thus its administration would lead to the development of the antiprotamine antibodies; such as IgG, IgE, etc. Moreover, protamine could also cross-react with anti-protamine IgG antibody, causing the socalled “anaphylactoid” reactions [11–16,37,52,53]. As known the nonimmunologic pathway mediated complement activation is the primary event of protamine-induced toxicity. To this end, measurement of complement consumption can serve as a key index to assess the toxicity of protamine and its derived LMWP analogs. Complement activation by the heparin/protamine and heparin/LMWP complexes were measured by using the CH50 assay, according to our previously established procedure. Results confirmed that the use of LMWP would significantly attenuate the attitude of complement activation caused by the use of native protamine [42]. Like most of the small peptides derived from their parent protein by enzymatic fragmentation, the LMWP was expected to possess a greatly diminished antigenicity, meaning a significantly reduced crossreactivity toward protamine-produced antibodies. Indeed, sera obtained from protamine-immunized mice and then placed on microtiterplates coated with either protamine or LMWP revealed that the ELISA absorbance readings for LMWP coated plates were only 1/10 of the

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The immunogenicity of protamine and TDSP-5 were examined in mice. The production of polyclonal antibodies was performed according to the method of Cooper and Paterson [55]. Each mouse was immunized with 50 μg of protamine or LMWP in the complete Freund's adjuvant. All sera were then tested for antibody levels using our modified ELISA method [56]. In brief, protamine or LMWP was used to coat the wells of the microplate in order to capture their related antibodies. The

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intensity of the protamine-coated plates. In a separate experiment, we further examined the cross-reactivity of LMWP toward human anti-protamine antibodies, using clinical blood samples obtained from diabetic patients who were previously exposed with protaminecontaining insulin. Results indicated that the EU values in these sera samples determined by using LMWP-coated plates were significantly lower than those determined using protamine-coated plates, as demonstrated in Fig. 3 [54].

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Time (min) Fig. 2. Elution profile of protamine fragments using a heparin column and NaCl gradient.

Fig. 3. Levels (in EU) of anti-protamine antibodies in variously diluted serum samples Q3 obtained from healthy control subjects (N = 52) and diabetic patients with positive anti-protamine antibody titers (N = 51) [54].

Please cite this article as: H. He, et al., Low molecular weight protamine (LMWP): A nontoxic protamine substitute and an effective cellpenetrating peptide, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.05.056

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3. In vitro/in vivo evaluation of the efficacy and toxicity of LMWP in neutralizing heparin and low molecular weight heparin (LMWH)

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Since the introduction of heparin in 1922 as an anticoagulant [57] and protamine in 1937 as its clinical antidote [58], these two drugs have become ubiquitous in cardiovascular operations. The definite need of protamine for heparin neutralization to avoid bleeding risks, along with the unpredictable propensity for dramatic and life threatening protamine allergy, however, have since also evolved as a major clinical dilemma. In this regard, the naturally derived, nontoxic LMWP peptides from protamine would seem be an ideal solution to such a dilemma.

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As reported, neither protamine nor platelet factor 4 (rPF4) could effectively neutralize the anticoagulant activity of LMWH [42]. To examine if LMWP could also be an effective antagonist to LMWH, neutralization of two LMWH preparations, Fragmin (5000 Da; a FDA-approved drug) and LMWH3000 (a 3000-Da laboratory product), was examined. It should be pointed out that reports from the literature indicated that neither of these two LMWH compounds could be effectively neutralized by protamine or rPF4. However as shown in Fig. 5B, TDSP-5 was able to completely neutralize the anti-Xa activity of both LMWH preparations; as 100% neutralization of the anti-Xa activity of both LMWH compounds was achieved at a TDSP-5 dose between 75 and 100 μg [41].

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3.3. In vivo efficacy of LMWP in heparin neutralization

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Dogs were known to magnify the typical human responses noted with protamine reversal of heparin. Hence dogs were deliberately selected as the animal models to evaluate the in vivo efficacy of LMWP in heparin neutralization [59]. The underlying rationale was that if LMWP did not elicit any significant hemodynamic response in the most sensitive animal model, it should be reasonable to assume that LMWP would not pose serious toxic effects in human beings. Neutralization of heparin anticoagulant activities was measured by using various biological and clinical assays including the anti-IIa, anti-Xa chromogenic assays, and the ACT, aPTT, TCT heparin clotting assays. Complete neutralization of all different heparin-induced anticoagulant activities was observed for both protamine and LMWP at the 3-min

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The pulmonary artery systolic pressure (PAS) and the mean arterial pressure (MAP), two outstanding parameters in signaling protamineinduced hypotensive responses, were monitored to assess the toxic effects of LMWP. As shown in Fig. 4, compared to the control animals in which the dogs received I.V. protamine displayed statistically significant changes in MAP and PAS between the 3- to 6-minute marks (Fig. 4A), indicating typical and remarkable hypotensive responses. In sharp contrast, the use of TDSP-5 for heparin reversal did not elicit detectable protamine-related hemodynamic responses. None of the MAP or PAS changes at any time interval were statistically significant, compared to the control animals or at the starting point of time zero. Another strong indicator for protamine-induced toxicity was to monitor its effects on blood cell counts. In general the maximum changes in white blood cell (WBC) and platelet (PLT) counts would predominantly occur at the 3-min mark following heparin reversal with protamine. As expected, animals exposed to protamine exhibited a significant and yet typical reduction of both the WBC and PLT counts. In contrast, a statistically insignificant reduction in WBC counts as well as a statistically significant but much alleviated reduction in PLT counts were observed in dogs receiving LMWP, when compared to the baseline values. These results suggested that LMWP was significantly deprived or lacks both the hemodynamic and hematological toxicity seen in protamine [51].

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A broad spectrum of biological and clinical assays, including the antiIIa, anti-Xa chromogenic assays, and the aPTT clotting assay, were used to evaluate the ability of protamine and LMWP in heparin neutralization. Our results demonstrated that both TDSP-4 and TDSP-5 were capable of completely neutralizing the heparin anticoagulant activities, under a relatively higher dose than that of protamine in heparin reversal (data are summarized in Fig. 5A.). Furthermore, by aligning amino acid sequence with the heparin neutralization potency of these protamine fragments, it was found that although interaction of positive protein with heparin is predominantly electrostatic, the presence of at least 2 arginine clusters (each containing 4 to 6 arginine residues) in the peptide was essential for the maintenance of full heparin neutralization affinity, indicating that the secondary structure of the cationic peptide might also be critical in heparin neutralization [41]. Among the three fractions examined, TDSP5 (also termed as LMWP) appeared to be the most effective heparin antidote [41,42].

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3.1. In vitro neutralization of heparin by LMWP

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goat-antimouse-IgG-alkaline phosphatase was then used to produce absorbance readings at 405 nm. A near 46% reduction in antibody production in the LMWP group was observed, and compared to that of the protamine group. Although this study was rather preliminary based on the number of animals, the results nevertheless suggested a trend of reduced immunogenicity for LMWP [39].

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Fig. 4. Hemodynamic changes in dogs. Parameters measured include (A) Mean Arterial Pressure (MAP); and (B) Pulmonary Artery Systolic Pressure (PAS) [51].

Please cite this article as: H. He, et al., Low molecular weight protamine (LMWP): A nontoxic protamine substitute and an effective cellpenetrating peptide, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.05.056

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4.1. Chemically and biologically synthesized protein drugs for PTD mediated 341 protein delivery 342

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Since the first discovery of the protein transduction domain (PTD) peptide – the TAT – in 1988 [60,61], and the PTD was widely used to deliver its linked macromolecular cargos into cells of all tissue/organ types. The PTD-mediated intracellular delivery of macromolecular compounds was so overwhelming that it has become a new paradigm in recent years to design innovative drug delivery systems [62–71]. Indeed, PTD was called the “Trojan horse”in drug delivery, primarily because of its ability to cross through various bio-barriers [72], including the blood brain barrier [73,74], member of the retina and neurons [65,75], intestinal wall [76,77], and even skin [70,78]. Because of this PTD-mediated drug delivery systems have drawn significant attention from both the medical and pharmaceutical communities [79–83]. Subsequently, various PTD peptides, including TAT, LMWP, and many other arginine-rich cationic peptides [64,74,82,84], have been discovered [50,66,68,75–77,80,85–96]. Both in vitro and in vivo studies showed that, by covalently linking PTD to almost any type of molecular species including proteins (MW N 150 kDa; more than 60 different proteins have already been tested) [62,65,69,77,94,97–100], nucleic acids [44,84–86,101–104], and nano-carriers (e.g. iron oxide nanoparticles, gold nanoparticles, quantum dot, and liposomes) [105–112], PTD was able to ferry the attached cargos across the cell membrane of all organ types, including the blood brain barrier. This PTD-mediated cell internalization was so overwhelming that it could not be matched by any other conventional cell entry method such as receptor-mediated endocytosis. Importantly, it was shown that PTDmediated cell entry did not cause any perturbation or alteration of the cell membrane. The findings from Dowdy's group in 1999 actually set up the tune and milestone for the PTD-based drug delivery, in which the recombinant TAT-β-galactosidase fusion protein was found to cross through the formidable blood–brain barrier (BBB) following intraperitoneal injection [73]. Nevertheless, most of the PTDs were derived from viral resources (e.g. TAT from HIV-1), and the absence of toxicological profiles for these PTD peptides cast serious concerns on their potential and applicability in clinical practice [45]. LMWP displayed a significant structural similarity to that of TAT yet with significantly reduced toxicities. Therefore it is of great importance to examine if LMWP, with the welldemonstrated safe pharmacological profiles, could also function like a PTD in facilitating intracellular protein or gene delivery. Preliminary in vitro and in vivo animal tests both revealed that LMWP could indeed

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translocate gelonin, a cell-impermeable protein toxin [113], across the tumor mass and subsequently inhibit the tumor growth as efficiently as that mediated by TAT [49]. The presence of an equivalent cell penetrating potency, absence of toxicity, and suitability for low-cost mass production could render LMWP to evolve as the primary PTD for enhancing protein or gene delivery in clinical practice.

4. LMWP functions as an effective protein transduction domain (PTD) peptide for protein therapeutics

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mark after their administration. None of the differences between protamine and TDSP-5 were statistically significant (p N 0.1).

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Fig. 5. A) Neutralization of heparin by protamine, TDSP5 (LMWP), orTDSP4, as measured by anti-IIa chromogenic assay. B) Neutralization of LMWH3000 and Fragmin by LMWP as measured by the anti-Xa chromogenic assay. Reproduced according to [40,41].

4.1.1. Chemically and biologically synthesized PTD-modified gelonin for enhanced anti-tumor activity Gelonin, a plant-derived toxin that inhibits protein translation has attracted significant attention in cancer therapy [113]. Recently studies reported that conjugation of folate to gelonin would retain the ribosomal-inactivating properties of gelonin and at the same time permit targeting to the folate receptor on the cell surface [114]. Thus, we modified gelonin with LMWP via both chemical conjugation and genetic recombination methods, to overcome the inability of gelonin to internalize cells [49]. Results confirmed that gelonin–LMWP chemical conjugate (cG-L) and recombinant gelonin–LMWP chimera (rG-L) possessed N-glycosidase activity equivalent to that of unmodified recombinant gelonin (rGel). However, unlike rGel, both gelonin-LMWP conjugates were able to internalize into cells. Cytotoxicity studies further demonstrated that both cG-L and rG-L exhibited significantly improved tumoricidal effects, with IC50 values being 120-fold lower than that of rGel [48]. Moreover, when tested against a CT26 s.c. xenograft tumor mouse model, significant inhibition of tumor growth was observed with rG-L doses as low as 2 mug/tumor, while no detectable therapeutic effects were seen with rGel at 10-fold higher doses. Overall, this study demonstrated the potential of utilizing PTD to overcome limitations of current cell-impermeable protein drugs, achieving potent anti-tumor efficacy (see Fig. 6) [49].

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4.1.2. LMWP enhanced intestinal absorption of the insulin The LMWP can enhance the intestinal absorption of insulin in two forms: LMWP/insulin formulation and a LMWP-conjugated insulin formulation. For the former, the polycationic protamine can combine with the polyanionic insulin to form aggregates, such as the neutral protamine hagedorn (NPH) insulin, which would prolong insulin absorption and thereby reducing frequent injections [43]. In our study, we demonstrated that the insulin preparations containing either protamine or LMWP significantly extended the time required to reach the minimum glucose level, as well as the overall time span to preserve

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4.1.4. Cell-penetrating peptide mediated encapsulation of protein therapeutics into intact red blood cells To reap the benefits of red blood cells (RBCs) as an ideal natural and long-lasting drug carrier, it is absolutely essential to retain both the structural and functional integrity of RBCs. Yet, all of the existing encapsulation techniques, such as drug-induced endocytosis [116], electroporation [117], and hypo-osmotic-based pre-swelling [118], rupture/ resealing [119,120], or dialysis [121], would be likely to create sufficiently large pores or perturbations in the cell membrane [122]. Herein,

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4.1.3. Cell permeable PTD-cocaine esterase Cocaine esterase (CocE) is by far the most efficient cocainemetabolizing enzyme being tested in vivo [115]. It displayed a rapid clearance of cocaine and a robust protection against cocaine's toxicity in animal models. However, the clinical application of the CocE is limited by two potential obstacles: proteolytic degradation of CocE and CocE-induced immune response [115]. To minimize these potential obstacles, we engineered a cell permeable form of CocE, by covalently linking a thermally stable disulfide-bridged CocE mutant (dmCocE)

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with a PTD. TAT and LMWP were employed in our study. Two types of chemical conjugates, LMWP–S–S–dmCocE and Tat–S–S–dmCocE, were synthesized and purified. For comparison, four recombinant PTD-dmCocE fusion proteins, Tat-N-dmCocE, LMWP-N-dmCocE, dmCocE-C-Tat, and dmCocE-C-LMWP, were also constructed (Fig. 8). To evaluate their cellular uptake behavior, a covalently linked fluorophore (FITC) was linked to these conjugates to visualize the cellular uptake event of all these six PTD-dmCocE variants in living HeLa cells. As expected, all of the six variants exhibited significant cellular uptake, yet with different intracellular distribution phenotypes. Further in vivo evaluation of the cell-encapsulated dmCocE-C-Tat in hydrolyzing cocaine and alleviating the immunogenicity and proteolytic degradation are currently in progress in our laboratory [103]. Overall, based on the results obtained thus far we can reach a general conclusion, i.e. covalent attachment of a PTD enables CocE to become cell-permeable while maintaining its enzymatic activity [115]. More importantly, our work also suggested that the in vitro properties, such as the cellular uptake phenotype of the PTD-attached CocE, can potentially be regulated by constructing the PTD-CocE conjugated via different points of attachments. Findings from this study are extremely valuable in terms of establishing a non-disruptive cell-encapsulation technology for PTD-CocE and virtually all other protein therapeutics [115].

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this low glucose level, when compared to preparations containing insulin alone [42]. In a further study, we reported that conjugation of low molecular weight protamine (LMWP) to insulin via the covalent linkage was capable of translocating insulin through the epithelial cell membranes of the intestinal mucosal layer. In order to avoid the automatic event of forming of tight LMWP/insulin aggregates through electrostatic interactions, we developed an innovative conjugation strategy to synthesize the 1:1 monomeric insulin:LMWP chemical conjugate, by using the NHS-PEG-MAL as an intermediate crosslinker during the chemical process of conjugation [49]. The formation of a homogenous, monomeric (1:1 ratio) insulin: LMWP conjugate without encountering the substrate aggregation was confirmed using MALDI-TOF mass spectroscopy and SDS-PAGE. In vitro evaluation showed that transport of the Insulin-PEG-LMWP conjugate across the intestinal mucosal monolayer was augmented by almost five folds, compared to that of native insulin (see Fig. 7). Furthermore, findings from the in situ loop absorption tests in rats demonstrated that systemic pharmacological bioavailability of insulin was significantly enhanced after its conjugation with LMWP [50]. Importantly, the presented chemical conjugation method could offer a reliable and novel means to conjugate compounds possessing opposite charges.

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Fig. 6. A) Scheme of gelonin-LMWP chemical conjugation via a disulfide bond. B) & C) In vivo tumoricidal effects of recombinant gelonin-LMWP chimera (rG-L) in a CT26 s.c. xenograft tumor mouse model. Reproduced according to [48].

Please cite this article as: H. He, et al., Low molecular weight protamine (LMWP): A nontoxic protamine substitute and an effective cellpenetrating peptide, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.05.056

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Fig. 7. The structure for LMWP–insulin and plots of the blood glucose level (BGL) against time after in situ loop administration of PBS; insulin solution; insulin and LMWP mixture; insulinPEG-Mal and LMWP; insulin-PEG-LMWP conjugate; and subcutaneous injection of insulin solution; and insulin-PEG-LMWP conjugate to male Wistar rats. Reproduced according to [50].

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In this section, we plan to discuss a recently invented targeted protein delivery system for potential treatment of brain tumor. This system is composed of the following components, including magnetic iron nanoparticles (MIONs) as carriers for brain tumor targeting, and CPP linked macromolecular drugs (e.g. siRNA, toxins) for potent anti-tumor drug therapy [110–112,124,125]. A heparin/protamineregulated prodrug protection and de-protection strategy was incorporated to introduce a tumor-specific drug therapy, leading to a potentially non-invasive, magnetic resonance imaging (MRI)-guided, clinically enabled yet safe treatment of brain tumors (see Fig. 10) [123]. By adopting this delivery strategy, we achieved by far the first experimental success in a rat model of delivering microgram quantity of the large β-galactosidase model protein, selectively into a brain tumor but not to the ipsi- or contra-lateral normal brain regions [123]. Since macromolecular therapeutics, such as toxin or siRNA drugs, possess the ability to fully (N99.9%) eradicate a tumor at the nano-molar dose

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we introduce a novel method for encapsulating proteins into intact red blood cells (Fig. 9). This new RBC encapsulation method calls for covalent conjugation of the protein drug with the PTD peptides via disulfide linkage [123]. Herein, we use L-asparaginase as a model protein to illustrate the process of LMWP-mediated red blood cell encapsulation. As shown, because of the universal and potent membranepenetrating activity of LMWP, the PTD-L-asparaginase conjugates were shown to be capable of internalizing erythrocytes without altering RBCs' structural and/or functional attributes [122].

range, the prospects of achieving a clinically enabled brain tumor drug 465 therapy become practically accomplishable. 466

4.3. Antibody targeted, protamine triggered, electrically modified prodrug-type strategy

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The novel drug delivery system for protein delivery, termed “ATTEMPTS”(antibody targeted, protamine triggered, electrically modified prodrug-type strategy) was developed by our research team based on the aforementioned heparin/protamine regulated prodrug protection/deprotection strategy [66,100]. This drug delivery system consists of the following components: (a) the drug compartment consisting of a conjugate of the polycationic LMWP and the protein therapeutics, and (b) the targeting compartment made of a heparin-linked tumortargeting antibody. The polyanionic heparin motif on the antibody forms a strong electrostatic binding with the poly-cationic peptideLMWP motif on the protein therapeutics forming a large charge-based prodrug, possessing both the active targeting function (due to the antibody) and the LMWP-mediated drug cell uptake ability. Following tumor targeting of the large complex by the attached antibody, the LMWP-toxin conjugate will be released from the complex by the administration of protamine, which possesses a stronger binding affinity to heparin than LMWP. The LMWP-toxin is then able to be internalized into tumor cells due to the cell-penetrating ability of LMWP, exerting the toxin's cyto-toxic effect within the tumor cells [71]. The formation of an electrostatic linkage between LMWP on the toxin and heparin on the antibody would also resolve the non-selectivity issue of LMWP on its mediated cell internalization, as the cell-entry function would not

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Please cite this article as: H. He, et al., Low molecular weight protamine (LMWP): A nontoxic protamine substitute and an effective cellpenetrating peptide, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.05.056

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Fig. 8. A) Chemical conjugation of CPP to dmCocE by using SPDP as the cross-linker; B) schematic design for the four recombinant CPP-dmCocE fusion proteins; C) internalization of the CPP-dmCocE constructs in HeLa cells. Reproduced according to [115].

be present until the heparin-induced inhibitory effect was released by the addition of protamine (see Fig. 11) [71].

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Both in vitro and animal studies showed that, by covalently or noncovalently linking PTD to almost any type of molecular species including proteins and DNAs, PTD was able to ferry the attached cargos across various cell membranes, without any perturbation or alteration of the membrane structure and functions [64,71,86,100,126]. Recent studies showed that PTD, like TAT, could increase the delivery of plasmid DNA and RNA to the cells in vivo, thereby overcoming the cell bioavailability issue caused by the lack of intracellular uptake of the plasmid DNA to achieve effective gene expression [80,84,95,127,128]. In this application, LMWP should be able to enhance the gene expression, by enhancing intracellular delivery of the LMWP/DNA complexes [45].

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5.1. Non-covalent LMWP/nucleic acid complex formed by electrostatic interactions

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Traditionally, the PTD-mediated gene delivery is either based on the non-covalent complexation between PTD and nucleic acid via

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electrostatic interactions (Fig. 12B), or the covalent conjugation of PTD to the plasmid DNA and RNA (Fig. 12A) [129]. Nevertheless, as discussed previously, covalent conjugation is far more different to achieve, due to the automatic aggregation of these two compounds with opposite charges. (See Fig. 13.) Our previous results showed that LMWP could inherit the full potential of protamine to function as an effective and yet non-toxic gene carrier. Using pSV-β-galactosidase plasmid as the model gene, the transfection efficiency of the pDNA/LMWP complex, measured by the expression of the β-galactosidase activity, was found to be augmented when protamine or polyethyleneimine (PEI) was used as the gene carrier. Cytotoxicity results, however, indicated that protamine or PEI was almost twice as toxic to cells as LMWP. These results clearly implied that LMWP could be a much safer carrier for the delivery of therapeutic DNAs [44]. Another research focused on the use of LMWP/siRNA complex for the treatment of cancers [47]. Our results show that the fluorescentlytagged siRNAs were localized in the cytoplasm shortly after incubation of the LMWP/siRNA complex with carcinoma cells. Cell uptake of siRNA was greatly enhanced by using the LMWP as the gene carrier, which resulted in a significant down-regulation of the expression of the model protein luciferase as well as of the therapeutic tumor marker,

Please cite this article as: H. He, et al., Low molecular weight protamine (LMWP): A nontoxic protamine substitute and an effective cellpenetrating peptide, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.05.056

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Although evidences suggested that through electrostatic condensation of the polyanionic siRNA (or DNA) by the polycationic PTD could enable cellular uptake of these nucleic acid drugs via the conventional endocytosis mechanism [84,86], problems still existed [103]. First,

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during the formation of the non-covalent PTD/DNA complexes by electrostatic interactions, the positive charges on PTD would be neutralized by the negative charges of DNA, causing a loss of biological functions of both compounds. Secondly, PTD possessed a nuclear-localization function and thus would ferry the attached cargos into the nucleus. However, the siRNA drug would have to work in the cytoplasm where the machinery RISC system located [130]. In addition, large drugs reportedly would remain enclosed within the lipid vesicles following endocytosis, and thus would be subjected to enzymatic degradation by proteases in the endosome, rendering only a small portion of the therapeutic agents to have the chance to access the cell cytoplasm [63]. Hence, the ideal CPP-mediated siRNA delivery would be to form a soluble, monomeric CPP-drug conjugate through a covalent but reversible linkage. Under such conditions, the CPP would mediate a potent cellular delivery of the covalently attached siRNA through its unique cell-penetrating mechanism. Once inside the cells, the disulfide linkage between CPP and siRNA would be automatically degraded by the cytoplasmic reductase activities, detaching with the PTD from siRNA and

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vascular endothelial growth factor (VEGF). In vivo studies using tumorbearing mice further demonstrated that, through systemic application the LMWP/siRNA complex could carry siRNA into tumor cells and inhibit the expression of VEGF, suppressing tumor growth [47]. In addition, no detectable increase of the inflammatory cytokines including interferon (IFN)-α and interleukin (IL)-12, was observed in the serum after treatment with the LMWP/siRNA complexes, suggesting that systemic delivery of the LMWP/siRNA complex would not trigger immune stimulatory effects. Hence, formation of the LMWP/siRNA complexes could be a reliable and safe means to maximize the effectiveness of therapeutic siRNA, for treatment of cancers and other diseases [47].

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Fig. 9. A cartoon illustrates the LMWP mediated encapsulation inside intact red blood cell. Reproduced according to [122].

Fig. 10. A cartoon illustrates the exemplary brain drug delivery system. Reproduced according to [124].

Please cite this article as: H. He, et al., Low molecular weight protamine (LMWP): A nontoxic protamine substitute and an effective cellpenetrating peptide, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.05.056

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6. LMWP mediated cell visualization using magnetic nanoparticles

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Magnetic cellular imaging is a new and attractive field of research that allows visualization of the implanted cells both in vitro and in vivo. In order to successfully monitor the labeled cells, a higher optimum labeling efficiency without any side effect is required. Herein, superparamagnetic iron oxide (SPIO) nanoparticles, the commonly used MRI agent were tested as a model, and were conjugated with LMWP to generate an efficient and non-toxic cell labeling tool for cell visualization [131]. The results show that cells labeled with LMWP–SPIO displayed the highest iron content compared to those labeled with naked SPIO or SPIO conjugated with the universally used transfection agent poly-Llysine. In addition, Prussian staining and confocal results indicated the highest intracellular location of LMWP–SPIO. Furthermore, the labeling procedure would not alter the cell differentiation capacity of mesenchymal stem cells [131]. Based on these findings, it was concluded that the LMWP-modified, cell-permeable magnetic nanoparticles could be

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7. Discussion and prospectives

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Recently, owing to the potential clinical benefits, there has been an increasing perception that LMWH could eventually replace heparin as the mainstream anticoagulant. Should this occur, it would certainly add further clinical value to LMWP products. As reported, neither protamine nor any new alternative under development (e.g. rPF4) could completely neutralize the anticoagulant activity of LMWH [40,41]. To this regard, LMWP could become the antagonist of choice to prevent the bleeding risk [133] associated with the use of LMWH. With the unparallelled feature of protecting the entire patient population from possible catastrophic protamine attacks, as well as the unique properties of serving as the sole antagonist for LMWH, it is anticipated that LMWP would eventually evolve as the wholesale substitute for protamine in its every possible clinical and biomedical application. To this regard, successful development of LMWP as a non-toxic protamine

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utilized as an effective labeling tool to provide efficient magnetic cell 598 visualization of the transplanted cells [132]. 599

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allowing the drug to remain in the cytosol and exert its therapeutic functions. Indeed, a recently published authoritative review of siRNA delivery has called this type of covalent, monomeric CPP-drug conjugate the “Holy Grail” of CPP-mediated drug delivery [72]. In our previous study, antisense-HIF1α-oligonucleotide (ASO) and mismatch-HIF1α-oligonucleotide (MMO) were both conjugated with LMWP, to enhance the ability of the ASO drug in blocking hypoxicangiogenesis, thereby eliciting the anti-obesity effect of the drug. The results revealed that the nano-sized ASO-LMWP conjugates greatly enhanced cellular uptake of ASO, yielding no cytotoxic effect and protecting ASO from attacks by enzymatic and chemical reactions. Compared with naked ASO and the complex of ASO with lipofectamine during hypoxic-differentiation, the ASO-LMWP conjugates displayed enhanced intra-cellular localization and induced significant downregulation of leptin and VEGF gene expressions, thereby reducing fat accumulation in the cell [131].

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Fig. 11. Schematic diagram of the ATTEMPS delivery system ([72]).

Fig. 12. PTD-mediated drug delivery for nucleic acid. A) Covalent PTD–siRNA conjugate. B) Non-covalent CPP/nucleic acid complex formed by electrostatic interactions. Drawing according to [129].

Please cite this article as: H. He, et al., Low molecular weight protamine (LMWP): A nontoxic protamine substitute and an effective cellpenetrating peptide, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.05.056

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Fig. 13. Cellular uptake of LMWP–SPIO into human mesenchymal stem cells (hMSCs). (A) Iron content in cells treated with SPIO, SPIO–NH2, PLL–SPIO and LMWP–SPIO. (B) FACS analysis of cell uptake by FITC-labeled LMWP–SPIO in hMSCs. (C) Cellular localization of FITC-labeled LMWP–SPIO in hMSCs. (D) Photomicrographs show Prussian blue staining of hMSCs incubated with low and high volumes of LMWP–SPIO [132]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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substitute could potentially bring about evolutional changes in current clinical practice in anesthesia and intensive care. On the other hand, the PTD-mediated cell internalization was so 618 overwhelming that it could not be matched by other existing cell 619 entry methods. Moreover, it was shown that PTD-mediated cell entry 620 did not cause any perturbation or alteration of the cell membrane. 621 This unprecedented and unparallelled cell-entry activity renders PTD 622 an ideal solution to the membrane barrier problem encountered in 623 protein or gene delivery. In this regard, more and more groups have 624 adopted LMWP as a PTD for the delivery of macromoleculars and topical 625 applications [134–137]. In this study, low molecular weight protamine 626 conjugated with epidermal growth factor (termed as rLMWP-EGF) 627 showed significantly improved burn wound healing efficacy, with 628 accelerated wound closure and minimized eschar and scar formation, 629 compared with EGF or no treatment [136]. Some other studies which 630 are based on the cell-penetrating ability of LMWP, a “smart” drug deliv631 ery system with enhanced permeability for facilitating site-specific 632 Q11 targeting delivery of anticancer drug was also developed [137]. In 633 short, with its powerful cell penetrating ability, the LMWP is now a 634 wide spread technology. 635 Yet, the absence of established pharmacological and toxicological 636 profiles for PTD, particularly since virtually all of these PTD peptides 637 are derived from viral resources (e.g. TAT from HIV-1), casts serious 638 concerns on the potential applicability of PTD in clinical practice. The 639 LMWP peptide under development displayed a significant structural 616 617

similarity to that of TAT, and also functioned as a potent cellpenetrating peptide like TAT in facilitating intracellular protein or gene delivery. With the well-demonstrated pharmacological profiles and in vivo safety, the presence of equivalent cell penetrating potency, absence of toxicity, and suitability for low-cost mass production, it was envisioned that LMWP could evolve as the primary PTD in achieving effective yet safe protein and gene therapy in real-time clinical practice. More effort should be put into making LMWP an FDA approved clinical drug.

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Acknowledgments

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This work was supported in part by the National Natural Science Foundation of China (NSFC, 81361140344) and Tianjin Municipal Science and Technology Commission (12JCZDJC34000). This research was also partially sponsored by the School of Pharmacy, Fudan University & the Open Project Program of Key Lab of Smart Drug Delivery (Fudan University), Ministry of Education, China (SDD2013-04) and National Key Basic Research Program of China (2013CB932502).

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References

Q13 657

[1] T. Ando, M. Yamsaki, K. Suzuki, Protamine, in: A. Kleinzeller (Ed.), Molecular Biology, Biochemistry, and Biophysics, Springer-Verlag, New York, 1973, pp. 1–109. [2] R. Balhorn, The protamine family of sperm nuclear proteins, Genome Biol. 8 (9) (2007) 227.

Please cite this article as: H. He, et al., Low molecular weight protamine (LMWP): A nontoxic protamine substitute and an effective cellpenetrating peptide, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.05.056

641 642 643 644 645 646 647 648

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658 659 660 661

H. He et al. / Journal of Controlled Release xxx (2014) xxx–xxx

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[37] M.E. Bollinger, R.G. Hamilton, R.A. Wood, Protamine allergy as a complication of insulin hypersensitivity: a case report, J. Allergy Clin. Immunol. 104 (2) (1999) 462–465. [38] C. Blanco, R. Castillo, J. Quiralte, J. Delgado, I. Garcia, P. dePablos, T. Carrillo, Anaphylaxis to subcutaneous neutral protamine Hagedorn insulin with simultaneous sensitization to protamine and insulin, Allergy 51 (6) (1996) 421–424. [39] Y. Byun, V.K. Singh, V.C. Yang, Low molecular weight protamine: a potential nontoxic heparin antagonist, Thromb. Res. 94 (1) (1999) 53–61. [40] Y. Byun, L.C. Chang, L.M. Lee, I.S. Han, V.K. Singh, V.C. Yang, Low molecular weight protamine: a potent but nontoxic antagonist to heparin/low molecular weight protamine, ASAIO J. 46 (4) (2000) 435–439. [41] L.C. Chang, H.F. Lee, Z. Yang, V.C. Yang, Low molecular weight protamine (LMWP) as nontoxic heparin/low molecular weight heparin antidote (I): preparation and characterization, AAPS PharmSci. 3 (3) (2001) E17. [42] L.C. Chang, J.F. Liang, H.F. Lee, L.M. Lee, V.C. Yang, Low molecular weight protamine (LMWP) as nontoxic heparin/low molecular weight heparin antidote (II): in vitro evaluation of efficacy and toxicity, AAPS PharmSci. 3 (3) (2001) E18. [43] H. He, A.E. David, J. Zhang, Y.S. Park, J. Wang, Y. Huang, J. Wang, V.C. Yang, Low molecular weight protamine/insulin formulation with potential to attenuate protamine-masqueraded insulin allergy, Macromol. Res. 19 (12) (2011) 1224–1226. [44] Y.J. Park, J.F. Liang, K.S. Ko, S.W. Kim, V.C. Yang, Low molecular weight protamine as an efficient and nontoxic gene carrier: in vitro study, J. Gene Med. 5 (8) (2003) 700–711. [45] Y.J. Park, L.C. Chang, J.F. Liang, C. Moon, C.P. Chung, V.C. Yang, Nontoxic membrane translocation peptide from protamine, low molecular weight protamine (LMWP), for enhanced intracellular protein delivery: in vitro and in vivo study, FASEB J. 19 (11) (2005) 1555–1557. [46] J.F. Liang, L. Zhen, L.C. Chang, V.C. Yang, A less toxic heparin antagonist—low molecular weight protamine, Biochemistry (Mosc) 68 (1) (2003) 116–120. [47] Y.S. Choi, J.Y. Lee, J.S. Suh, Y.M. Kwon, S.J. Lee, J.K. Chung, D.S. Lee, V.C. Yang, C.P. Chung, Y.J. Park, The systemic delivery of siRNAs by a cell penetrating peptide, low molecular weight protamine, Biomaterials 31 (6) (2010) 1429–1443. [48] Y.S. Park, A.E. David, Y. Huang, J.B. Park, H. He, Y. Byun, V.C. Yang, In vivo delivery of cell-permeable antisense hypoxia-inducible factor 1alpha oligonucleotide to adipose tissue reduces adiposity in obese mice, J. Control. Release 161 (1) (2012) 1–9. [49] M.C. Shin, J. Zhang, A.E. David, W.E. Trommer, Y.M. Kwon, K.A. Min, J.H. Kim, V.C. Yang, Chemically and biologically synthesized CPP-modified gelonin for enhanced anti-tumor activity, J. Control. Release 172 (1) (2013) 169–178. [50] H. He, J. Sheng, A.E. David, Y.M. Kwon, J. Zhang, Y. Huang, J. Wang, V.C. Yang, The use of low molecular weight protamine chemical chimera to enhance monomeric insulin intestinal absorption, Biomaterials 34 (31) (2013) 7733–7743. [51] L.M. Lee, L.C. Chang, S. Wrobleski, T.W. Wakefield, V.C. Yang, Low molecular weight protamine as nontoxic heparin/low molecular weight heparin antidote (III): preliminary in vivo evaluation of efficacy and toxicity using a canine model, AAPS PharmSci. 3 (3) (2001) E19. [52] J.C. Horrow, Protamine — a review of its toxicity, Anesth. Analg. 64 (3) (1985) 348–361. [53] J.M. Weiler, P. Freiman, M.D. Sharath, W.J. Metzger, J.M. Smith, H.B. Richerson, Z.K. Ballas, P.C. Halverson, D.J. Shulan, S. Matsuo, R.L. Wilson, Serious adverse reactions to protamine sulfate — are alternatives needed?, J. Allergy Clin. Immunol. 75 (2) (1985) 297–303. [54] B. Tsui, V.K. Singh, J.F. Liang, V.C. Yang, Reduced cross-reactivity towards antiprotamine antibodies of a low molecular weight protamine analogue, Thromb. Res. 101 (2001) 417–420. [55] H.N. Cooper, Y. Paterson, Production of antibodies, in: J.E. Coligan, A.M. Kruisbbeck, D.H. Margulies (Eds.), Current Protocols in Immunology, Green Publishing Associates & Wiley-Interscience, NY., 1991 [56] Y. Byun, L.C. Chang, L.M. Lee, I.S. Han, V.K. Singh, V.C. Yang, Low molecular weight protamine: a potent but nontoxic antagonist to heparin/low molecular weight protamine, ASAIO J. 6 (4) (2000) 435–439. [57] W.H. Howell, Heparin, an anticoagulant: preliminary communication, Am. J. Physiol. 63 (1922) 434–435. [58] E. Chargaff, Studies on the chemistry of blood coagulation. VII. Protamines and blood clotting, J. Biol. Chem. 125 (1938) 671–676. [59] V.C. Yang, F.K. Port, J.S. Kim, C.L.C. Teng, G.O. Till, T.W. Wakefield, The use of immobilized protamine in removing heparin and preventing protamine-induced complications during extracorporeal blood-circulation, Anesthesiology 75 (2) (1991) 288–297. [60] A.D. Frankel, C.O. Pabo, Cellular uptake of the tat protein from human immunodeficiency virus, Cell 55 (6) (1988) 1189–1193. [61] M. Green, P.M. Loewenstein, Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein, Cell 55 (6) (1988) 1179–1188. [62] S. Fawell, J. Seery, Y. Daikh, C. Moore, L.L. Chen, B. Pepinsky, J. Barsoum, Tatmediated delivery of heterologous proteins into cells, Proc. Natl. Acad. Sci. 91 (2) (1994) 664–668. [63] H. Brooks, B. Lebleu, E. Vives, Tat peptide-mediated cellular delivery: back to basics, Adv. Drug Deliv. Rev. 57 (4) (2005) 559–577. [64] S. Deshayes, M. Morris, F. Heitz, G. Divita, Delivery of proteins and nucleic acids using a non-covalent peptide-based strategy, Adv. Drug Deliv. Rev. 60 (4–5) (2008) 537–547. [65] G.P. Dietz, E. Kilic, M. Bahr, Inhibition of neuronal apoptosis in vitro and in vivo using TAT-mediated protein transduction, Mol. Cell. Neurosci. 21 (1) (2002) 29–37. [66] Y.M. Kwon, Y.T. Li, J.F. Liang, Y.J. Park, L.C. Chang, V.C. Yang, PTD-modified ATTEMPTS system for enhanced asparaginase therapy: a proof-of-concept investigation, J. Control. Release 130 (3) (2008) 252–258.

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C

O

R

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C

T

[3] I.J. Welsby, M.F. Newman, B. Phillips-Bute, R.H. Messier, E.D. Kakkis, M. StaffordSmith, Hemodynamic changes after protamine administration — association with mortality after coronary artery bypass surgery, Anesthesiology 102 (2) (2005) 308–314. [4] L.B. Jaques, Protamine — antagonist to heparin, Can. Med. Assoc. J. 108 (10) (1973) 1291–1297. [5] Protamine sulfate, American Hospital Formulary Service (AHFS) Drug Information, American Society of Hospital Pharmacists Inc., Bethesda, 1989, pp. 711–713. [6] Insulins, American Hospital Formulary Service (AHFS) Drug Information, American Society of Hospital Pharmacists Inc., Bethesda MD, 1989, pp. 1725–1736. [7] M.M. Levinson, W. Ohm, The pathophysiology and treatment of severe protamine reactions in cardiovascular surgery, The Heart Surgery Forum, 1995. [8] R. Porsche, Z.R. Brenner, Allergy to protamine sulfate, Heart Lung 28 (6) (1999) 418–428. [9] J.M. Weiler, M.A. Gellhaus, J.G. Carter, R.L. Meng, P.M. Benson, R.A. Hottel, K.B. Schillig, A.B. Vegh, W.R. Clarke, A prospective-study of the risk of an immediate adverse reaction to protamine sulfate during cardiopulmonary bypass-surgery, J. Allergy Clin. Immunol. 85 (4) (1990) 713–719. [10] S. Metz, J.C. Horrow, Protamine and newer heparin antagonists, in: R.K. Stoelting (Ed.), Pharmacology & Physiology in Anesthetic Practice, 1994, pp. 1–15. [11] M.E. Weiss, N.F. Adkinson, Allergy to protamine, Clin. Rev. Allergy Immunol. 9 (3–4) (1991) 339–355. [12] J.C. Horrow, Protamine allergy, J. Cardiothorac. Anesth. 2 (1988) 225–242. [13] B. Lindblad, Protamine sulfate: a review of its effects: hypersensitivity and toxicity, Eur. J. Vasc. Surg. 3 (1989) 195–201. [14] J.G. Cormack, J.H. Levy, Adverse reactions to protamine, Coron. Artery Dis. 4 (5) (1993) 420–425. [15] J.A. Carr, N. Silverman, The heparin–protamine interaction — a review, J. Cardiovasc. Surg. (Torino) 40 (5) (1999) 659–666. [16] G. Blaise, Heparin and protamine interaction, Can. J. Anaesth. 45 (12) (1998) 1144–1147. [17] N.M. Katz, Y.D. Kim, R. Siegelman, S.A. Ved, S.W. Ahmed, R.B. Wallace, Hemodynamics of protamine administration — comparison of right atrial, left atrial, and aortic injections, J. Thorac. Cardiovasc. Surg. 94 (6) (1987) 881–886. [18] E. Ovrum, H. Lindberg, E.A. Holen, M. Abdelnoor, Hemodynamic-effects of intraaortic versus intravenous protamine administration after cardiopulmonary bypass in man, Scand. J. Thorac. Cardiovasc. Surg. 26 (2) (1992) 113–118. [19] J.K. Kirklin, D.E. Chenoweth, D.C. Naftel, E.H. Blackstone, J.W. Kirklin, D.D. Bitran, J. G. Curd, J.G. Reves, P.N. Samuelson, Effects of protamine administration after cardiopulmonary bypass on complement, blood-elements, and the hemodynamic state, Ann. Thorac. Surg. 41 (2) (1986) 193–199. [20] E. Lowenstein, W.E. Johnston, D.G. Lappas, M.N. Dambra, R.C. Schneider, W.M. Daggett, C.W. Akins, D.M. Philbin, Catastrophic pulmonary vasoconstriction associated with protamine reversal of heparin, Anesthesiology 59 (5) (1983) 470–473. [21] J.O. Justviera, C.R. Fischer, O. Gago, J.D. Morris, Acute reaction to protamine — its importance to surgeons, Am. Surg. 50 (1) (1984) 52–60. [22] F.E. Ralley, The use of nitric oxide for managing catastrophic pulmonary vasoconstriction arising from protamine administration, Anesth. Analg. 88 (3) (1999) 505–507. [23] M. Hurby, B. Skrovina, I. Vanek, Anaphylactic reaction to protamine in cardiovascular surgery, Rozhl. Chir. 74 (1995) 282–283. [24] G.N. Olinger, R.M. Becker, L.I. Bonchek, Noncardiogenic pulmonary-edema and peripheral vascular collapse following cardiopulmonary bypass — rare protamine reaction, Ann. Thorac. Surg. 29 (1) (1980) 20–25. [25] C.A. Cobb, D.L. Fung, Shock due to protamine hypersensitivity, Surg. Neurol. 17 (4) (1982) 245–246. [26] M.D. Sharath, W.J. Metzger, H.B. Richerson, R.K. Scupham, R.L. Meng, B.H. Ginsberg, J.M. Weiler, Protamine-induced fatal anaphylaxis — prevalence of antiprotamine immunoglobulin-E antibody, J. Thorac. Cardiovasc. Surg. 90 (1) (1985) 86–90. [27] P.P. Neidhart, B. Meier, B.S. Polla, J.A. Schifferli, D.R. Morel, Fatal anaphylactoid response to protamine after percutaneous transluminal coronary angioplasty, Eur. Heart J. 13 (6) (1992) 856–858. [28] C. Kindler, A. Bircher, P. Stulz, Protamine-induced fulminating non-cardiogenic pulmonary edema following cardiopulmonary bypass, Eur. J. Cardiothorac. Surg. 10 (6) (1996) 463–466. [29] R.B. Hird, T.W. Wakefield, R. Mukherjee, B.U. Jones, F.A. Crawford, P.C. Andrews, J.C. Stanley, F.G. Spinale, Direct effects of protamine sulfate on myocyte contractile processes — cellular and molecular mechanisms, Circulation 92 (9) (1995) 433–446. [30] T.W. Wakefield, S.K. Wrobleski, B.J. Nichol, A.M. Kadell, J.C. Stanley, Heparinmediated reductions of the toxic effects of protamine sulfate on rabbit myocardium, J. Vasc. Surg. 16 (1) (1992) 47–53. [31] W.K. Park, J.J. Pancrazio, C. Lynch, Mechanical and electrophysiological effects of protamine on isolated ventricular myocardium — evidence for calcium overload, Cardiovasc. Res. 28 (4) (1994) 505–514. [32] J.R. Adkins, J.D. Hardy, Sodium heparin neutralization and anticoagulant effects of protamine sulfate, Arch. Surg. 94 (2) (1967) 175–177. [33] R.J. Cobelgeard, H.I. Hassouna, Interaction of protamine sulfate with thrombin, American, Am. J. Hematol. 14 (3) (1983) 227–233. [34] A.H. Kuitunen, M.T. Salmenpera, J. Heinonen, V.P. Rasi, G. Myllyla, Heparin rebound: a comparative study of protamine chloride and protamine sulfate in patients undergoing coronary artery bypass surgery, J. Cardiothorac. Vasc. Anesth. 5 (1991) 221–226. [35] N. Ellison, C.P. Beatty, D.R. Blake, H.A. Wurzel, H. Macvaugh, Heparin rebound — studies in volunteers and patients, Transfusion 13 (5) (1973) 364-364. [36] R. Kim, Anaphylaxis to protamine masquerading as an insulin allergy, Del. Med. J. 65 (1993) 17–23.

U

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N C O

R

R

E

C

D

P

R O

O

F

[99] S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y. Sugiura, Argininerich peptides — an abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery, J. Biol. Chem. 276 (8) (2001) 5836–5840. [100] Y. Huang, Y.S. Park, J. Wang, C. Moon, Y.M. Kwon, H.S. Chung, Y.J. Park, V.C. Yang, ATTEMPTS system: a macromolecular prodrug strategy for cancer drug delivery, Curr. Pharm. Des. 16 (21) (2010) 2369–2376. [101] M.E. Martin, K.G. Rice, Peptide-guided gene delivery, AAPS J. 9 (1) (2007) E18–E29. [102] S.R. Schwarze, S.F. Dowdy, In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA, Trends Pharmacol. Sci. 21 (2) (2000) 45–48. [103] M.A. Zanta, P. Belguise-Valladier, J.P. Behr, Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus, Proc. Natl. Acad. Sci. 96 (1) (1999) 91–96. [104] T.S. Zatsepin, J.J. Turner, T.S. Oretskaya, M.J. Gait, Conjugates of oligonucleotides and analogues with cell penetrating peptides as gene silencing agents, Curr. Pharm. Des. 11 (28) (2005) 3639–3654. [105] C. Rudolph, U. Schillinger, A. Ortiz, K. Tabatt, C. Plank, R.H. Muller, J. Rosenecker, Application of novel solid lipid nanoparticle (SLN)-gene vector formulations based on a dimeric HIV-1 TAT-peptide in vitro and in vivo, Pharm. Res. 21 (9) (2004) 1662–1669. [106] V.P. Torchilin, Cell penetrating peptide-modified pharmaceutical nanocarriers for intracellular drug and gene delivery, Biopolymers 90 (5) (2008) 604–610. [107] V.P. Torchilin, Tatp-mediated intracellular delivery of pharmaceutical nanocarriers, Biochem. Soc. Trans. 35 (Pt4) (2007) 816–820. [108] J.M. de la Fuente, C.C. Berry, Tat peptide as an efficient molecule to translocate gold nanoparticles into the cell nucleus, Bioconjug. Chem. 16 (5) (2005) 1176–1180. [109] C. Walther, K. Meyer, R. Rennert, I. Neundorf, Quantum dot-carrier peptide conjugates suitable for imaging and delivery applications, Bioconjug. Chem. 19 (12) (2008) 2346–2356. [110] J. Zhang, M.C. Shin, A.E. David, J. Zhou, K. Lee, H. He, V.C. Yang, Long-circulating heparin-functionalized magnetic nanoparticles for potential application as a protein drug delivery platform, Mol. Pharm. 10 (10) (2013) 3892–3902. [111] J. Zhou, J. Zhang, A.E. David, V.C. Yang, Magnetic tumor targeting of betaglucosidase immobilized iron oxide nanoparticles, Nanotechnology 24 (37) (2013) (3751029, 12 pp.). [112] J. Zhang, M.C. Shin, V.C. Yang, Magnetic targeting of novel heparinized iron oxide nanoparticles evaluated in a 9l-glioma mouse model, Pharm. Res. 31 (3) (2013) 579–592. [113] F. Stirpe, S. Olsnes, A. Pihl, Gelonin, a new inhibitor of protein synthesis, nontoxic to intact cells. Isolation, characterization, and preparation of cytotoxic complexes with concanavalin A, J. Biol. Chem. 255 (14) (1980) 6947–6953. [114] S.F. Atkinson, T. Bettinger, L.W. Seymour, J.P. Behr, C.M. Ward, Conjugation of folate via gelonin carbohydrate residues retains ribosomal-inactivating properties of the toxin and permits targeting to folate receptor positive cells, J. Biol. Chem. 276 (30) (2001) 27930–27935. [115] T.Y. Lee, Y.S. Park, G.A. Garcia, R.K. Sunahara, J.H. Woods, V.C. Yang, Cell permeable cocaine esterases constructed by chemical conjugation and genetic recombination, Mol. Pharm. 9 (5) (2012) 1361–1373. [116] L.M. Matovcik, I.G. Junga, S.L. Schrier, Drug-induced endocytosis of neonatal erythrocytes, Blood 65 (5) (1985) 1056–1063. [117] C. Lizano, S. Sanz, J. Luque, M. Pinilla, In vitro study of alcohol dehydrogenase and acetaldehyde dehydrogenase encapsulated into human erythrocytes by an electroporation procedure, Biochim. Biophys. Acta 1425 (2) (1998) 328–336. [118] M. Hamidi, N. Zarei, A. Zarrin, S. Mohammadi-Samani, Preparation and validation of carrier human erythrocytes loaded by bovine serum albumin as a model antigen/protein, Drug Deliv. 14 (5) (2007) 295–300. [119] G.M. Ihler, R.H. Glew, F.W. Schnure, Enzyme loading of erythrocytes, Proc. Natl. Acad. Sci. 70 (9) (1973) 2663–2666. [120] S.J. Updike, R.T. Wakamiya, E.N. Lightfoot Jr., Asparaginase entrapped in red blood cells: action and survival, Science 193 (4254) (1976) 681–683. [121] M.I. Garin, R.M. Lopez, S. Sanz, M. Pinilla, J. Luque, Erythrocytes as carriers for recombinant human erythropoietin, Pharm. Res. 13 (6) (1996) 869–874. [122] Y.J. Chen, B.R. Liu, Y.H. Dai, C.Y. Lee, M.H. Chan, H.H. Chen, H.J. Chiang, H.J. Lee, A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides, Gene 493 (2) (2012) 201–210. [123] Y.M. Kwon, H.S. Chung, C. Moon, J. Yockman, Y.J. Park, S.D. Gitlin, A.E. David, V.C. Yang, L-Asparaginase encapsulated intact erythrocytes for treatment of acute lymphoblastic leukemia (ALL), J. Control. Release 139 (3) (2009) 182–189. [124] H. He, A. David, B. Chertok, A. Cole, K. Lee, J. Zhang, J. Wang, Y. Huang, V.C. Yang, Magnetic nanoparticles for tumor imaging and therapy: a so-called theranostic system, Pharm. Res. 30 (10) (2013) 2445–2458. [125] F. Yu, L. Zhang, Y. Huang, K. Sun, A.E. David, V.C. Yang, The magnetophoretic mobility and superparamagnetism of core-shell iron oxide nanoparticles with dual targeting and imaging functionality, Biomaterials 31 (22) (2010) 5842–5848. [126] E.L. Snyder, S.F. Dowdy, Protein/peptide transduction domains: potential to deliver large DNA molecules into cells, Curr. Opin. Mol. Ther. 3 (2) (2001) 147–152. [127] M.J. Gait, Peptide-mediated cellular delivery of antisense oligonucleotides and their analogues, Cell. Mol. Life Sci. 60 (5) (2003) 844–853. [128] L.N. Johnson, S.M. Cashman, R. Kumar-Singh, Cell-penetrating peptide for enhanced delivery of nucleic acids and drugs to ocular tissues including retina and cornea, Mol. Ther. 16 (1) (2008) 107–114. [129] B.R. Meade, S.F. Dowdy, Exogenous siRNA delivery using peptide transduction domains/cell penetrating peptides, Adv. Drug Deliv. Rev. 59 (2–3) (2007) 134–140. [130] M. Tijsterman, R.H.A. Plasterk, Dicers at RISC: the mechanism of RNAi, Cell 117 (1) (2004) 1–3.

E

T

[67] M. Zhao, R. Weissleder, Intracellular cargo delivery using tat peptide and derivatives, Med. Res. Rev. 24 (1) (2004) 1–12. [68] E. Vives, Present and future of cell-penetrating peptide mediated delivery systems: is the Trojan horse too wild to go only to Troy? J. Control. Release 109 (1–3) (2005) 77–85. [69] E. Vives, P. Brodin, B. Lebleu, A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus, J. Biol. Chem. 272 (25) (1997) 16010–16017. [70] Y. Huang, Y.S. Park, C. Moon, A.E. David, H.S. Chung, V.C. Yang, Synthetic skinpermeable proteins enabling needleless immunization, Angew. Chem. Int. Ed. Engl. 49 (15) (2010) 2724–2727. [71] H. He, J. Ye, Y. Wang, Q. Liu, H.S. Chung, Y.M. Kwon, M.C. Shin, K. Lee, V.C. Yang, Cell-penetrating peptides meditated encapsulation of protein therapeutics into intact red blood cells and its application, J. Control. Release 176C (2014) 123–132. [72] Y. Huang, Y. Jiang, H. Wang, J. Wang, M.C. Shin, Y. Byun, H. He, Y. Liang, V.C. Yang, Curb challenges of the “Trojan horse” approach: smart strategies in achieving effective yet safe cell-penetrating peptide-based drug delivery, Adv. Drug Deliv. Rev. 65 (10) (2013) 1299–1315. [73] S.R. Schwarze, A. Ho, A. Vocero-Akbani, S.F. Dowdy, In vivo protein transduction: delivery of a biologically active protein into the mouse, Science 285 (5433) (1999) 1569–1572. [74] G.P. Dietz, M. Bahr, Peptide-enhanced cellular internalization of proteins in neuroscience, Brain Res. Bull. 68 (1–2) (2005) 103–114. [75] E.M. Barnett, B. Elangovan, K.E. Bullok, D. Piwnica-Worms, Selective cell uptake of modified Tat peptide-fluorophore conjugates in rat retina in ex vivo and in vivo models, Invest. Ophthalmol. Vis. Sci. 47 (6) (2006) 2589–2595. [76] N. Kamei, M. Morishita, Y. Eda, N. Ida, R. Nishio, K. Takayama, Usefulness of cellpenetrating peptides to improve intestinal insulin absorption, J. Control. Release 132 (1) (2008) 21–25. [77] N. Kamei, M. Morishita, K. Takayama, Importance of intermolecular interaction on the improvement of intestinal therapeutic peptide/protein absorption using cell-penetrating peptides, J. Control. Release 136 (3) (2009) 179–186. [78] L.B. Lopes, C.M. Brophy, E. Furnish, C.R. Flynn, O. Sparks, P. Komalavilas, L. Joshi, A. Panitch, M.V. Bentley, Comparative study of the skin penetration of protein transduction domains and a conjugated peptide, Pharm. Res. 22 (5) (2005) 750–757. [79] M.C. Shin, J. Zhang, K.A. Min, K. Lee, Y. Byun, A.E. David, H. He, V.C. Yang, Cellpenetrating peptides: achievements and challenges in application for cancer treatment, J. Biomed. Mater. Res. A 102 (2) (2014) 575–587. [80] F. Wang, Y. Wang, X. Zhang, W. Zhang, S. Guo, F. Jin, Recent progress of cellpenetrating peptides as new carriers for intracellular cargo delivery, J. Control. Release 174 (2014) 126–136. [81] F. Heitz, M.C. Morris, G. Divita, Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics, Br. J. Pharmacol. 157 (2) (2009) 195–206. [82] R. Brasseur, G. Divita, Happy birthday cell penetrating peptides: already 20 years, Biochim. Biophys. Acta 1798 (12) (2010) 2177–2181. [83] S. Deshayes, M.C. Morris, G. Divita, F. Heitz, Cell-penetrating peptides: tools for intracellular delivery of therapeutics, Cell. Mol. Life Sci. 62 (16) (2005) 1839–1849. [84] T. Lehto, K. Kurrikoff, U. Langel, Cell-penetrating peptides for the delivery of nucleic acids, Expert. Opin. Drug Deliv. 9 (7) (2012) 823–836. [85] P. Jarver, K. Langel, S. El-Andaloussi, U. Langel, Applications of cell-penetrating peptides in regulation of gene expression, Biochem. Soc. Trans. 35 (Pt4) (2007) 770–774. [86] Y.J. Chen, B.R. Liu, Y.H. Dai, C.Y. Lee, M.H. Chan, H.H. Chen, H.J. Chiang, H.J. Lee, A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides, Gene 493 (2) (2012) 201–210. [87] A. Chugh, F. Eudes, Cellular uptake of cell-penetrating peptides pVEC and transportan in plants, J. Pept. Sci. 14 (4) (2008) 477–481. [88] G.P. Dietz, M. Bahr, Synthesis of cell-penetrating peptides and their application in neurobiology, Methods Mol. Biol. 399 (2007) 181–198. [89] S.T. Henriques, J. Costa, M.A. Castanho, Translocation of beta-galactosidase mediated by the cell-penetrating peptide pep-1 into lipid vesicles and human HeLa cells is driven by membrane electrostatic potential, Biochemistry 44 (30) (2005) 10189–10198. [90] T. Jiang, E.S. Olson, Q.T. Nguyen, M. Roy, P.A. Jennings, R.Y. Tsien, Tumor imaging by means of proteolytic activation of cell-penetrating peptides, Proc. Natl. Acad. Sci. 101 (51) (2004) 17867–17872. [91] C. Dohmen, E. Wagner, Multifunctional CPP polymer system for tumor-targeted pDNA and siRNA delivery, Methods Mol. Biol. 683 (2011) 453–463. [92] K. Splith, I. Neundorf, Antimicrobial peptides with cell-penetrating peptide properties and vice versa, Eur. Biophys. J. 40 (4) (2011) 387–397. [93] J.W. Hu, B.R. Liu, C.Y. Wu, S.W. Lu, H.J. Lee, Protein transport in human cells mediated by covalently and noncovalently conjugated arginine-rich intracellular delivery peptides, Peptides 30 (9) (2009) 1669–1678. [94] J.S. Wadia, S.F. Dowdy, Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer, Adv. Drug Deliv. Rev. 57 (4) (2005) 579–596. [95] S.B. Fonseca, M.P. Pereira, S.O. Kelley, Recent advances in the use of cellpenetrating peptides for medical and biological applications, Adv. Drug Deliv. Rev. 61 (11) (2009) 953–964. [96] G.P. Dietz, M. Bahr, Delivery of bioactive molecules into the cell: the Trojan horse approach, Mol. Cell. Neurosci. 27 (2) (2004) 85–131. [97] M. Chang, J.C. Chou, C.P. Chen, B.R. Liu, H.J. Lee, Noncovalent protein transduction in plant cells by macropinocytosis, New Phytol. 174 (1) (2007) 46–56. [98] Y.W. Hou, M.H. Chan, H.R. Hsu, B.R. Liu, C.P. Chen, H.H. Chen, H.J. Lee, Transdermal delivery of proteins mediated by non-covalently associated arginine-rich intracellular delivery peptides, Exp. Dermatol. 16 (12) (2007) 999–1006.

U

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[135] J.S. Suh, J.Y. Lee, Y.J. Choi, H.K. You, S.D. Hong, C.P. Chung, Y.J. Park, Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis, Int. J. Nanomed. 9 (2014) 1153–1166. [136] J.H. Lee, I.H. Bae, J.K. Choi, J.W. Park, Evaluation of a highly skin permeable lowmolecular-weight protamine conjugated epidermal growth factor for novel burn wound healing therapy, J. Pharm. Sci. 102 (11) (2013) 4109–4120. [137] H. Xia, G. Gu, Q. Hu, Z. Liu, M. Jiang, T. Kang, D. Miao, Q. Song, L. Yao, Y. Tu, H. Chen, X. Gao, J. Chen, Activatable cell penetrating peptide-conjugated nanoparticles with enhanced permeability for site-specific targeting delivery of anticancer drug, Bioconjug. Chem. 24 (3) (2013) 419–430.

N

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E

C

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E

D

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O

F

[131] Y.S. Park, Y. Huang, Y.J. Park, A.E. David, L. White, H. He, H.S. Chung, V.C. Yang, Specific down regulation of 3T3-L1 adipocyte differentiation by cell-permeable antisense HIF1alpha-oligonucleotide, J. Control. Release 144 (1) (2012) 82–90. [132] J.S. Suh, J.Y. Lee, Y.S. Choi, F. Yu, V.C. Yang, S.J. Lee, C.P. Chung, Y.J. Park, Efficient labeling of mesenchymal stem cells using cell permeable magnetic nanoparticles, Biochem. Biophys. Res. Commun. 379 (3) (2009) 669–675. [133] S.B. McDonald, M. Renna, E.L. Spitznagel, M. Avidan, C.W. Hogue, M.R. Moon, B. Barzilai, R. Saleem, J.M. McDonald, G.J. Despotis, Preoperative use of enoxaparin increases the risk of postoperative bleeding and re-exploration in cardiac surgery patients, J. Cardiothorac. Vasc. Anesth. 19 (1) (2005) 4–10. [134] N. Do, G. Weindl, L. Grohmann, M. Salwiczek, B. Koksch, H.C. Korting, M. SchäferKorting, Cationic membrane-active peptides — anticancer and antifungal activity as well as penetration into human skin, Exp. Dermatol. 23 (5) (2014) 326–331.

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