Pluronic® block copolymers: novel functional molecules for gene therapy

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Advanced Drug Delivery Reviews 54 (2002) locate /drugdelivPluronic block copolymers: novel functional molecules forgene therapya , b a b*Alexander V. Kabanov , Pierre Lemieux , Sergey Vinogradov , Valery AlakhovaDepartment of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha,NE 68198, USAbSupratek Pharma Inc., 531 Blvd. des Prairies, Build. 18, Laval, Quebec H7B 1B7, CanadaAbstractPluronic block copolymers are recognized pharmaceutical excipients listed in the US and British Pharmacopoeia. Theyhave been used extensively in a variety of pharmaceutical formulations including delivery of low molecular mass drugs andpolypeptides. This review describes novel applications of Pluronic block copolymers in gene therapy. In particular, thesemolecules can modify the biological response during gene therapy in the skeletal muscle, resulting in an enhancement of thetransgene expression as well as an enhancement of the therapeutic effect of the transgene. Furthermore, Pluronic blockcopolymers are versatile molecules that can be used as structural elements of the polycation-based gene delivery systems(polyplexes). Based on these studies, the use of block copolymers in gene delivery is a promising area of research, in whichnew and important developments are expected. 2002 Elsevier Science B.V. All rights reserved.Keywords: Gene delivery; Gene therapy; DNA; Polycation; Block copolymer; Poly(ethylene glycol)Contents1. Introduction ............................................................................................................................................................................ 2232. Structure and synthesis of Pluronic block copolymers .............................................................................................................. 2243. Self-assembly of Pluronic block copolymers ........................................................................................................................... 2244. Pluronic block copolymers as biological adjuvants .................................................................................................................. 2255. Enhancement of transgene expression in skeletal muscle by Pluronic .............................................................................. 2256. Pluronic -polycation conjugates for gene delivery ..................................................................................................................... 2277. Pluronic -polycation conjugates for delivery of oligonucleotides ................................................................................................ 2298. Conclusion ............................................................................................................................................................................. 230Acknowledgements ...................................................................................................................................................................... 230References .................................................................................................................................................................................. 2311. IntroductionThe field of non-viral gene therapy has recently*Corresponding author. Tel.: 11-402-559-9364; fax: 11-402-gained increased interest [1]. It is widely believed559-9543.E-mail address: (A.V. Kabanov). that non-viral gene therapy can overcome some0169-409X/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0169-409X( 02 )00018-2224 A.V. Kabanov et al. / Advanced Drug Delivery Reviews 54 (2002) 223 233problems inherent to current viral-based therapies, results in an amphiphilic copolymer, in which theincluding immune and toxic reactions as well as the number of hydrophilic EO (x) and hydrophobic POpotential for viral recombination [2]. One major ( y) units can be altered to vary the size, hydro-approach in non-viral gene therapy is based on philicity and lipophilicity. The structure formula ofpolyplexes, complexes formed by mixing DNA Pluronic block copolymers is presented in Fig. 1.with synthetic polycations [3,4]. The polyplexes Copolymers with various x and y values are char-form spontaneously as a result of electrostatic inter- acterized by distinct hydrophiliclipophilic balanceactions between the positively charged groups of the (HLB). The nomenclature of Pluronic block co-polycation and the negatively charged phosphate polymers is explained in Appendix A.groups of the DNA. This results in DNA condensa- Pluronic block copolymers are synthesized bytion, protection from the nuclease digestion, and sequential polymerization of PO and EO monomersmore efficient delivery within a cell. A variety of in the presence of an alkaline catalyst, such aspolycation molecules have been proposed for poly- sodium or potassium hydroxide [8]. The initial stageplex formation [3,4]. These molecules differ in the of the synthesis includes growth of the PO blockchemical composition and the number of the repeat- followed by the growth of EO chains at both ends ofing units, as well as in the architecture of the the PO block. Anionic polymerization usuallypolymer backbone, which may be linear, randomly produces polymers with a low polydispersity indexbranched, dendrimeric, block- or graft copolymer. (M /M ). However, the commercially availablen wAn alternative approach for gene delivery evaluates Pluronic preparations may contain admixtures ofnonionic polymers, such as poly(vinyl pyrrolidone), the PO homopolymer as well as di- and triblockwhich enhance gene expression of naked DNA in copolymers, exhibiting lower degrees of polymeri-select tissues, such as skeletal muscle [5,6]. In zation than expected. Chromatographic fractionationcontrast to polyplexes such nonionic polymers en- can be employed in procedures for the manufacturehance gene expression through mechanisms, which of highly purified block copolymers [9]. This reducesmost likely do not involve DNA condensation and the presence of admixtures, particularly, of the POfacilitated transport within cells [7]. The current homopolymer and of the block copolymers con-review describes a novel nonionic polymer class, taining less of the EO block than expected.Pluronic block copolymers, which appear to bevery valuable for gene delivery in skeletal muscle.Furthermore, Pluronic block copolymers haveproven to be useful elements in polyplexes on the 3. Self-assembly of Pluronic block copolymersbase of the polycation and DNA complexes that havea potential in a variety of gene therapy applications. A defining property of Pluronic is the ability ofBoth types of applications of the Pluronic blockcopolymers for gene delivery are considered alongwith some major aspects of self-assembly and bio-logical properties of these block copolymers, whichare essential for the development of such genedelivery systems.2. Structure and synthesis of Pluronic blockcopolymersPluronic block copolymers (also termed Polox-amer or Synperonic) consist of ethylene oxide Fig. 1. Pluronic block copolymers available from BASF (Wyan-(EO) and propylene oxide (PO) blocks arranged in a dotte, MI), contain two hydrophilic EO blocks and a hydrophobictriblock structure: EO PO EO . This arrangement PO block.x y xA.V. Kabanov et al. / Advanced Drug Delivery Reviews 54 (2002) 223 233 225individual block copolymer molecules, termed un- 4. Pluronic block copolymers as biologicalimers, to self-assemble into micelles in aqueous adjuvantssolutions. These unimers form a molecular disper-In addition to the use of Pluronic block co-sion in water at block copolymer concentrationspolymers as structural components of micellar drugbelow the critical micelle concentration (CMC). Atformulations, Pluronic -based systems exhibit aconcentrations of the block copolymer above thevariety of useful biological properties. For example,CMC, the unimer molecules aggregate, formingwater-in-oil, oil-in-water and water-in-oil-in-watermicelles through a process called micellization. Theemulsions formulated with select Pluronic blockdriving force for the micellization is the hydrophobiccopolymers have been used extensively as immuno-interactions of the PO blocks. The PO blocks self-adjuvants [2031]. These studies suggested signifi-assemble into the inner core of the micelles covered cant enhancement of both cell-mediated and humoralby the hydrophilic corona from EO blocks. Pluronicimmune response induced by addition of the blockmicelles are commonly pictured as spheres com-copolymer formulations with respect to a very broadposed of a PO core and an EO corona. This portrayalspectrum of antigens. Selected block copolymers,is correct for most block copolymers, which have ansuch as Pluronic F127 have been found to sig-EO content above 30%, especially in relatively dilutenificantly enhance the rate of wound and burnsolutions at body temperature. However, additionalhealing, and therefore have been included in creammicelle morphologies, including lamella and rods formulations and skin substitutes for the treatment of(cylinders), can also form in Pluronic systems [10].burns and for other tissue engineering applicationsWhen spherical micelles are formed, depending on [3239]. Several Pluronic -based formulations werethe Pluronic type the micelles commonly have anshown to effectively prevent postoperative adhesionsaverage hydrodynamic diameter ranging from aboutor at least to reduce adhesion area after surgery20 to about 80 nm [10]. The number of block[40,41]. Furthermore, Pluronic block copolymerscopolymer unimers forming one micelle is referredcan enhance sealing of cell membranes permeabil-to as the aggregation number. Usually this numberized by ionizing radiation and electroporation thusranges from several to over a hundred.preventing cellular necrosis, which can be helpful forThe process of transfer of water-insoluble com-improving drug and gene delivery in skeletal musclepounds into the PO core of the micellar solution is [4244]. Recent studies have demonstrated thatreferred to as solubilization. Pluronic micellesselect Pluronic block copolymers can interact withcontaining solubilized low molecular mass drugs andmultidrug resistant (MDR) cancer cells resulting inpolypeptides are being actively investigated as po-chemosensitization of these cells [4549]. Thesetential drug delivery systems [1119]. The core-shellinteractions involve alteration of the structure of cellarchitecture of polymeric micelles is essential formembranes by block copolymer molecules as well astheir utility for these applications. The core formedenergy depletion in MDR cells leading to the inhibi-by the PO chains is a water-incompatible compart-tion of various energy-dependent drug resistancement that is segregated from the aqueous exterior bymechanisms. Therefore, formulation of many anti-the hydrophilic chains of the EO corona, therebycancer drugs with such block copolymers results informing, within the core, a cargo hold for theenhancement of the chemotherapy of cancer [45,50].incorporation of various therapeutic reagents. As aThese examples show that the block copolymersresult, polymeric micelles can be used as efficientexhibit valuable biological activities, which can be ofcarriers for compounds, which alone exhibit poorconsiderable importance for various therapeutic ap-solubility, undesired pharmacokinetics and lowplications.stability in a physiological environment. The hydro-philic shell contributes greatly to the pharmaceuticalbehavior of block copolymer formulations by main- 5. Enhancement of transgene expression intaining the micelles in a dispersed state, as well as by skeletal muscle by Pluronicdecreasing undesirable drug interactions with cellsand proteins through steric-stabilization effects. One of the current and potentially successful226 A.V. Kabanov et al. / Advanced Drug Delivery Reviews 54 (2002) 223 233approaches in gene therapy involves the direct either the myoblast or the myofiber developmentalinjection of Pluronic block copolymers in combina- stages of the murine muscle cell line, C2C12, whention with a plasmid DNA in skeletal muscle tissue, compared to plasmid DNA alone. However, forfor example, to enhance immunization during DNA intramuscular administration, SP1017 was shown tovaccination. It is well known that plasmid DNA can promote expression of both reporter and therapeuticbe injected into skeletal muscle and generate thera- genes [61,62]. Importantly, the dose-dependencypeutically meaningful levels of gene expression [51]. study suggested that maximal stimulation of geneThis application of naked DNA was demonstrated expression with this formulation was observed at ausing therapeutic genes encoding (1) systemically relatively low concentration of the block copolymeracting secreted proteins, such as erythropoietin (0.01% wt.), which provides for at least a 500-fold(EPO) and interleukin-5 [52,53], and (2) locally safety margin in animals. Recent data demonstratedacting proteins, such as basic fibroblast growth that plasmid DNAs driven by either CMV- or NFkB-factor, vascular endothelial growth factor and responsive elements were considerably more respon-dystrophin [5456]. However, in many cases the sive to the SP1017 effect than plasmid DNAs thatrelatively low level of gene expression achieved were under the control of either SV-40- or an AP-1-limits the applicability of naked DNA as a therapeu- response element cassette [62]. Such promoter de-tic agent. Furthermore, commercially available cat- pendence is unusual and it suggests that SP1017 mayionic carriers, such as cationic dendrimers and lipids, act as a synthetic biological response modifier,that are known to improve gene expression in other probably by affecting mechanisms of the transcrip-tissues, significantly inhibit intramuscular gene ex- tional control of the transgene expression. Comparedpression (unpublished data). to poly(vinyl pyrrolidone), SP1017 is more efficientTherefore, an alternative approach has been to and requires less DNA to produce the same amountidentify compounds that can enhance gene expres- of transgene [61].sion in the muscle. One such compound, poly(vinyl The capacity of SP1017 to enhance plasmid-drivenpyrrolidone) has been widely discussed in the litera- expression of secreted therapeutic genes was clearlyture as a potential booster of the gene expression illustrated in experiments carried out using theusing the naked DNA [57,5760]. This polymer murine EPO gene [61]. The physiological effect thatdoes not condense the DNA at the concentrations resulted from the expression of the transgeneused, which is a marked distinction compared to the (mEPO) was readily quantifiable by simply measur-cationic polymers, which all bind electrostatically to ing the hematocrit levels from the blood of thethe DNA molecule and cause its condensation. treated mice. In this study, the hematocrits of miceConsequently, the mechanism of the poly(vinyl injected with 10 mg of the mEPO-expressing plasmidpyrrolidone) appears to be quite different from that formulated with SP1017, increased to 5864% com-of the polycations [7]. pared to 4562% observed in the control non-treatedIt was recently discovered that certain Pluronic mice [61]. In contrast, the hematocrits of miceblock copolymers significantly increase expression of injected with the same dose of naked pCMV-mEPOplasmid DNA in skeletal muscle in mice [61]. A (without SP1017 formulation) appeared slightly high-formulation based on the mixture of the block er than those of the control non-treated animals,copolymers, Pluronic L61 and Pluronic F127, has however the results were not statistically significantbeen identified that increases gene expression by in this study.520-fold compared to naked DNA. This formula- Furthermore, the hematocrits of mice injected with1tion was termed SP1017. Unlike cationic DNA pCMV-mEPO formulated with SP1017 were sus-carriers, and like poly(vinyl pyrrolidone), SP1017 tained for a long period of time, from 21 to 50 daysdoes not condense DNA. Furthermore, this formula- post-injection. The increase in hematocrits obtainedtion does not improve the in vitro transfection of with pCMV-mEPO/SP1017-treated mice correlatedwith an increase in mEPO levels in both the serumand in the muscle, as detected by ELISA on day 71Supratek Pharma Inc., Montreal, Canada. post-injection. In contrast, animals treated withA.V. Kabanov et al. / Advanced Drug Delivery Reviews 54 (2002) 223 233 227naked pCMV-mEPO showed a small, but significant EL and peanut oil. It is noteworthy that the vehiclesincrease in the mEPO level in muscle, while in the tested in this study contained 25% wt. Pluronicserum of these animals, the levels of mEPO re- gels, which is over a thousand times higher than themained below the sensitivity of the detection assay. concentrations of block copolymers used in the geneThe results of the dose escalation study showed that delivery applications considered in this section.the hematocrits of mice treated with 50 mg of Therefore, as far as intramuscular administration ofpCMV-mEPO/SP1017 reached 7362%, which is block copolymers is concerned, the Pluronic -basedcomparable to the level achievable with adeno-asso- gene delivery systems may have a very substantialciated virus [63]. safety margin. These studies demonstrate thatWhile the mechanism of the effects of SP1017 in Pluronic block copolymers are promising agents forthe applications described above is still being ex- non-viral gene therapy. These agents provide for aplored, it is noteworthy that the effects of SP1017 on simple and efficient gene transfer method potentiallytransgene expression are observed in vivo only, applicable in multiple gene therapy protocols involv-using an animal model, but not in vitro, in cell ing intramuscular injection of plasmid DNA in orderculture. This suggests that SP1017 may exhibit its to synthesize therapeutic proteins or to vaccinateactivity through interactions with some molecules or against a particular gene product.cells involved in the control of gene expression inthe body, which are not present in vitro. Futurestudies should identify such targets of Pluronic 6. Pluronic -polycation conjugates for geneaction. Furthermore, the fact that SP1017 exhibits deliverymaximal activity for intramuscular administration ata concentration that is close to the CMC values of Some recent reports suggest that Pluronic blockboth of the block copolymers present in the SP1017 copolymers can be useful as the components of novelmixture indicates that Pluronic unimers are in- self-assembling gene delivery systems. Astafieva etvolved in this activity. In this respect, the molecular al. [65] have demonstrated that Pluronic blockinteractions underlying the activity of Pluronic in copolymers can enhance polycation-mediated genegene therapy might be similar to the interactions transfer in vitro. In this study, a synthetic polycation,involved in the block copolymer activity in MDR poly(N-ethyl-4-vinylpyridinium bromide) was usedcells, such as effects on energy conservation mecha- to prepare complexes with a plasmid DNA, and thennisms, changes in the structure and dynamics of these complexes were evaluated for DNA intracellu-biological membranes and effects on intracellular lar uptake and transgene expression in cell culturetransport systems. models. Poly(N-ethyl-4-vinylpyridinium bromide) isThe toxicological aspects of injecting Pluronic a relatively low efficient gene transfer vector com-block copolymers in muscle tissue is of considerable pared to some more recently developed polycations,interest in view of the gene delivery applications. such as polyethyleneimine (e.g. ExGen 500) orThe muscle toxicity, caused by injection of both polyamidoamine dendrimers (e.g. SuperfectE) [66].single and multiple doses of various block copoly- However, when the poly(N-ethyl-4-vinylpyridiniummers, was evaluated by morphological examination bromide) and DNA were mixed with 1% Pluronicof the muscle tissue and by monitoring creatine P85 and then the cells were exposed to the resultingphosphokinase levels [64]. This study concluded that formulation, both the DNA uptake in the cells asthe toxicity of the block copolymers was propor- well as the transgene expression were significantlytional to their lipophilicity; the more lipophilic the increased compared to the cells treated with thecopolymer, the more severe the lesions. Overall, poly(N-ethyl-4-vinylpyridinium bromide) and DNA Pluronic F88 and Pluronic F127 were considered complex alone [65]. A recent study reported that thesuitable for incorporation into gel formulations, receptor-mediated gene delivery to hepatic cell line,while Pluronic P105 and Pluronic P123 appeared HepG2 using complexes of a plasmid DNA with anto be more toxic than other accepted vehicles for asialo-oroso-mucoid-poly(L-lysine) conjugate was in-intramuscular use, such as normal saline, Cremophor creased fourfold in the presence of Pluronic F127228 A.V. Kabanov et al. / Advanced Drug Delivery Reviews 54 (2002) 223 233[67]. Similarly, an additional increase of the transfec- organs, allowing for a significant improvement ofgene expression in liver.tion efficiency was demonstrated in the presence of A recent study evaluated several non-viral trans-Pluronic F127 when the cervical cancer cell line,fection systems based on the complexes of DNA andC-33A was transfected with the polycation /DNA polycations (polyplexes) with respect to their effec-complex [68]. It is interesting that Pluronic F127tiveness, toxicity and cell type dependence in aenhanced the transgene expression exhibited by thevariety of in vitro models [66]. The panel of polyca-complexes whereas the agents that are frequentlytions examined included branched and linear poly-used to boost the DNA release from the endosomes,ethyleneimines (25 kDa and 50 kDa), poly(N-ethyl-fusogenic peptide HA2 and chloroquine, had no4-vinylpyridinium bromide), polyamidoamine den-effects on the transgene expression [67].drimer (SuperfectE), poly(propyleneimine) dendri-In another study, a block-graft copolymer syn-mer (AstramolE) and a P123-g-PEI(2K) graft-blockthesized by covalent conjugation of Pluronic P123copolymer. Using a panel of cell lines the linearand branched polyethyleneimine (P123-g-PEI(2K))polyethyleneimine ExGenE 500, SuperfectE, bran-was evaluated as a gene transfer vector in vitro andched polyethyleneimine 25 kDa, and P123-g-in vivo [69]. This conjugate mixed with free PEI(2K) were determined as systems displayingPluronic P123 formed small and stable complexeshighest transfection activity while exhibiting rela-with DNA (110 nm) that exhibited high transfectiontively low cytotoxicity. These systems had activityactivity in vitro, comparable to that of SuperfectE.higher than or comparable to lipid transfectionFurthermore, gene expression was observed inreagents (Lipofectin , LipofectAMINEE,spleen, heart, lungs and liver 24 h after intravenousinjection of this complex in mice. Compared to CeLLFECTIN and DMRIE-C) but did not reveal1,2-bis(oleoyloxy)-(trimethylammonio) propane serum dependence and were less toxic than the(DOTAP)-cholesterol and branched poly- lipids. This study demonstrated good potential ofethyleneimine (25 kDa) transfection systems, the P123-g-PEI(2K) graft-block copolymer based poly-block-graft copolymer system revealed a more uni- plex system as transfection reagents with relativelyform distribution of gene expression between these low cytotoxicity. As is shown in Fig. 2 this systemFig. 2. Relative transfection activity of various polyplexes in Cos-7 cells using luciferase reporter gene. Data represents the range from thelowest to the highest values of luciferase expression (ng/mg) obtained following transfection with a given polyplex over a series ofexperiments [66]. PEI, polyethyleneimines; PEVP, poly(N-ethyl-4-vinylpyridinium bromide), polyamidoamine dendrimer (SuperfectE);Am-64, AstramolE.A.V. Kabanov et al. / Advanced Drug Delivery Reviews 54 (2002) 223 233 229ranks among the best in vitro transfection systems. [7176]. In particular, cationic copolymers, such as,One major advantage of this system is that the for example, poly(ethylene oxide)-g-poly-complexes formed by P123-g-PEI(2K) and DNA in ethyleneimine, PEO(8K)-g-PEI(2K), spontaneouslythe presence of the free Pluronic are stable in a form polyelectrolyte complexes with phosphorothio-variety of conditions particularly in the presence of ate and phosphodiester oligonucleotides. These struc-serum proteins [69]. In contrast, the complexes tures are described as coreshell micelle-like par-formed by homopolymer polyethyleneimine or poly- ticles with a core from the neutralized oligonucleo-amidoamine dendrimers reveal the tendency for tide /polycation complex and a shell from poly-aggregation particularly in the presence of serum (ethylene oxide) chains attached to poly-(Table 1). ethyleneimine. This type of system has been success-High stability of the block-graft copolymer-based fully used for the in vivo delivery of oligonucleotidescomplexes is evidently due to formation of the [74]. More recently, a block-graft copolymer ofmicelle-like structures, which include the DNA, Pluronic P85 and polyethyleneimine, P85-g-polycation and block copolymer chains. Electrostatic PEI(2K), was used for preparing oligonucleotidebinding of the DNA and polyethyleneimine chains of formulations [77]. Like in the case of the plasmidP123-g-PEI(2K) results in neutralization of the DNA described in the previous section the oligo-polyion charges. The zeta-potential of resulting nucleotide formulation also contained free Pluroniccomplexes is close to zero, yet the complexes form P85 to stabilize the dispersion. The organ accumula-stable dispersions. Therefore, it is likely that these tion of antisense oligonucleotides formulated withsystems are stabilized in dispersion by the EO corona P85-g-PEI(2K) was recently examined. Followingin a manner similar to regular Pluronic micelles. i.v. administration oligonucleotide formulated withFurthermore, the self-assembly of these materials is PEO(8K)-g-PEI(2K) accumulated mainly in kidneys,likely to involve the hydrophobic interactions of the while the same oligonucleotide formulated with P85-PO chain segments. It is noteworthy, that the free g-PEI(2K) was found almost exclusively in the liver.Pluronic , a component shown to be essential for Furthermore, in the case of the animals injected withexhibiting high transfection activity, is also neces- the P85-g-PEI(2K)-based complexes most of thesary for formation of a stable dispersion from DNA ODN was found in hepatocytes, while only a minorand P123-g-PEI(2K) [70]. portion of oligonucleotide was found in the lympho-cyte /monocyte populations. The results of this studysuggest that formulating oligonucleotide with7. Pluronic -polycation conjugates for delivery PEO(8K)-g-PEI(2K) and P85-g-PEI(2K) carriersof oligonucleotides allows to redirect ODN to the liver or kidneys,respectively. The variation in the tissue distributionBeside plasmid DNA, block copolymers have of ODN observed with the two carriers is probablybeen used to formulate and deliver oligonucleotides due to different hydrophiliclipophilic balances ofTable 1Effect of buffer components and serum on the size of the polyplexes [69]Polycation Buffer Particle diameter, nm1 Serum 2 SerumPolyethyleneimine, 25 kDa 10 mM phosphate 850 1100PBS 460 380DMEM 580 140P123-g-PEI(2K) 10 mM phosphate 260 250PBS 270 270DMEM 320 215230 A.V. Kabanov et al. / Advanced Drug Delivery Reviews 54 (2002) 223 233the polyether chains grafted to polyethyleneimine in already suggest that Pluronic block copolymers arethese molecules. Therefore, cationic block copoly- promising agents for utilization in gene therapymers provide a simple way to enhance oligonucleo- applications. Firstly, it appears that a least some oftide accumulation to selected compartments without these molecules can modify the biological responsethe need of a specific targeting moiety. during gene therapy, resulting in an enhancement ofThus these systems appear to be similar to polyion the transgene expression as well as an enhancementcomplex micelles from DNA and cationic block of the therapeutic effect of the transgene. Secondly,copolymers proposed for gene delivery by several Pluronic block copolymers are versatile moleculesinvestigators, which have a core from neutralized that can be used as structural elements in novelDNA and polycation chains and a corona from self-assembling gene delivery systems that may behydrophilic nonionic chains such as EO. However, in superior compared to currently known vectors. Basedthe case of P123-g-PEI(2K)-based complexes, the on these studies, the use of block copolymers in genenonionic Pluronic chains also carry hydrophobic delivery is a very exciting area of research in whichPO components. The PO chains in Pluronic mole- new and important developments are expected in thecules are known to interact with lipid membranes near future.and induce structural rearrangements in the mem-branes. Furthermore, due to the presence of POchains Pluronic molecules exhibit the ability to Acknowledgementstranslocate within cells, while homopolymer EOdoes not exhibit this ability. Thus, P123-g-PEI(2K)- AVK acknowledges support from National Sciencebased complexes contain sticky elements, which, in Foundation (BES-9907281) and the Nebraska Re-essence, are similar to fusogenic sequences in the search Initiative Gene Therapy program. The authorsviruses, that allow for effective delivery of genetic have been co-founders, shareholders and/or em-material within a cell. Due to the presence of such ployees of Supratek Pharma Inc. (Montreal, PQ,elements P123-g-PEI(2K) copolymer is much more Canada).efficient in transporting DNA within the cells com-pared to the block- and graft-copolymers formed bypolycation and EO chains [70]. Furthermore, the Appendix A. Nomenclature of Pluronic blockpolycations modified by EO chains, e.g. poly- copolymers(ethylene oxide)-g-polyethyleneimine, are usuallymuch less efficient transfection agents than the Pluronic nomenclature includes one letter, F,unmodified polycations, e.g. polyethyleneimine. In P, or L, followed by a two- or three-digit numericcontrast, the polyplexes formed by P123-g-PEI(2K) code. The letters stand for solid (F), paste (P) orcopolymer have practically the same or comparable liquid (L). The numeric code defines the structuralactivity as the unmodified polycation. Overall parameters of the block copolymer. The last digit ofPluronic -based polyplexes, such as the P123-g- this code approximates the weight content of EOPEI(2K) graft-block copolymer complexes, are block in tens of weight percent (for example, 80%promising vectors for gene delivery, which have wt. if the digit is 8 or 10% wt. if the digit is 1). Thesignificant advantages compared to many other poly- remaining first one or two digits encode the molecu-plex systems. These advantages result from the fact lar mass of the central PO block. To decipher thethat the Pluronic -based polyplexes combine (1) code, one should multiply the corresponding numberhigh stability in dispersion with (2) high transfection by 300 to obtain the approximate molecular mass inactivity. Da. Therefore Pluronic nomenclature provides aconvenient approach to estimate the characteristics ofthe block copolymer in the absence of reference8. Conclusion literature. For example, the code F127 defines theblock copolymer, which is a solid, has a PO block ofIn conclusion, although the studies described in 3600 Da (123300) and 70% wt. of EO. The precisethis paper are relatively recent, the results obtained molecular characteristics of each Pluronic blockA.V. Kabanov et al. / Advanced Drug Delivery Reviews 54 (2002) 223 233 231carriers as versatile vehicles for drug delivery, Expert Opin.copolymer are provided by the manufacturer and canInvest. Drugs 7 (1998) 14531473.also be located in the literature [19]. [17] N. Rapoport, Stabilization and activation of Pluronic mi-celles for tumor-targeted drug delivery, Colloids Surf. B:Biointerfaces 16 (1999) 93111.[18] N.Y. Rapoport, J.N. Herron, W.G. Pitt, L. Pitina, MicellarReferences delivery of doxorubicin and its paramagnetic analog, rubox-yl, to HL-60 cells: effect of micelle structure and ultrasoundon the intracellular drug uptake, J. Control. Release 58[1] P. Felgner et al., Nomenclature for synthetic gene delivery(1999), Hum. Gene Ther. 8 (1997) 511512.[19] A.V. Kabanov, V.Y. Alakhov, Pluronic block copolymers in[2] R. Christiano, Viral and non-viral vectors for cancer genedrug delivery: from micellar nanocontainers to biologicaltherapy, Anticancer Res. 18 (1998) 32413246.response modifiers, Crit. Rev. Ther. Drug Carrier Syst., in[3] A.V. Kabanov, Taking polycation gene delivery systems vitro to in vivo, Pharm. Sci. Tech. Today 2 (1999)[20] R. Bomford, The adjuvant activity of fatty acid esters. The365372.role of acyl chain length and degree of saturation, Immunol-[4] S. De Smedt, J. Demeester, W. Hennink, Cationic polymerogy 44 (1981) 187192.based gene delivery systems, Pharm. Res. 5 (2000) 1425[21] R. Hunter, F. Strickland, F. Kezdy, The adjuvant activity of1433.nonionic block polymer surfactants. I. The role of hy-[5] A.P. Rolland, R.J. Mumper, Plasmid delivery to muscle:drophilelipophile balance, J. Immunol. 127 (1981) 1244recent advances in polymer delivery systems, Adv. Drug1250.Deliv. Rev. 30 (1998) 151172.[22] R.L. Hunter, B. Bennett, The adjuvant activity of nonionic[6] R.J. Mumper, J.G. Duguid, K. Anwer, M.K. Barron, H.block polymer surfactants. II. Antibody formation andNitta, A.P. Rolland, Polyvinyl derivatives as novel interactiveinflammation related to the structure of triblock and octab-polymers for controlled gene delivery to muscle, Pharm. Res.lock copolymers, J. Immunol. 133 (1984) 31673175.13 (1996) 701709.[23] R.L. Hunter, B. Bennett, The adjuvant activity of nonionic[7] R.J. Mumper, J. Wang, S.L. Klakamp, H. Nitta, K. Anwer, F.block polymer surfactants. III. Characterization of selectedTagliaferri, A.P. Rolland, Protective interactive noncondens-biologically active surfaces, Scand. J. Immunol. 23 (1986)ing (PINC) polymers for enhanced plasmid distribution and287300.expression in rat skeletal muscle, J. Control. Release 52[24] R. Hunter, M. Olsen, S. Buynitzky, Adjuvant activity of(1998) 191203.non-ionic block copolymers. IV. Effect of molecular weight[8] I.R. Schmolka, A review of block polymer surfactants, J.and formulation on titre and isotype of antibody, Vaccine 9Am. Oil Chem. Soc. 54 (1977) 110116.(1991) 250256.[9] M.R. Emanuele, M. Balasubramanian, H.S. Alludeen, Poly-[25] R.L. Hunter, J. McNicholl, A.A. Lal, Mechanisms of actionoxypropylene /polyoxyethylene Copolymers with Improvedof nonionic block copolymer adjuvants, AIDS Res. Hum.Biological Activity, CytRx Corporation, US, 1996.Retroviruses 10 (1994) S95S98.[10] R. Nagarajan, Solubilization of hydrocarbons and resulting[26] A.C. Allison, N.E. Byars, An adjuvant formulation thataggregate shape transitions in aqueous solutions of Pluronicselectively elicits the formation of antibodies of protective(PEOPPOPEO) block copolymers, Colloids Surf. B:isotypes and of cell-mediated immunity, J. Immunol. Meth-Biointerfaces 16 (1999) 5572.ods 95 (1986) 157168.[11] A.V. Kabanov et al., The neuroleptic activity of haloperidol[27] A.C. Allison, N.E. Byars, Adjuvant formulations and theirincreases after its solubilization in surfactant micelles. Mi-mode of action, Semin. Immunol. 2 (1990) 369374.celles as microcontainers for drug targeting, FEBS Lett. 258[28] N.E. Byars, A.C. Allison, Adjuvant formulation for use in(1989) 343345.vaccines to elicit both cell-mediated and humoral immunity,[12] A.V. Kabanov et al., A new class of drug carriers: micelles ofVaccine 5 (1987) 223228.poly(oxyethylene)poly(oxypropylene) block copolymers as[29] Y. Ke, C.L. McGraw, R.L. Hunter, J.A. Kapp, Nonionicmicrocontainers for drug targeting from blood in brain, J.triblock copolymers facilitate delivery of exogenous proteinsControl. Release 22 (1992) 141157.into the MHC class I and class II processing pathways, Cell[13] P.L. Wang, T.P. Johnston, Enhanced stability of two modelImmunol. 176 (1997) 113121.proteins in an agitated solution environment using poloxamer[30] P. Millet, M.L. Kalish, W.E. Collins, R.L. Hunter, Effect of407, J. Parenter. Sci. Technol. 47 (1993) 183189.adjuvant formulations on the selection of B-cell epitopes[14] P.L. Wang, T.P. Johnston, Thermal-induced denaturation ofexpressed by a malaria peptide vaccine, Vaccine 10 (1992)two model proteins: effect of poloxamer 407 on solution547550.stability, Int. J. Pharm. 96 (1993) 4149.[31] K. Takayama, M. Olsen, P. Datta, R.L. Hunter, Adjuvant[15] E.V. Batrakova et al., Anthracycline antibiotics non-cova-activity of non-ionic block copolymers. V. Modulation oflently incorporated into the block copolymer micelles: inantibody isotype by lipopolysaccharides, lipid A and pre-vivo evaluation of anti-cancer activity, Br. J. Cancer 74cursors, Vaccine 9 (1991) 257265.(1996) 15451552.[32] I.R. Schmolka, Artificial skin. I. Preparation and properties[16] V.Y. Alakhov, A.V. Kabanov, Block copolymeric biotransport232 A.V. Kabanov et al. / Advanced Drug Delivery Reviews 54 (2002) 223 233of pluronic F-127 gels for treatment of burns, J. Biomed. tion effect in MDR cancer cells, Pharm. Res. 16 (1999)Mater. Res. 6 (1972) 571582. 13731379.[33] G. Rodeheaver, V. Turnbull, M.T. Edgerton, L. Kurtz, R.F. [49] A. Venne, S. Li, R. Mandeville, A. Kabanov, V. Alakhov,Edlich, Pharmacokinetics of a new skin wound cleanser, Am. Hypersensitizing effect of pluronic L61 on cytotoxic activity,J. Surg. 132 (1976) 6774. transport, and subcellular distribution of doxorubicin inmultiple drug-resistant cells, Cancer Res. 56 (1996) 3626[34] G.T. Rodeheaver, L. Kurtz, B.J. Kircher, R.F. Edlich,3629.Pluronic F-68: a promising new skin wound cleanser, Ann.Emerg. Med. 9 (1980) 572576. [50] V. Alakhov, E. Klinski, S. Li, G. Pietrzynski, A. Venne, E.Batrakova, T. Bronitch, A.V. Kabanov, Block copolymer-[35] R.M. Nalbandian, R.L. Henry, K.W. Balko, D.V. Adams, N.R.based formulation of doxorubicin. From cell screen toNeuman, Pluronic F-127 gel preparation as an artificial skinclinical trials, Colloids Surf. B: Biointerfaces 16 (1999)in the treatment of third-degree burns in pigs, J. Biomed.113134.Mater. Res. 21 (1987) 11351148.[51] J.A. Wolff, R.W. Malone, P. Williams, W. Chong, G. Acsadi,[36] A.J. Gear, T.B. Hellewell, H.R. Wright, P.M. Mazzarese, P.B.A. Jani, P.L. Felgner, Direct gene transfer into mouse muscleArnold, G.T. Rodeheaver, R.F. Edlich, A new silver sul-in vivo, Science 247 (1990) 14651468.fadiazine water soluble gel, Burns 23 (1997) 387391.[52] M. Tokui et al., Intramuscular injection of expression[37] F. Follis, B. Jenson, K. Blisard, E. Hall, R. Wong, R. Kessler,plasmid DNA is an effective means of long-term systemicT. Temes, J. Wernly, Role of poloxamer 188 during recoverydelivery of interleukin-5, Biochem. Biophys. Res. Commun.from ischemic spinal cord injury: a preliminary study, J.233 (1997) 527531.Invest. Surg. 9 (1996) 149156.[38] M.S. Agren, An amorphous hydrogel enhances epithelialisa- [53] S.K. Tripathy, E.C. Svensson, H.B. Black, E. Goldwasser,tion of wounds, Acta Derm. Venereol. 78 (1998) 119122. M. Margalith, P.M. Hobart, J.M. Leiden, Long-term expres-sion of erythropoietin in the systemic circulation of mice[39] Y.L. Cao, E. Lach, T.H. Kim, A. Rodriguez, C.A. Arevalo,after intramuscular injection of a plasmid DNA vector, Proc.C.A. Vacanti, Tissue-engineered nipple reconstruction, Plast.Natl. Acad. Sci. USA 93 (1996) 1087610880.Reconstr. Surg. 102 (1998) 22932298.[54] R.J. Laham et al., Intracoronary basic fibroblast growth[40] A. Vlahos, P. Yu, C.E. Lucas, A.M. Ledgerwood, Effect of afactor (FGF-2) in patients with severe ischemic heart dis-composite membrane of chitosan and poloxamer gel onease: results of a phase I open-label dose escalation study, J.postoperative adhesive interactions, Am. Surg. 67 (2001)Am. Coll. Cardiol. 36 (2000) 21322139.1521.[55] K.G. Lathi, P.R. Vale, D.W. Losordo, R.M. Cespedes, J.F.[41] R.E. Leach, R.L. Henry, Reduction of postoperative adhe-Symes, D.D. Esakof, M. Maysky, J.M. Isner, Gene therapysions in the rat uterine horn model with poloxamer 407, Am.with vascular endothelial growth factor for inoperable cor-J. Obstet. Gynecol. 162 (1990) 13171319.onary artery disease: anesthetic management and results,[42] J. Hannig, D. Zhang, D.J. Canaday, M.A. Beckett, R.D.Anesth. Analg. 92 (2001) 1925.Astumian, R.R. Weichselbaum, R.C. Lee, Surfactant sealing[56] S. Braun et al., Immune rejection of human dystrophinof membranes permeabilized by ionizing radiation, Radiat.following intramuscular injections of naked DNA in mdxRes. 154 (2000) 171177.mice, Gene Ther. 7 (2000) 14471457.[43] R.C. Lee, D.J. Canaday, S.M. Hammer, Transient and stableionic permeabilization of isolated skeletal muscle cells after [57] J.G. Fewell, F. MacLaughlin, V. Mehta, M. Gondo, F. Nicol,electrical shock, J. Burn Care Rehabil. 14 (1993) 528540. E. Wilson, L.C. Smith, Gene therapy for the treatment ofhemophilia B using PINC-formulated plasmid delivered to[44] R.C. Lee, J. Hannig, K.L. Matthews, A. Myerov, C.T. Chen,muscle with electroporation, Mol. Ther. 3 (2001) 574583.Pharmaceutical therapies for sealing of permeabilized cellmembranes in electrical injuries, Ann. NY Acad. Sci. 888 [58] M.A. Morse, Technology evaluation: VEGF165 gene(1999) 266273. therapy, Valentis Inc, Curr. Opin. Mol. Ther. 3 (2001) 97101.[45] V.Y. Alakhov, E.Y. Moskaleva, E.V. Batrakova, A.V. Kabanov,Hypersensitization of multidrug resistant human ovarian [59] S.K. Mendiratta, A. Quezada, M. Matar, J. Wang, H.L.carcinoma cells by pluronic P85 block copolymer, Biocon- Hebel, S. Long, J.L. Nordstrom, F. Pericle, Intratumoraljug. Chem. 7 (1996) 209216. delivery of IL-12 gene by polyvinyl polymeric vector systemto murine renal and colon carcinoma results in potent[46] E.V. Batrakova, S. Li, V.Y. Alakhov, A.V. Kabanov, Selectiveantitumor immunity, Gene Ther. 6 (1999) depletion and sensitization of multiple drug resistantcancer cells by Pluronic block copolymers, Polym. Prepr. 41 [60] S.K. Mendiratta, A. Quezada, M. Matar, N.M. Thull, J.S.(2000) 16391640. Bishop, J.L. Nordstrom, F. Pericle, Combination of inter-leukin 12 and interferon alpha gene therapy induces a[47] E.V. Batrakova, S. Li, V.Y. Alakhov, A.V. Kabanov, Mecha-synergistic antitumor response against colon and renal cellnism of sensitization of MDR cancer cells by Pluronic blockcarcinoma, Hum. Gene Ther. 11 (2000) 18511862.copolymers: selective energy depletion, Br. J. Cancer, inpress. [61] P. Lemieux, N. Guerin, G. Paradis, R. Proulx, L.[48] E.V. Batrakova, S. Lee, S. Li, A. Venne, V. Alakhov, A. Chistyakova, A. Kabanov, V. Alakhov, A combination ofKabanov, Fundamental relationships between the composi- poloxamers increases gene expression of plasmid DNA intion of pluronic block copolymers and their hypersensitiza- skeletal muscle, Gene Ther. 7 (2000) 986991.A.V. Kabanov et al. / Advanced Drug Delivery Reviews 54 (2002) 223 233 233[62] V. Alakhov, E. Klinski, P. Lemieux, G. Pietrzynski, A. [71] A.V. Kabanov, S.V. Vinogradov, Yu.G. Suzdaltseva, V.Yu.Kabanov, Block copolymeric biotransport carriers as ver- Alakhov, Water-soluble block polycations for oligonucleotidesatile vehicles for drug delivery, Expert Opin. Biol. Ther. 1 delivery, Bioconjug. Chem. 6 (1995) 639643.(2001) 583602. [72] S.V. Vinogradov, T.K. Bronich, A.V. Kabanov, Self-assembly[63] S. Zhou, J.E. Murphy, J.A. Escobedo, V.J. Dwarki, Adeno- of polyaminepoly(ethylene glycol) copolymers with phos-associated virus-mediated delivery of erythropoietin leads to phorothioate oligonucleotides, Bioconjug. Chem. 9 (1998)sustained elevation of hematocrit in nonhuman primates, 805812.Gene Ther. 5 (1998) 665670. [73] S.V. Vinogradov, E. Batrakova, S. Li, A. Kabanov, Polyion[64] T.P. Johnston, S.C. Miller, Toxicological evaluation of complex micelles with protein-modified corona for receptor-poloxamer vehicles for intramuscular use, J. Parenter. Sci. mediated delivery of oligonucleotides into cells, Bioconjug.Technol. 39 (1985) 8389. Chem. 10 (1999) 851860.[65] I. Astafieva, I. Maksimova, E. Lukanidin, V. Alakhov, A. [74] S. Roy, K. Zhang, T. Roth, S. Vinogradov, R.S. Kao, A.Kabanov, Enhancement of the polycation-mediated DNA Kabanov, Reduction of fibronectin expression by intravitrealuptake and cell transfection with Pluronic P85 block co-administration of antisense oligonucleotides, Nat. Biotech-polymer, FEBS Lett. 389 (1996) 278280.nol. 17 (1999) 476479.[66] C.L. Gebhart, A.V. Kabanov, Evaluation of polyplexes as[75] K. Kataoka, H. Togawa, A. Harda, K. Yasugi, T. Matsumoto,gene transfer agents, J. Control. Release 73 (2001) 401416.S. Katayose, Spontaneous formation of polyion complex[67] C.W. Cho, Y.S. Cho, H.K. Lee, Y.I. Yeom, S.N. Park, D.Y.micelles with narrow distribution from antisense oligonu-Yoon, Improvement of receptor-mediated gene delivery tocleotide and cationic copolymer in physiological saline,HepG2 cells using an amphiphilic gelling agent, Biotechnol.Macromolecules 29 (1996) 85568557.Appl. Biochem. 32 (2000) 2126.[76] A. Harada, H. Togawa, K. Kataoka, Physicochemical prop-[68] C.W. Cho, Y.S. Cho, B.T. Kang, J.S. Hwang, S.N. Park, D.Y.erties and nuclease resistance of antisense-oligodeoxynucleo-Yoon, Improvement of gene transfer to cervical cancer celltides entrapped in the core of polyion complex micelleslines using non-viral agents, Cancer Lett. 162 (2001) 7585.composed of poly(ethylene glycol)poly(L-lysine) block[69] H.K. Nguyen et al., Evaluation of polyetherpoly-copolymers, Eur. J. Pharm. Sci. 13 (2001) 3542.ethyleneimine graft copolymers as gene transfer agents,Gene Ther. 7 (2000) 126138. [77] B. Ochietti, N. Guerin, S.V. Vinogradov, Y. St-Pierre, P.[70] C. Gebhart, S. Sridibahatla, S.V. Vinogradov, A.V. Kabanov, Lemieux, A.V. Kabanov, Yvu. Alakhov, Altered organ ac-Pluronicpolyethyleneimine conjugates for gene delivery: cumulation of oligonucleotides using polyethyleneiminecell transport and transgene expression, Polym. Preprints 42 grafted with poly(ethylene oxide) or pluronic as carriers. J.(2001) 119120. Drug Target., in press.


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