stimuli-responsive polymers in gene delivery

Review

10.1586/17434440.2.4.501 © 2005 Future Drugs Ltd ISSN 1743-4440 501www.future-drugs.com

Stimuli-responsive polymers in gene deliveryErhan Piskin

Hacettepe University, Chemical Engineering Department, Bioengineering Division,and TÜBTAK-ÜSAM-BiyomedtekBeytepe, 06800 Ankara, TurkeyTel.: +90 532 707 9468Fax: +90 312 440 [email protected]

KEYWORDS: gene therapy, nonviral vectors, stimuli-responsive polymers

Recent interest in clinical therapy has been directed to deliver nucleic acids (DNA, RNA or short-chain oligonucleotides) that alter gene expression within a specific cell population, thereby manipulating cellular processes and responses, which in turn stimulate immune responses or tissue regeneration, or blocks expression at the level of transcription or translation for treatment of several diseases. Both ex vivo and in vivo gene delivery can be achieved mostly by using a delivery system (vector). Viral vectors exhibit high gene expression, but also have very significant side effects. Mainly cationic polymeric systems are used as nonviral vectors, although usually with low levels of transfection. Through the use of stimuli-responsive polymers as novel vectors for gene delivery, two benefits can be obtained: high gene expression efficiency and more selective gene expression.

Expert Rev. Med. Devices 2(4), 501–509 (2005)

Gene delivery for therapyConventionally, gene therapy is used to treatgenetic disorders such as cystic fibrosis,hemophilia and cancer, by introducing newgenes in the target cells for the productionof therapeutic proteins necessary for thenormalization of cellular processes [1–3].Clinical gene therapy is currently limited byessential nonreproductive somatic cells, inwhich alterations are not heritable. How-ever, germline gene therapy is also possible,which involves alteration of the DNA of agamete or fertilized egg. Here, changes areheritable and pass from treated individual tooffspring. Currently, germline gene therapyis not allowed to be used in humans due tosocial and ethical problems; in addition,generally, gene therapy is neither sufficientlyeffective – or well controlled. Recent interesthas been expanded to the applications ofnucleic acid-based therapies to treat diseaseby delivering nucleic acids (e.g., DNA frag-ments, antisense oligonucleotides and shortinterfering RNA [siRNA]) that alter geneexpression within a specific cell population,thereby manipulating cellular processes andresponses, which in turn stimulates immuneresponses or tissue regeneration, or blocksexpression at the level of transcription or

translation for the treatment of several cardio-vascular, inflammatory, metabolic, infectiousand bone diseases, as well as in oncology [4–6].

There are three components in current genetherapy. First, a gene that encodes a specifictherapeutic protein. Second, a gene expressionplasmid DNA that controls the functioning ofa gene within a target cell. Gene expressionplasmids contain both the therapeutic proteinencoding the gene and several other geneticelements, including introns, polyadenylationsequences and transcript stabilizers to controltranscription, translation and protein stabilityand secretion from the host cells. Third, a genedelivery system that delivers the gene expres-sion plasmid DNA to specific locations withinthe body and into the target cells [2].

Delivery of plasmid DNA can be performedeither ex vivo or in vivo as schematicallydepicted in FIGURE 1. The ex vivo approachincludes the following steps:

• Isolation of healthy cells (specific target cells,or even cells differentiated from stem cells)from either the patient or a donor

• Intracellular uptake of plasmid DNA intothese cells in cell culture media

• Subsequently implanting the transfectedcells back to the patient

CONTENTS

Gene delivery for therapy

Rationale for a delivery system

Vectors for gene delivery

Gene delivery with stimuli-responsive polymers

Expert commentary & five-year view

Key issues

References

Affiliation

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Piskin

502 Expert Rev. Med. Devices 2(4), (2005)

In this approach, only internalization of the naked plasmidDNA or the delivery system that carries the plasmid throughthe target cell membrane and intracellular transport for nucleartranslocation should be considered for an effective delivery.

Ex vivo delivery of naked plasmid DNA can be achieved with-out using a carrier system. Numerous techniques are being inves-tigated for ex vivo delivery of naked plasmids, such as the genegun, hydrostatic pressure, electroporation, continuous infusion,sonication and photochemical internalization [7–10]. They pro-vide relatively high transfections; however, these procedures arecostly and may not be appropriate for all situations.

In the in vivo delivery, the delivery system carrying the trans-fecting DNA is directly introduced to the patient through sev-eral routes such as direct injection intravascularly or into severaltissues including skeletal muscle, liver, thyroid, heart muscle,urologic organs, skin and tumor sites. In this approach thedelivery systems are designed to control the effective distribu-tion and access of the system to the target cell, and/or preferen-tial recognition by a cell-surface receptor followed by intra-cellular uptake and nuclear translocation. The delivery systemsshould protect plasmid DNA from premature degradation inthe extracellular media and deliver it specifically to the targetcells. Other elements in a delivery system may facilitate theintracellular trafficking of the delivery system [9].

Systemic injection is also a convenient route for gene admin-istration. However, it is not a very successful approach as yet,owing to rapid degradation of DNA by nucleases in the serumin the case of delivery without a carrier system, and rapid clear-ance from the circulation mainly owing to extensive uptake byrestriction endonucleases (RES), especially by the liver via scav-enger receptors (for naked plasmid DNA). This results in theprevention of extravasation to any organ or tissue, which thenresults in low levels of transfection in all major organs [9].

In the in vivo approach, the delivery system can be adminis-trated directly to the target site or as close to the site of pathol-ogy as possible. Barriers not only at the intracellular, but also atthe extracellular level should be seriously considered. In addi-tion, the naked plasmid DNA injected directly into a tissuedrains rapidly into the lymphatics. Nucleases easily degradeunprotected DNA. Mechanical techniques are also capable of

transfecting cells in vivo, possibly by compromising the integ-rity of the cell membrane, thus allowing entry of naked DNAor the delivery system into the cell. However, the details of themechanism of internalization for the various systems are notwell understood. In addition, these techniques may create pos-sible difficulties with patient compliance since target tissuesmust be surgically exposed for this type of delivery.

Note that the targeting of gene delivery systems to a desiredcell population in vivo is an important subject, and is in factone of the major challenges in the field of gene therapy. Someexamples to targeting ligands are as follows: lactose, mannose,galactose and derivatives, insulin-based ligands, transferin,low-density lipoprotein, monoclonal antibodies, Fab frag-ments of immunoglobulin G (IgG), folic acid andarginine–glycine–aspartate (RGD) peptides [11–14]. Most stud-ies focus on the effect of targeting ligands that are covalentlyattached to the delivery system and have all been demonstratedto enhance the uptake and expression of DNA by takingadvantage of receptor-mediated endocytosis

Finally, it should be carefully noted that there is almost no cor-relation between ex vivo and in vivo transfections. An approachof a carrier is characteristic; favoring efficient transfection ex vivomay be ineffective in vivo.

Rationale for a delivery systemIn gene therapy it is possible to obtain local transient transgeneexpression when naked plasmid DNA is administered to tissue,but not very effectively [10,15,16]. Naked plasmid DNA is usuallyunable to cross biologic membranes, such as the endothelium,plasma membrane and nuclear membrane. Plasmid DNA is typ-ically 103–104 bp in length (molecular weight ~106–107 bp), hasa supercoiled tertiary structure in aqueous media, and has aneffective hydrodynamic diameter of greater than 100 nm. Thesurface charge density of naked DNA, which has zeta potentialsranging from -30 to -70 mV, creates repulsion between the DNAand the negatively charged cell surface.

This large size and negative surface charge density most likelylimits the uptake of DNA by cells. In addition, as mentionedabove, DNA injected directly into a tissue drains rapidly intothe lymphatics, and/or easily degraded by nucleases both in

intra- and extravascular media. In order toincrease the uptake of plasmid DNA andprevent its degradation in extracellular com-partments, a delivery system, or a so-calledvector, is required.

Note that the delivery system is intra-cellularly uptaken by endocytotic processes,meaning that these carriers will enter theendosomal/lysosomal pathway. This path-way may lead to death of the plasmid; or inother words degradation of plasmid DNA bylysosomal enzymes that are activated at lowpH, before it reaches the final destination(usually nuclear translocation). Therefore,the delivery system should prevent the

Intravascular

Injection ofvector/plasmid

DNA

Into tissue

Vector/plasmidDNA

Transfected cells

Cellculture

Cells from the patient

Figure 1. Gene delivery approaches. (A) Ex vivo and (B) in vivo.

Stimuli-responsive polymers in gene delivery

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degradation of DNA when it is in the endosomes, which maybe achieved by buffering the pH (inhibiting pH drop) in theendosomes and therefore preventing lysosomal activity, or bydisrupting the endosomal membrane that may then be bro-ken and release its contents into the cytoplasm before enzy-matic degradation takes place. Alternatively, plasmid DNAcan be released in the cytoplasma and then may enter thenucleus for effective gene expression. Therefore, a deliverysystem is again required for safe intracellular trafficking ofplasmid DNA.

Vectors for gene deliveryViruses are reported to be comparatively much more efficaciousin transferring genes into mammalian cells both ex vivo andin vivo [17–19]. The first clinical trials for gene therapyapproaches to combat disease were performed in 1990 usingviral vectors. Hundreds of gene therapy trials have been com-pleted to date, with most using viral vectors. Note that theviruses used have all been disabled of any pathogenic effects.The use of viruses is a powerful technique since many haveevolved specific machinery to deliver DNA to cells. They caneffectively transfect both dividing and nondividing cells. Someof the viral vectors (e.g., retroviruses) can easily transfect bonemarrow and other dividing cells, and the transfecting gene isintegrated into the host genome, which is a stable and long-term. Some vectors (e.g., adenoviruses) effectively transductboth dividing and nondividing cells, exhibit some kind of tar-geting ability (e.g., adenoviruses can target site-specific cellsbearing capsid receptors) and gene delivery is episomal (nointegration to host genome), which means expression is tran-sient (or short-term) [17]. However, humans have an immunesystem to fight off the virus, which may reduce the effectivenessof the viral vectors. There are also several recurring issues thathave led to a reconsideration of their use in human clinical tri-als. These include the ability of some viral vectors to integratetheir DNA with the host genome and permanently alter itsgenetic structure, which may also be a random integration intothe host chromosome. This could lead to activation of onco-genes or inactivation of tumor suppressor genes; in otherwords, insertional mutagenesis that has been already observedin some applications. It should also be noted that there are stillconsiderable immunologic problems with viral vectors.Another current drawback is the carrying capacity of the viralvectors; in other words, the limitation on the amount ofgenomic information that can be introduced into these vec-tors. It is believed that all of these existing viral vectors needto be re-evaluated and further modified with novel targetingmolecules to generate a safer, more specific and efficient viralgene delivery, and most importantly with sustained expression.

Several lipid-, peptide- and polymer-based nonviral vectorsare currently under investigation for gene delivery [20–21]. Wehave come a long way since the first nonviral gene therapy clin-ical trial in 1992 by Nabel and coworkers. However, onlyapproximately 20% of clinical trials reported the use of non-viral vectors. Nonviral vectors are receiving increasing attention

due to higher biosafety, compound stability and potential easeof chemical modification, low cost and consistent standards ofproduction. However, to date, nonviral vectors are generallyless efficient in delivering DNA and initiating gene expressionwhen compared with their viral counterparts, particularlywhen used in vivo.

Felgner and coworkers pioneered gene delivery with cationicliposomes in 1987 [22]. Since then, several cationic lipid systemswere proposed. Lipofection is the first cationic lipid formulationcomposed of cationic lipids, N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoyl phosphatidylethanolamine with a ratio of 1:1. Thus far, several cationic lipid systemshave been investigated to deliver plasmid DNA to the lungs,brain, tumors and skin by local administration, or to vascularendothelial cells [23–28]. Some cationic lipid–DNA complexes havealso been utilized in clinical trials for the treatment of cancer andcystic fibrosis. However, there are still serious limitations of cat-ionic lipid systems including low transfection efficiency similar toother nonviral vectors, high toxicity on repeated use; potent anti-inflammatory activity in vivo, high price and so on. Although theaddition of polyethylene glycol (PEG), known as PEGylation, canreduce the levels of compliment activation and binding to plasmaproteins, subsequent gene expression levels of PEGylated cationiclipids are significantly reduced [29].

The cationic polymers (polycations), which are frequentlyinvestigated as nonviral vectors, generally bear protonableamines, such as poly(L-lysine), poly(L-oronithin), both linearand branched poly(ethylene imine), diethylaminoethyl-dextran, poly(amidoamine) dendimers, poly(dimethylamino-ethyl methacrylate) and chitosan [30–34]. These polymers varywidely in their structures, which influence their complexationwith nucleic acids and transfection efficiency. The positivecharge of polycations is important as it allows complex forma-tion with negatively charged plasmid DNA. This is also knownas a condensation reaction since polycation chains squeeze in theplasmid DNA and create nanosize particulate form condensates,which can then be easily endocytosed by the cells. An extra posi-tive charge after complex formation is usually requested, whichincreases the interaction between the vector particles and nega-tively charged cell surface, which in turn triggers intercellularuptake. Note also that condensing DNA with polycations gen-erally improves the resistance of DNA against enzymatic break-down. In addition the primary amines also serve as functionalgroups which can be further chemically modified to bind spe-cific ligands including targeting molecules that can enhance oneor more of the steps in the transfection process.

However, high positive charge on the polycationic vectors isalso a limitation which may cause cytotoxicity. Jeong andcoworkers investigated the influence of cationic charge densityof the polyethylenimine (PEI)s on cell viability, and concludedthat the charge density could be an important factor for cellviability [35]. Fischer and coworkers have studied the effects ofthe type of PEIs on cytotoxicity, and suggested that high cat-ionic charge densities and a compact and highly branchedstructure, affect the biocompatibility in a negative sense [36–38].

Piskin


Similar observations were reported for poly(L-lysine) [39]. Therehave seen attempts to prepare carriers that have lower toxicity.For example, recent evidence shows that low molecular weightpreparations of polycations such as chitosan, PEI and β-cyclo-dextrin-containing polymers are significantly less toxic thanhigh-molecular-weight polycations, both in cultured cells andanimals [38,40,41]. Additionally, the distance between charge cent-ers along the backbone of a polycation and has been shown toaffect the toxicity [41]. Thus, the molecular architecture of thenonviral delivery system can modulate the toxicity, and thesedata suggest that the toxicity should be controllable. Intelligentpolymers have much lower toxicities which are discussed later.

Water soluble polymers such as polyvinyl alcohol or poly-vinylpyrrolidone, which do not condense DNA, have been alsoevaluated to protect naked genes from extracellular nuclease deg-radation and to retain them better at the site of injection [42].These polymers are amphiphilic molecules, having both ahydrophilic and hydrophobic portion. The hydrophilic portionof these polymers may interact with plasmid DNA by hydrogenbond, van der Waals and/or by electrostatic interactions due topolarity on the polymer chains. The interaction between thehydrophobic monomer units via van der Waals interaction canform a hydrophobic coating of vinyl backbone around the plas-mid DNA, and this causes a significant increase in the uptake ofthe final condensate. The cellular uptake is due to hydrophobicinteraction between the condensate and the cell wall. Commer-cially available surfactants poly(ethylene oxide)-b poly(propyleneoxide)-b-poly(ethylene oxide) copolymer (e.g., Pluronic P85)have also been shown to increase to enhance the transfection.Moreover, polymeric nano- or microparticles (e.g., based onpolycyanoacrylates, poly[D,L-lactic acid], poly[D,L-lactic acid-co-glycolic acid], gelatin, alginate, chitosan), which adsorb orencapsulate oligonucleotides or genes, are under investigationas sustained release matrices for genetic information [10,42–46].

Gene delivery with stimuli-responsive polymersStimuli-responsive polymers, also known as intelligent or smartpolymers, exhibit fast, macroscopically observable and reversiblephysical changes in response to external stimuli such as pH, tem-perature, light, ionic strength, solvents, electrical or magneticfield and so on [47–49]. These polymers undergo microstructuralchanges stimulated by small changes in the environment. Thesemicroscopic changes are apparent at the macroscopic level asphase, size/shape, reactivity, permeability, surface wettabilitychanges and so on. The changes are reversible and the systemreturns to its initial state when the stimuli are removed. Thesepolymers may be as water-soluble chains, immobilized on solidsurfaces or in solid gel form. A stimulus, for instance, increasingtemperature over a certain critical value, causes a response such asa polymer chain change from water-soluble coils to water-insoluble globules in aqueous media, collapse of the polymerchains on the solid surface or shrinkage of the solid gel.

Several pH-responsive polymers have been described. They areusually composed of a hydrophobic monomer and an ionizable,more hydrophilic comonomer. The net charge on the polymer

chain can be decreased by changing the pH to neutralize thecharges on the polymer chains, which in turn causes reductionin their hydrophilicity (or increase the hydrophobicity) andtherefore, the phase change. Typical examples are thecopolymers of methylmethacrylate (MMA) with methacrylicacid (MAc) or dimethylaminoethyl methacrylate (DMAEMA).MMA is the hydrophobic section, while MAc is thehydrophilic part of the chain. MAc is hydrophilic at high pHwhen COOH groups are deprotonated, although it becomesmore hydrophobic when COOH groups are protonated. Thephase change occurs at the pKa value of COOH groups, whichis approximately 4.5–5.5. The copolymers of MMA withDMAEMA are hydrophilic at low pH when amino groups areprotonated, although more hydrophobic when amino groupsare deprotonated. These copolymers are soluble at low pH butprecipitate at slightly alkaline conditions.

Another important class of intelligent polymers are the so-called thermoresponsive polymers, which are uncharged poly-mers and soluble in water due to the hydrogen bonding withwater molecules. The efficiency of hydrogen bonding reduceswith increases in temperature. The phase separation of polymertakes place when the efficiency of hydrogen bonding becomesinsufficient for the solubility of the polymer. When the temper-ature is increased above a certain critical temperature, which isknown as lower critical solution temperature (LCST), also referredto as the cloud point, phase separation takes place, and the poly-mer chains change from water-soluble coils to water-insolubleglobules. Poly(N-isopropylacrylamide) (PNIPAAm) is a well-known and widely studied temperature-sensitive polymer that hasa LCST value of 32°C. Copolymerization of NIPAAm withhydrophilic comonomers (e.g., acrylic acid [AAc] or DMAEMA)not only results in an increase in LCST, but also renders the poly-mer both temperature and pH sensitive. In addition, several mole-cules including the targeting bioligands mentioned above can becovalently bound onto the polymer backbone via carboxyl groupsto create more intelligent carriers.

Light can also be applied as a stimulus. Various photo-responsive polymers have been extensively studied to date. Theconformational changes of the polymers induced by the photo-promoted or thermally promoted isomerization enable it to tailorthe physical and chemical properties, including viscosity, refrac-tive index, conductivity, pH, solubility, wettability, mechanicalproperties, polymer morphology and so on. This concept hasstimulated many chemists to create a wide variety of photo-responsive polymers, especially those with azobenzene moietieseither in the side or main chains, with the motivation of produc-ing the light-controllable functional materials [50]. Azobenzene isa well-known photoresponsive chromophore, and its photo-induced and thermal geometric isomerizations have been exten-sively studied. Azobenzene and its derivatives take both trans andcis structures with respect to the azo linkage and normally exist inthe more stable trans form. The transition from trans to cis isomeris observed when ultraviolet (UV) light is applied (365 nm),while the cis configuration returns back to the trans form uponphotoirradiation with visible light around 436 nm.



When using stimuli-responsive polymers as novel vectors forgene delivery, two benefits can be obtained [51]. The first bene-fit is a high gene expression efficiency. In an ideal nonviral vec-tor system, the polymer/plasmid complex needs to fulfill thetwo functions simultaneously: protection from extracellularand intracellular enzymatic degradation and allow trafficking ofthe plasmid (both through the cell membrane and intra-cellular), and safe dissociation of the gene expression plasmid inthe cell for free access to RNA polymerase. However, it isalmost impossible to fulfill the two opposing phenomena: tightcomplex formation, which allows a favorable cell uptake andprotection of DNA degradation, and ease of complex dissocia-tion for favorable transcription by RNA polymerase. To reachan optimal intermediate tightness with conventional nonviralvectors is difficult, and cannot afford the maximum efficiency forboth functions. The aim with the intelligent vector systems is tofulfill both functions simultaneously, in which a tight complexcan be formed to ensure evading DNA degradation and highcellular uptake, but at the later step, by introducing a stimulusthat can maximize by complex dissociation for high tran-scription. The second benefit of stimuli-responsive vectors isselective gene expression. Site-, timing- and duration period-spe-cific gene expression can be achieved with the intelligent syn-thetic vectors by releasing DNA from the DNA-carrier com-plex by inducing a stimuli (e.g., local changes in temperature,pH, applying light) in a controllable manner.

Pioneering studies by the group of Hennink and coworkerssuggested the possible use of poly(N-isopropylacrylamide)-co-dimethylaminoethyl methacrylate) (PNIPAAm-co-DMAEMA)as pH- and temperature-sensitive nonviral vectors for gene ther-apy [30,52]. They have synthesized a series of copolymers ofNIPAAm and DMAEMA with various monomer ratios andmolecular weights. All copolymers, even with a low DMAEMAcontent of 15 mol%, were able to bind to DNA at 25°C. Thecomplexes using high-molecular-weight copolymers or lowerratios of NIPAAm with plasmid DNA were relatively stable at37°C, when compared with the others. They claimed that forma-tion of stable copolymer/plasmid condensates with a size ofaround 200 nm, which was considered as optimal average size,and is a prerequisite for efficient transfection. The cytotoxicity ofthe copolymers decreased with increase in concentration ofNIPAAm. The copolymer/plasmid ratio at which the transfec-tion efficiency was maximal increased with increasing NIPAAmcontent, while the maximum transfection efficiency stronglydecreased with increasing NIPAAm content of the copolymer. Itwas concluded that besides particle size, the zeta potential playsan important role in transfection. With decreasing zeta potentialboth transfection efficiency and cytotoxicity strongly decrease.

Yokoyama and coworkers introduced hydrophobic comono-mers for example butylmethacrylate (BMA) into PNIPAAm-co-DMAEMA copolymers, evaluated their transfection efficiencyat different incubation temperatures and reported enhancedDNA binding, release and transfection as aconsequence [51,53–54]. In the terpolymer, BMA is the hydro-phobic component, and thus the solubility of terpolymer/DNA

complexes is most likely regulated by both ionic and hydrophobicinteractions. A terpolymer containing 8 mol% of DMAEMA and11 mol% of BMA had a phase transition temperature of 21°C,which was found to be the same after complex formation withDNA. The terpolymer/DNA complexes showed partial dissocia-tion at 20°C, although no dissociation at 37°C, suggesting that theformation/dissociation of the complexes was also modulated bytemperature. The transfection efficiencies of terpolymer/DNAcomplexes incubated at lower temperatures were much higher thanfor those incubated at higher temperatures even for longer times.Terpolymer/DNA complex can easily be dissociated for tran-scription below the LCST, while above the LCST, these complexesare tightly formed by additional hydrophobic interactions. Theenhancement ratio (activity with the cooling procedure/activitywithout the cooling procedure) reached 8.6-times in the 3 h incu-bation period case. It was concluded that such an enhancementcould be useful in obtaining site- and timing-specific expression offoreign genes in biologic and medical applications.

Nakasaki and colleagues synthesized a novel water-solublepolyazobenzene dendimer modified with L-lysine at the periph-ery and investigated interaction of this polycation with plasmidDNA that is photoregulated by radiation [55]. Light scatteringand gel-filtration chromatography studies showed that particlesize is controllable by UV and visible light irradiation. Theaffinity of this cationic dendrimer toward DNA was photo-controllable due to changes of the charge on the dendimer’ssurfaces. Interestingly, in the transfection studies, UV light irra-diation after the polymer/plasmid condensate was uptaken inthe cells caused approximately a 50% increase in the transfectionefficiency compared with the negative control, which wasexplained by the fact that UV radiation promoted dissociationof the complex in the cytoplasm.

More recently, Twaites and coworkers prepared a range ofcationic polymers including derivatives of branched PEI con-taining both short hydrophobic side chains and end-graftedPNIPAAm homopolymers, and reported the behavior ofthese polymers in DNA-binding assays in vitro in compari-son with linear polycations synthesized from NIPAAm,DMAEMA and hydrophobic side chain monomers [56]. Theyclaimed that complexation behavior of the copolymers withplasmid DNA is dependent upon responsive polymer struc-ture and composition, and therefore temperature. The affin-ity was low with high-molecular-weight linear cationic PNI-PAAm above LCST, whereas a branched PNIPAm-co-PEIcopolymer bound with a higher affinity above the PNI-PAAm phase transition. The thermoresponsive polymersalso exhibited changes in particle morphology across thesame temperature ranges with polymer DNA complexes pre-pared at N/P ratios of 2:1 generating spherical particles vary-ing in radius between 30–70 nm at 25°C and 60–100 nm at40–45°C. Preliminary transfection experiments indicate thatall the polymers in this study were effective in transportingplasmid DNA to cell nuclei while thermoresponsive poly-mers also achieved low levels of protein expression in mousemuscle cells.

Piskin


Recently, a series of water-soluble poly(N-isopropylacrylamide-block-polyethyleneimine) copolymers have been synthesized andapplied in ex vivo transfection of both HeLa cell lines and pri-mary cells in the author’s laboratories, which are briefly providedhere [57–62]. Recently, different types of polyethyleneimine andtheir block copolymers with N-isopropylacrylamide as tempera-ture sensitive polycationic nonviral vectors have been investi-gated. Representative STM micrographs of PNIPAAm homopol-ymer, PNIPAAm-co-PEI copolymer and the conjugate of thiscopolymer with plasmid DNA are demonstrated in FIGURE 2. Therod-like structures in FIGURE 2A demonstrate the PNIPAAmhomopolymer chains in the extended form (below its LCST).PNIPAAm (rod-like chains) form block copolymers with thebranched PEI (2 kDa) as seen in FIGURE 2B, in which the globularpart of the structures represent the branched PEI blocks (belowits LCST). Moreover, when the temperature is raised above theLCST of the copolymer, the rod-like PNIPAAm blocks collapseforming globular copolymeric particles (FIGURE 2C). PlasmidDNA also forms globular structures both below and above theLCST of the copolymer as exemplified in FIGURE 2D.

In the studies by the author’s group, first carboxyl-terminatedPNIPAAm was synthesized and then copolymerized with PEIsbranched or linear and with two different molecular weights(2 and 25 kD). Addition of PEI units to the PNIPAAm chainsincreased the LCST values up to body temperature. Zetapotentials of the copolymers were significantly lower than cor-responding PEI homopolymers. A green fluorescent protein-expressing plasmid was used as a model. Complexes of thisplasmid both with PEIs and their copolymers were formed.The zeta potentials of these complexes were between -3.1 and

+21.3. Higher values were observed for the complexes preparedwith branched and higher molecular weight PEIs. Copoly-merization caused a profound decrease in the positive charges.Particle sizes of the complexes were in the range of190–1235 nm. Using high polymer/plasmid ratios caused aggre-gation. The smallest complexes were obtained with the copoly-mer prepared with branched PEI with molecular weight 25 kDa.Copolymers were able to squeeze plasmid DNA more at bodytemperature. Cytotoxicity was observed with PEIs, especiallywith the branched higher molecular weights. Copolymerizationreduced cytotoxicity. Cell viability was maintained over 80%.Transfection of a cell line (i.e., HeLa cells) and primary cells(both smooth muscle and endothelial cells) were studied in cellculture. The best transfection efficiency (70%) was achieved withthe complex prepared with PNIPAAm-co-PEI-25B with HeLacells. However, PNIPAAm-co-PEI-25L was the most successfulwith regard to gene expression. The best gene expression effi-ciency achieved with smooth muscle cells was approximately30% with the complex prepared at polymer/plasmid ratio of 6.Temperature change from 37 to 28°C enhanced the gene expres-sion efficiency by up to 50%. The linear copolymer (PNIPAAm-co-PEI-25L) showed successful DNA uptake and gene expres-sion in vitro for primary smooth muscle cells. Copolymercontribution to increased efficiency was also proved.

Expert commentary & five-year viewToday, there is a strong tendency in modern therapies for severalbiologic molecules including nucleic acids (e.g., plasmid DNA,antisense oligonucleotides, therapeutic RNAs and siRNAs) andproteins (e.g., monoclonal antibodies, growth factors, hormones,therapeutic enzymes and synthetic oligopeptides) to be used ashighly specific pharmaceutical agents. Gene therapy aims totreat diseases by intracellular delivery of nucleic acids thatalter gene expression within a specific cell population, therebymanipulating cellular processes and responses. Although itwas originally devised for the treatment of inherited geneticdisorders, recent work has expanded the applications of genetherapy to develop strategies for the treatment of a wide rangeof metabolic, infectious and inflammatory diseases.

All of these novel therapeutics, including nucleic acids, arefragile; in other words, they are faced with biodegradationwithin the body before they reach their target. A carrier vehicleor vector is needed to allow targeted and intracellular deliverythat is certainly one of the most important and exciting futur-istic challenges. There are many major barriers for these vehi-cles to reach the target cells and for intracellular trafficking. Forex vivo gene delivery, mechanical techniques including genegun, hydrostatic pressure, electroporation, continuous infusion,and sonication can be used. They provide relatively high genetransfer efficiency ex vivo; however, these procedures are costlyand may not be appropriate for all situations. Application ofthese techniques in vivo is certainly difficult, in most cases it isnot possible, or even if it is possible surgery would be required,which is of course not very desirable. Reaching the target cells,especially in in vivo gene delivery, is currently an unmet need.

A B

C D

Figure 2. Representative STM images. (A) PNIPAAm homopolymers (scan area: 30 × 30 nm), (B) its block copolymer with PEI (scan area: 12 × 12 nm), (C) for the same copolymer above its LCST (scan area: 30 × 30 nm) and (D) conjugate of this copolymer with plasmid DNA (scan area: 1200 × 1200 nm). LCST: Lower critical solution temperature; PEI: Polyethylenimine; PNIPAAm: Poly(N-isopropylacrylamide).



There are a number of approaches under investigation, and themost important and futuristic uses targeting molecules (e.g.,bioligands) that are attached to the carrier vehicle and allow itto be directed to the target cells via several body compartments.Oligopeptides are among the most attractive bioligands, havingvery specific biorecognition ability to the specific receptorswhich exist (or may be created) on the target cell population.These targeting peptides can be selected from the peptidelibraries by novel techniques such as phage display, and are syn-thesized synthetically. They may offer not only the ability todirect the vehicles to the target cells, but also their effective andspecific uptake by the cells.

Viruses are relatively effective gene delivery vectors. Sometypes have the ability to find specific cell populations withinthe body, or targeting bioligands can be attached to theirsurfaces for better and more specific delivery. They haveevolved a specific machinery to deliver DNA into cells andeven into to the nucleus. Some of them leave the gene ofinterest in the nucleus, in which it can be translated episo-mally. However, this gene modification is transient. Othersintegrate the gene into the host genome, which may allowstable gene modification, but unfortunately not to targetedlocations, which may cause mutagenesis. There is certainly alarge interest in developing new generations of viral vectorsthat will not only find the target cells, but will also insert thegene into the correct position in the host genome. We maybe witness to the design of much safer and effective viralvectors in the coming years.

Nonviral vectors are much safer but less effective than viralvectors. There is a great interest in developing these vehiclesfor more effective gene delivery. Besides the targeting strategiesmentioned above for reaching the target cells, they have to bespecifically uptaken by these cells, which occurs mainly byendocytosis. The targeting ligands that are attached onto thevectors may allow or trigger this specific uptake. In some cases,such as polycationic carriers, the positive charge may cause orenhance the uptake. Endosomal escape is necessary beforelysosomal activity, which causes the loss (degradation) of thegene (nucleic acid). Avoiding lysosomal activity is anotherimportant problem in intracellular delivery that needs to besolved, in order to increase the gene delivery, especially byusing nonviral vectors. Note that viruses know how to escapeendosomal degradation. For instance, it is known that influ-enza viruses use their fusogenic peptides to disturb the endo-somal membrane. There are several approaches to avoid lyso-somes. As mentioned above, polycations exhibit a protonation(buffering) effect due to their amino groups which stop a pHdrop in the endosome, and therefore lysosomic activity can bereduced or eliminated. Other novel approaches have beenalready proposed including the use of an endosomal releasingagent such as the synthetic peptides mimicking the fusogenicpeptides of viruses that enable the vector and its cargo toescape the endosome. Several chemicals and also polymericforms have been developed that will disrupt the endosomalmembrane for an effective endosomal escape. Smart polymers,

especially pH responsive ones will certainly contribute tothis area. For instance, polycationic and pH-sensitive poly-meric carriers form compact complexes with plasmid DNA,and not only allow for effective uptake (due to positivecharge and also smaller size) but also prevent early (undesir-able) endosomal degradation due to positive charge (proto-nation) and compact structure. We will hopefully see betterdesigns and more intelligent vectors carrying other endo-somal membrane disrupting groups in this direction in thenear future. More mechanistic approaches, for instancephotochemical rupture of endocytic vesicles (lysosomes andendosomes) with photosensitising compounds, have alreadybeen proposed and will be developed further.

Vectors should also safely release their content (e.g., plas-mid DNA) within the cytosol, to carry it into the final desti-nation, which is the nucleus in the case of gene therapy. Asnonviral vectors, polycations may work well for efficient celluptake and endosomal escape, and pH- and/or temperature-sensitive smart polycations squeeze the plasmid DNA andenhance even further the effectiveness of these steps as men-tioned due to more compact and smaller complexformation. However, there is a disadvantage in releasing theplasmid DNA within the cytosol. Introducing temperature-and light-sensitive groups onto the polymer chains of thesmart polymers may have a critical role even at this releasingstage – which has been exhibited recently by some of thegroups working in this area (including the author’s group),the results of which are very promising. Further develop-ments are certainly needed in this direction, especially onlocal control of the environmental conditions (e.g., pH,temperature and light intensity), which will allow the use ofthe ability of the intelligent polymers, or in other words,their intelligence, which will certainly appear in thecoming years.

AcknowledgementErhan Piskin was supported by the Turkish Academy ofSciences as a full member.

Key issues

• Gene therapy is one of the most promising therapies, especially for the treatment of genetic disorders.

• Both ex vivo and in vivo gene delivery can be achieved mostly by using a delivery system (vector). Viral vectors exhibit high gene expression, but also have very significant side effects.

• Several lipid-, peptide- and polymer-based nonviral vectors are currently under investigation for gene delivery. Cationic polymeric systems are used as nonviral vectors, but usually with low levels of transfection.

• Through the use of stimuli-responsive polymers as novel vectors for gene delivery, two benefits can be obtained: high gene expression efficiency and more selective gene expression.

Piskin


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Affiliation• Erhan Piskin, MD

Hacettepe University, Chemical Engineering Department, Bioengineering Division,and TÜBTAK-ÜSAM-BiyomedtekBeytepe, 06800 Ankara, TurkeyTel.: +90 532 707 9468Fax: +90 312 440 [email protected]

stimuli-responsive polymers in gene delivery

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