Localized,non-viral delivery of nucleic acids:Opportunities,challenges and current strategies

Oliver Germershaus,Kira Nultsch

Institute for Pharma Technology,School of Life Sciences,University of Applied Sciences and Arts Northwestern Switzerland,Gru¨ndenstrasse 40,CH 4132,Muttenz,Switzerland

Review

Localized,non-viral delivery of nucleic acids:
Opportunities,challenges and current strategies

Oliver Germershaus＊,Kira Nultsch

Institute for Pharma Technology,School of Life Sciences,University of Applied Sciences and Arts Northwestern Switzerland,Gru¨ndenstrasse 40,CH 4132,Muttenz,Switzerland

ARTICLEINFO

Article history:

Received 8 September 2014

Received in revised form

8 October 2014

Accepted 10 October 2014

Available online 24 December 2014

Gene delivery

Topical application

Tissue engineering

Controlled release

SiRNA

DNA

Localized delivery of drugs is an emerging f i eld both with regards to drug delivery during disease as well as in tissue engineering.Despite signif i cant achievements made in the last decades,the eff i cient delivery of proteins and peptides remains challenging,especially in cases requiring long-term release of proteins after application.The localized delivery of nucleic acids(NA)represents an interesting alternative due to higher physicochemical stability of NA,increased eff i ciency by harnessing cells as bioreactors for the production of required proteins and improved versatility with regards to expression of specif i c proteins through plasmid DNA or repression of gene products through siRNA.However,unlike most proteins and peptides,NA must be delivered to the cytoplasm or nucleus to be eff i cacious, resulting in signif i cant delivery challenges.We herein describe frequently used non-viral vectors for the delivery of NA including polyplexes,lipoplexes and lipopolyplexes and summarize recent developments in the f i eld of nucleic acid delivery systems for local application based on hydrogels,solid scaffolds and physical delivery methods.The challenges associated with the different approaches are identif i ed and options to address these challenges are discussed.

©2014 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

1.Introduction

The eff i cient delivery of nucleic acids(NA)is one of the most exquisite challenges to formulation scientists nowadays. Unlike small chemical drugs and most biologic drugs,NA must be delivered into the cytoplasm or into the nucleus to be effective.Small interfering RNA(siRNA)and antisense oligonucleotides interact with mRNA and therefore must be transported to the cytoplasm.However,transport of NA across the cell membrane is challenging due to its negative charge and high molecular weight[1].Plasmid DNA(pDNA) additionally requirestranslocation into the nucleus where it is transcribedintomRNA,eventuallyresultinginprotein synthesis.

From a physicochemical and formulation point of view NA are almost ideal drug molecules.They possess high stability against both chemical and physical degradation,which isexemplif i ed by the possibility to extract and sequence DNA from fossils of extinct species and the remarkable half-life of DNA of 521 years[2,3].It is therefore not surprising that several studies investigating the stability of formulated NA yielded promising results,especially when nucleic acids were protected from hydrolytic and enzymatic degradation,e.g. through freeze-drying[4-6].It is also important to note that NA potentially require signif i cantly less formulation development work once an optimal formulation has been found compared to other macromolecular drugs such as proteins and peptides.The relatively simple structure of NA results in minimal changes of physicochemical properties of NA when changing the sequence.In contrast,exchange of a single amino acid may result in signif i cantly changed structure and physicochemical properties in the case of proteins and peptides[7,8].Therefore,it is reasonable to assume that reformulationefforts to fulf i ll the needsof NA with a new sequence will be minimal.Furthermore,NA,especially siRNA and antisense oligonucleotides can be simply produced by chemical synthesis with good yield and in high purity.In contrast, production of proteins such as monoclonal antibodies or growth factors requires a cellular expression system and extensive purif i cation and/or refolding[9].Finally,NA possess high specif i city,resulting in expression of a specif i c protein (plasmid DNA)or inhibition of a specif i c target gene(siRNA, antisense oligonucleotides),potentially maximizing the ratio of desired to undesired effects compared to alternative therapies[10].

Apart from the numerous advantageous properties of NA, the requirement for delivery into the cytoplasm or nucleus represents a signif i cant challenge.This challenge is addressed using attenuated viral vectors as delivery systems or non-viral delivery strategies.A discussion of the general advantages and disadvantages of viral versus non-viral vectors is beyond the scope of this review and the reader is referred to the review by Guo et al.for detailed information on this topic[11]. We herein focus on non-viral delivery strategies,which are often preferredbecause of safety concerns associate with viral vectors and versatility and ease of modif i cation of non-viral vectors[11].

In most studies,systemic administration of NA delivery systems through the parenteral route is investigated,primarily because of its versatility,e.g.with regards to treatment of distant or disseminated cells,mostly in the context of tumor therapy.However,achieving eff i cient delivery of NA to target cells after parenteral application requires overcoming numerous extracellular and intracellular barriers.Stability and compatibility of the carrier system in the blood stream must be maintained to achieve prolonged circulation,allowing the delivery system to reach its target site.This entails surface modif i cation with hydrophilic polymers to avoid uptake of the delivery system by the reticulo-endothelial system (RES),crosslinking to prevent premature release of the NA cargo and control of delivery system size to avoid blocking of capillaries and to allow extravasation at the target site,e.g.in the tumor or liver[12,13].Additionally,active or passive targeting strategies may facilitate the eff i cient extravasation and cellular uptake at the site of action.In recent years,stimuliresponsive systems have been developed,further improving accumulation of the delivery system at the site of action. These systems change their physicochemical properties in response to extracellular stimuli such as changes of the pH value,enzymatic environment etc.[14,15].It is therefore obvious that the successful development of NA delivery systems for systemic administration requires an optimal combination of different properties within the delivery system, representing a signif i cant formulation challenge.Furthermore,the clinical application of these delivery systems has been signif i cantly limited because of concerns with regards to toxicity of the vehicle and unsatisfactory delivery eff i ciency.

While the majority of studies has focused on improving the eff i ciency,stability and compatibility of delivery systems for systemic application,localized delivery of NA represents an interestingbutlargelyuntappedalternative,worthyof increased exploration.While it is certainly not possible to achieve the versatility of systemic application,e.g.regarding transfection of distant or disseminated cells,localized delivery avoids several major barriers associated with systemic delivery and is therefore more likely to result in safe and eff i cacious therapy.Achieving suff i ciently high drug concentrations at the target site is a common challenge in drug therapy.In the case of NA delivery this general challenge is aggravated by the necessity of intracellular delivery of NA[9]. Because of the sensitivity of NA towards enzymatic degradation,encapsulation is often required but on the other hand eff i cient release must be maintained[1].These requirements increase the complexity of delivery systems,especially in the case of systemic delivery.

Within this review,we seek to give an overview of the current status of localized delivery of NA using non-viral delivery systems.The discussion is however focused to topical delivery in the broadest sense,excluding oral,nasal and pulmonary delivery as these delivery routes have additional special requirements to the delivery system.We shortly portray non-viral vectors that are employed for localized delivery of NA and review recent systems for localized NA delivery based on various matrices.Our aim is to highlight challengesbutalsoopportunitiesassociatedwiththis approach to NA delivery and discuss potential f i elds of application.

2.Non-viral nucleic acid delivery systems employed for localized delivery

The high molecular weight and negative charge of NA represents a major hurdle to eff i cient cellular uptake.For most cell types,the size requirement for particle uptake is in the rage of up to 200 nm,signif i cantly smaller than the hydrodynamic diameter of DNA of a few thousand base pairs[16].Furthermore,negatively charged moieties anchored to the cell membrane result in electrostatic repulsion of negatively charged NA.Non-viral delivery agents are designed to improve cellular uptake eff i ciency either through complexation and charge reversal of NA or through physical methods allowing NA to directly enter the cell.However,the challenge with such delivery systems is on the one hand to prevent the NA degradation within the micro-or nanoparticles and on the other hand to achieve eff i cient intracellular release[1].

2.1.Polymeric systems(Polyplex)Cationic polymers such as polyethylenimine(PEI),chitosan or dendrimers are among the most studied non-viral vectors for NA[17-20].These polymers proved to be highly eff i cient in vitro,and several are commercially available as transfection agents(JetPEI,Polyplus transfection SA;ExGen500, Biomol GmbH).

Positively charged polymeric nanocomplexes(polyplexes) in the size range of tens to hundreds of nanometers are spontaneously formed when NA and suitable cationic polymers are mixed under appropriate conditions.Polyplexes facilitate cell attachment through electrostatic interaction, internalizationbyendocytosisandendosomalescape [18,21,22].One of the main advantages of polymeric systems for non-viral delivery is their ease of modif i cation,allowing straightforward design and synthesis of multifunctional delivery systems[11].Some of these modif i cations include the shielding of surface charge through modif i cation with hydrophilic polymers prior to[23]or after complex formation [24]with NA.Furthermore,conjugation of targeting moieties can be performed in a similar fashion either directly[25]or via grafted hydrophilic polymers[26].Biodegradable crosslinking,e.g.using bioreducible linkers,is another potential modif i cation allowing tailoring the stability of polyplexes in different environments to specif i c needs[27,28].However, cytotoxicity of polycations is one of the major problems, which was frequently found to correlate with transfection eff i ciency[29,30].Several groups addressed this issue,e.g. through bioreducible crosslinks in the polymer backbone, resulting in improved degradation and potentially improved excretion[31,32].A detailed overview of the current status of polymeric non-viral vectors can be found in[33]and advantages and disadvantages of polymeric non-viral vectors are discussed in[17].

2.2.Lipid-based systems(Lipoplex)

Lipoplexes are formed through the interaction of anionic NA with the surface of cationic liposomes,resulting in aggregated and multilamellar complexes.The challenges associated with cationic liposomes as a vector for NA delivery are their high zeta potential,low transfection eff i ciencies and often an ineff i cient protection against lysosomal NA degradation[4]. Lipid-based systems were among the f i rst non-viral vectors used to transfer NA into cells.Felgner et al.showed in 1987 that N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride(DOTMA),a synthetic cationic lipid,is able to condense plasmid DNA(pDNA)and to transfect different cell lines in vitro[34].The complexation eff i ciency with pDNA was found to be close to 100%and it was observed that complexes fuse with the cell membrane,resulting in eff i cient intracellular delivery of the cargo[34,35].The interaction with the cell surface is thought to be primarily due to electrostatic interaction of positively charged lipoplexes with negatively charged residues at the cell surface,such as sialic acid. In addition,hydrophobic interactions contribute to fusion with the cell membrane and cargo delivery.In this regard, theadditionofneutralhelperlipidslikedioleoylphosphatidylethanolamine(DOPE)or cholesterol results in stabilization of liposomes and improved membrane fusion and hence improves transfection eff i ciency[36].Later studies foundthateff i cienttransfectiondependsonclathrinmediated endocytosis and direct translocation of NA across the cell membrane is of only minor importance[22,37].However,similar to the polymeric systems,cytotoxicity is a major concern.Toxicity was frequently found to be associated with the charge ratio between NA and cationic lipid,the dose administered and the type of reagent or cell type[38].One way to deal with these toxicity issues is the local administration of lipoplexes,reducing the effects on non-target cells.Numerous improvements and modif i cations to the basic concept of lipofection as described above have been published,including stabilizedliposomes,immunoliposomesandstimuliresponsive liposomes.The reader is referred to[39,40]for a detailed discussion of current lipoplex systems and to references[38,39]for an in depth discussion of advantages and disadvantages of lipoplexes as vectors for NA.

2.3.Lipopolyplex

Lipopolyplexes,representing ternary complexes composed of cationic polymers,cationic liposomes and NA,were developed in an attempt to combine the advantageous properties of polyplexes and lipoplexes.Besides improvements with regards to stability in the presence of serum, particlesizeandreproducibilityoftransfection,lipopolyplexes were shown to signif i cantly increase transfection eff i ciency compared to polyplexes[41,42].Through further supplementation with hyaluronic acid or heparin, lipopolyplexes with very low immunostimulatory potential compared to traditional lipopolyplexes were obtained[43]. Recently,structurally stabilized lipopolyplexes consisting of lipoplexes which were alternatively coated with poly(acrylic acid)and polyethylenimine were prepared[44].These lipopolyplexes showed improved transfection eff i ciency and reduced cytotoxicity compared to various polyplexes and lipoplexes and commercial lipofectamine.Lipopolyplexes are therefore an eff i cient alternative to traditional lipo-and polyplexes with reduced cytotoxicity.Additional information on the advantages and disadvantages of lipopolyplexes can be found in[44].

3.Delivery systems for localized release of nucleic acids

3.1.Local delivery of naked nucleic acids

The direct administration of NA as a liquid is certainly the simplest form of local delivery,it is highly attractive due to simple formulation development and administration and, under appropriate conditions,results in eff i cient NA delivery. As an example,the treatment of choroidal neovascularization by direct injection of stabilized siRNAs was investigated by Shenet al.[45].In this study,Sirna-027,a chemicallystabilized siRNA against vascular endothelial growth factor receptor 1 (VEGFR1),was administered locally through intravitreal or periocular injections in a murine model of choroidal or retinal neovascularization.Upon injection of Sirna-027,VEGFR1mRNA levels were signif i cant reduced by 40-57%.Labeled siRNA was detected in retinal cells for 4-5 days after intravitreal injection,siRNA was also able to penetrate the sclera and choroidea after periocular injection and was detectable in the retina for at least 5 days.Finally,treatment with Sirna-027 resulted in eff i cient suppression of ocular neovascularization in murine models of choroidal and retinal neovascularization. The safety and tolerability of direct intravitreal injection of Sirna-027 was later tested in a phase 1 clinical study in 26 patients with choroidal neovascularization resulting from neovascular age-related macular degeneration[46].Intravitreal injections of 100-1600 μg of Sirna-027 were well tolerated and no dose-limiting toxicity was observed.Secondary objectives including retinal thickness and visual acuity were found to be stabilized or improved after treatment, respectively.Further phase 1 and phase 2 studies evaluating stabilized siRNA targeting the RTP801 gene were performed in patients with neovascular age-related macular degeneration [47]and diabetic macular edema[48],respectively.Similar to the study on Sirna-027,intravitreal injections of siRNA in these two studies were generally well tolerated and safe.Only few mild to moderate treatment-related adverse events were observed.However,Kleinman et al.demonstrated that inhibition of VEGF expressionand reduction of neovascularization might be due to unspecif i c activation of cell surface Toll-like receptor 3(TLR3)[49].In addition,it was shown that cellular uptake of siRNA was not required for this effect.These observations as well as failure to meet eff i cacy requirements in clinical studies resulted in discontinuation of the development of ocular injection of Sirna-027.

Meuli et al.reported that direct injection of naked plasmid DNA into full-thickness wounds in a murine model resulted in eff i cient local transfection of macrophages,f i broblasts and adipocytes[50].Interestingly,transfection eff i ciency of naked DNA was comparable to eff i ciency of optimized cationic liposome/DNA complexes.Similar results were obtained in pig skin,in rats and another murine model before[51-53]. Local transfection eff i ciency after administration of naked DNA in the skin can be further improved through oscillating solid microneedles[54,55].

3.2.Composite systems

The development of drug delivery systems for the localized release of NA,apart from application of naked NA described above and physical methods described below,frequently requires the combination of a component that condenses NA andallowsthe transfection oftarget cells(vector)anda matrix that controls release of the vector and provides physical support.In the simplest case,NA or NA/vector combinations are locally administered as a solution without the need for a supporting matrix.If a matrixis used,NAmay be incorporated into the matrix(encapsulation)or may be attached to the matrix surface(immobilization).

3.2.1.Hydrogels

Hydrogels are among the most frequently used systems in the fi eld of localized therapy,including tissue engineering.The well hydrated,tissue-like environment of hydrogels,their variability with regards to the chemical composition and modif i cation as well as the typically mild,aqueous fabrication conditions all render hydrogels ideally suited as scaffolds for localized NA delivery.Hydrogels are typically formed eitherby crosslinking or by self-assembly from hydrophilic polymers of natural,synthetic or semi-synthetic origin.The release of encapsulated NA can be controlled via the pore size of the hydrogel,its degradation rate or through physical interaction or chemical conjugation of the vector system with hydrogel components.In the following sections,we will highlight the advantages and challenges associated with various hydrogelbased delivery of non-viral vectors.For an in depth discussion of properties and application of natural and semi-synthetic polymers for drug delivery the reader is referred to[56].

3.2.1.1.Hydrogels based on natural polymers.Natural polymers like collagen,f i brin or alginate were frequently used to prepare NA releasing hydrogels.Natural hydrogels in this regard may serve as a reservoirfor NAvectors with release being controlled by the hydrogel pore size,vector-matrix interactions and/or degradation.Such systems have been developed based on collagen[57-59],f i brin[60,61],alginate [62,63],chitosan[64]and hyaluronic acid[61].The sustained release of NA from these biocompatible scaffolds frequently resulted in improved transfection eff i ciency both in vitro and in vivo[65,66].Furthermore,sustained release of polyplexes or lipoplexes was often found to signif i cantly reduce the cytotoxicity of non-viral vectors[59,67].Retention of the vector within the hydrogel and hence release rate can be modif i ed by variations of the vector and hydrogel chemistry.As an example,Cohen-Sacks et al.modif i ed a collagen hydrogel matrix with poly-L-lysine to improve the retention of plasmid DNA through non-specif i c electrostatic interactions[57]. Alternatively,NA or vector,i.e.polyplexes can be chemically crosslinked with the hydrogel matrix.Capito et al.showed by direct comparison of a)simple soaking of a pre-crosslinked hydrogel and b)crosslinking of plasmid DNA with the scaffold,that the latter method allows to reduce burst release and to link DNA release to hydrogel degradation[58].Similarly, control over sustained release of siRNA/PEI complexes was achieved by covalent attachment of PEI via ester linkages to photocrosslinked dextran hydrogel[68].However,aggregation of non-viral vectors during incorporation into hydrogels associated with loss of activity was identif i ed as a major limitation.Lei et al.recently developed a procedure termed caged nanoparticle encapsulation allowing loading of concentrated non-viral vector nanoparticles into various hydrogels avoiding aggregation[61,69].The encapsulation process consisted of lyophilization of polyplexes using agarose and sucrose and subsequent reconstitution of the lyophilized powder with hydrogel precursor,followed by crosslinking for hydrogel formation(Fig.1).

3.2.1.2.Hydrogels based on synthetic polymers.While natural hydrogelsoftenexcelwithregardstobiocompatibility, biodegradability and can be obtained from renewable resources[56],synthetic polymers allow simple chemical modif i cation and functionalization,improved control over composition and properties and often carry lower risk of immunogenicity than natural polymers[70].It is therefore not surprising that numerous hydrogel systems for the delivery of NA based on synthetic polymers have been described.As with natural polymers,NA incorporation may be achieved by simple soaking of the hydrogel with NA,encapsulation of vectors during hydrogel formation or physical or chemical conjugation of NA or vector to the hydrogel matrix[71-74]. Furthermore,in situ forming hydrogels[75,76]and stimuliresponsive systems[77,78]have been developed based on synthetic polymers.Release properties may therefore be varied according to the specif i c needs through the mode of NA incorporation or by variation of scaffold properties.Takahashi et al.varied the crosslinking density of PEG hydrogels to modify the diffusion-controlled release of siRNA/PEI polyplexes[71].NA release can be sustained even further through conjugation of NA/vector complexes to the scaffold.Li et al. used a triblock copolymers consisting of methoxy-PEG,poly(εcaprolactone)and poly[2-(dimethylamino)ethyl methacrylate (MPEG-PCL-PDMAEMA)that eff i ciently condensed plasmid DNA to generate hydrogels by crosslinking mPEG blocks using α-cyclodextrin[74].Using this supramolecular hydrogel,sustained release of DNA for up to 6 days was achieved.

Eff i cient cell inf i ltration is another critical property for hydrogel delivery system employed for tissue engineering and therefore control over the macroporous structure is crucial.Orsi et al.developed hydrogels with variable and controllable pore size distribution based on PEG hydrogels usinggelatinmicroparticlesfortemplating,achieving improved and controllable cell migration into the scaffold [79].Similarly,matrix stiffness of hydrogels affects NA delivery performance and was shown to be inversely related to transfection eff i ciency in hydrogels[80,81].Keeney et al. synthesized hydrogels with a tunable matrix stiffness between 2 and 175 kPa using PEG-dimethacrylate(PEG-DMA) andgelatin-methacrylate(Gelatin-MA)[82].Cellswere coencapsulated with polyplexes inside hydrogels and cell proliferation and transfection eff i ciency was investigated. While cell proliferation decreased with matrix stiffness, transfection eff i ciency of optimized polyplexes was found to be high across a broad stiffness range,but being strongly reduced at high gel stiffness(Fig.2).Therefore it is important to note that besides optimization of vector eff i ciency and release rate,mechanical and physical properties of the matrix such as porosity and stiffness may be important optimizationparameters,especiallyincasewherecell inf i ltration into the scaffold is intended.

3.2.1.3.Bioresponsive hydrogels.Despite the progress made using hydrogel-based delivery,release of NA vectors for such composite systems is controlled solely by hydrogel and vector properties,such as diffusion-and degradation rate or vector/ hydrogel interactions.In contrast,bioresponsive drug delivery systems have been developed in recent years.These systems change their properties in response to a biological trigger such as changes of the pH,temperature,light or presence of biomolecules such as enzymes[83].Such functions are highly desirable when designing hydrogels for NA delivery,allowing novel modes of drug release,e.g.self-regulating systems that release the drug only in a well-def i ned disease state.Among the numerous bioresponsive systems described to date, hydrogels that release their payload in the presence of elevated levels of enzymes will be discussed in more detail. Cell-matrix interactions represent an interesting trigger for drug release from hydrogels,especially for tissue engineering applications.Matrix metalloproteinases(MMPs)are enzymes that degrade both,matrix and non-matrix proteins,and are important for remodeling of the extracellular environment of cells[84].MMP-degradable hydrogels support cell growth and allow cell migration and have been intensively studied by Hubbell and coworkers and others[85-87].The signif i cant potential of MMP-degradable hydrogels as drug delivery matrix was recently highlighted by Purcell et al.,incorporating recombinant tissue inhibitor of MMPs(rTIMP-3)into MMP-sensitive hydrogels[88].MMP activity in a porcine model of myocardial infarction was signif i cantly reduced after targeted delivery of hydrogel/rTIMP-3 and left ventricular remodeling was attenuated.However,both the loading capacity of hydrogels as well as the stability of proteins within hydrogels is limited,representing a signif i cant hurdle to eff i cient,longterm treatment in many f i elds.Therefore the targeted, stimuli-responsive delivery of plasmid DNA or siRNA through MMP-sensitive hydrogels may represent an attractive alternative,allowing long-term bioavailability since cells inf i ltrating the scaffold act as bioreactors for the required proteins.NAdeliveryfromMMP-degradablehydrogel matrices hasbeenrecentlyinvestigatedby TatianaSeguraand coworkers[78,80,89].In these studies,it was shown that hydrogels loaded with non-viral vectors and allowing cell inf i ltration by MMP-sensitive crosslinking resulted in longlastingtransgeneexpressionincombinationwithlow cytotoxicity.

3.2.2.Porous structures

In contrast to hydrogels,microporous structures as discussed herein are solid,porous or foam-like matrices typically composed of polymers like polyurethans or poly(lactic-coglycolic acid)(PLGA)and intended for implantation or topical application as opposed to injection.Such scaffolds may be prepared through a variety of methods,including top-down approaches such assimple foamforming and electrospinning,or bottom-up approaches such as microparticle composites.In the following paragraphs we will focus on foam-like structures and electrospun scaffolds,representing frequently applied technologies.One of the main challenges associated with solid porous structures for localized NA delivery is to achieve both,optimal cell inf i ltration as well as appropriate release of NA.To fulf i ll the requirement of optimalcellinf i ltration,structures ofsuff i ciently highporosity are fabricated.On the other hand,such highly porous structures are frequently characterized by very thin walls separating the pores,which results in short diffusion distance and fast release if drugs are simply encapsulated into the scaffold. However,despite these challenges,solid structures allow for substantial variation of material composition,structure and surface modif i cation and because of this f l exibility are highly interesting for the development of multifunctional NA delivery systems.

3.2.2.1.Foam-likescaffolds.Biodegradablepolyurethans (PUR)are promising foam-like biomaterials with proven performance as injectable drug delivery system for various small chemical and biologic drugs[90-94].PURs suitability for NA delivery has recently been evaluated by Nelson et al.[95,96]. SiRNA was complexed into nanoparticles using a diblock copolymer composed of 2-(diethylamino)ethyl methacrylate, butyl methacrylate and 2-propyl acrylic acid and nanocomplexes were encapsulated into the matrix during PUR foam preparation.These scaffolds were shown to release nanocomplexes over 3 weeks based on a diffusion-controlled mechanism and bioactivity of siRNA was maintained for 4-21 days depending on the model used[95,96].The therapeutic potential of PUR scaffolds was evaluated in vivo after subcutaneous implantation of scaffolds into balb/c mice.SiRNA targeting prolyl hydroxylase domain protein 2(PHD2)was encapsulated into scaffolds as described above.During normoxia PHD2 activity results in degradation of pro-angiogenic hypoxia inducible factor 1α(HIF1α).Suppression of PHD2 therefore results in enhanced activity of HIF1α,mediating expression of pro-angiogenic vascular endothelial growth factor(VEGF),f i broblast growth factor 2(FGF-2),and others [96].After 14 days,VEGF and FGF-2 expression increased approximately 200%and 300%,respectively and a signif i cantly improved development of functional vascular structures was imaged and quantif i ed by micro-CT(Fig.3),providing preliminary evidence for therapeutic eff i ciency of such scaffolds in vivo.

An alternative approach was followed by Rives et al., applying a sandwich approach where the structure of the polymeric material was varied in an attempt to uncouple scaffold structural requirements from requirements of drug delivery[97].The authors prepared layered poly(lactic-coglycolic acid)(PLGA)scaffolds consisting of porous layers allowing cell inf i ltration,and non-porous layers containing plasmid DNA and allowing slow release.The non-porous layer was fabricated from PLGA microparticles and loaded with plasmid DNA.It was then sandwiched between two porous layers,which were fabricated from PLGA microparticles using gas foaming in combination with particle leaching.These scaffolds allowed eff i cient cell inf i ltration into the outer porous layers.Pronounced burst release of plasmid DNA was observed within the f i rst 3 days,resulting in declining transgene expression after 3 days.However,after implantation of scaffolds loaded with plasmid DNA encoding FGF-2 into intraperitoneal fat of C57BL/6 mice,a signif i cant increase in the total vascular volume fraction was achieved,in part being a result of an increase in vessel size.Nevertheless,achieving more sustained plasmid release kinetics would potentially extend transgene expression and reduce plasmid-associated inf l ammatory response[97].

Finally,additive manufacturing(also known as 3D printing or rapidprototyping)isanother approachfollowedto fabricate scaffolds with just the right combination of properties.In contrast to traditional scaffold manufacturing methods rapid prototyping allows the fabrication of complex structures composed of different materials and does not require the use of toxic solvents.Andersen et al.recently presented scaffolds composed of an NA-loaded hydrogel component and polycaprolactone for structural stability fabricated by additive manufacturing[98].The hydrogel composition was optimized to achieve printability by hydroxyethylcellulose,stability by alginate and cell attachment and survival by hyaluronan and the gels were loaded with siRNA and used for 3D printing as shown in Fig.4.

In order to improve load-bearing ability of scaffolds, hydrogels were co-printed with PCL f i bers,resulting in scaffolds that were impossible to crush by hand.These composite scaffolds could be seeded with stem cells and encapsulated siRNA was shown to stay localized and being taken up by nearby cells.Image analysis further revealed that localized knockdown of green f l uorescent protein(GFP)used as reporter gene could be achieved in checker patterned scaffolds with alternating patches with siRNA targeting GFP and mismatched siRNA(Fig.5)[98].

3.2.2.2.Electrospun matrices.The structural similarity of electrospun nonwovens to the extracellular matrix and their wide variability with regards to composition and dimension of individual fi bers as well as structure of nonwovens have resulted in signi fi cant attention on the electrospinning technique in the biomaterials and tissue engineering fi elds[99]. Numerous polymers,such as PLGA,PCL and PEG as well as various fabrication techniques such as emulsion electrospinning,coaxialelectrospinningortwo-nozzleelectrospinning may be used and combined[99].Furthermore, subsequent modi fi cation of the fi ber surface opens additional options for the fabrication of complex composite materials. Herein we will highlight some recent developments in the fi eld of electrospun nonwovens for nucleic acid delivery, focusing on novel techniques that go beyond diffusion-based release of NA from electrospun polymer fi bers representing the fi rst generation of electrospun NA delivery systems [100-102].Burst release of large amounts of loaded NA was a major challenge associated with some of these early systems [100,101].However,this problem could at least partly be solved through encapsulation of NA/polycation complexes into nonwoven scaffolds[102,103].Saraf et al.prepared core/ shell fi bers using PCL,PEI and hyaluronic acid(HA)as shell material,while PEG and DNA were located in the core of the fi bers[103].The effects of variation of four processing parameters,on fi ber diameter,release kinetics of PEI-HA and transfection ef fi ciency were evaluated based on a fractional factorial design.The average transfection ef fi ciency was affected by PEG molecular weight and concentration and lasted for up to 60 days under optimized conditions in vitro.An alternative approach to encapsulation of NA into polymer fibers was presented by Sakai et al.,who used a layer-by-layer assembly to load NA onto the fi ber surface[104].In this study,plasmid DNA and PEI were employed as polyanion and polycation,respectively to achieve successive deposition andmultilayer buildup.Transfection eff i ciency of scaffolds prepared using this coating technique depended both on the amount of DNA loaded onto the scaffolds as well as on incubation time with cells.

Interestingly,several studies reported that signif i cant cytotoxicity of polyplexes was observed despite encapsulation or adsorption of polyplexes to electrospun nonwovens,which was at least in part attributed to signif i cant burst release [100-102].In a recent study the cytotoxicity and transfection eff i ciency of polyplexes encapsulated into the innermost or the outermost layer of microcapsules consisting of silk f i broin was directly compared in vitro[105].From these experiments it became evident that encapsulation of polyplexes signif icantly reduced cytotoxicity while maintaining transfection eff i ciency.Furthermore,both,cytotoxicity and transfection eff i ciency were reduced when polyplexes were incorporated into the innermost layer compared to polyplexes being present in the outermost layer.One might therefore conclude that despite a signif i cant reduction of cytotoxicity of polyplexes can be achieved through encapsulation and sustained release,delivery systems must still be optimized to achieve a proper balance of transfection eff i ciency versus cytotoxicity.

Recent reports showed that electrospun scaffolds are quite suitable for designing bioresponsive NA delivery systems [106-109].Kim et al.developed a strategy where PEI was coupled via an MMP-sensitive linker onto the surface of electrospun f i bers consisting of PCL-PEG copolymer[106,108,109]. Upon incubation with plasmid DNA or siRNA,eff i cient complexation of NA with surface-bound PEI was observed and MMP activity resulted in release of PEI/DNA complexes and bioresponsive transfection(Fig.6).The therapeutic eff i ciency of this approach was evaluated in an in vivo model of diabetic ulcers,either using plasmid DNA encoding for human epidermal growth factor(hEGF)[108]or employing siRNA targeted at MMP-2[109].In both cases,accelerated wound recovery was observed,either through increased expression of hEGF leading to enhanced re-epithelialization or through reduction of MMP-2 expression resulting in normalization of the wound milieu.These studies show the potential of bioresponsive delivery systems based on electrospun scaffolds with regards to achieving and maintaining just the right drug concentration in response to and controlled by a specif i c diseasestate.Furtherdevelopmentswilllikelyfocuson improved protection of NA during storage and after application,increased loading capacity for NA and simplif i ed synthesis and fabrication of these systems.

Finally,electrospun scaffolds allow the combination of various delivery systems and drugs within one device as well as signif i cant structural variability.The ability to combine different NA within one scaffold is exemplif i ed by studies conducted by Li and colleagues who coencapsulated plasmid DNA coding for VEGF(pVEGF)and FGF(pFGF)into core/shell fi bers by emulsion electrospinning[110,111].In a fi rst study the core contained plasmid DNA/PEI polyplexes while the shell consisted of poly(DL-lactide)-poly(ethylene glycol)(PELA) [111].The release kinetics of plasmid DNA from fi bers containing only pVEGF was very similar to fi bers containing both plasmids and both types showed low burst release of approximately10%(Fig.7A).Cytotoxicityofpolyplexes released from scaffolds was evident but was reduced when polyplexes were encapsulated into the core of the fi bers compared to fi ber mats where polyplexes were adsorbed to the surface(Fig.7B).The authors also observed that cell proliferation after approximately 3 days was more pronounced for scaffolds with encapsulated polyplexes than for adsorbed polyplexes.This was attributed to sustained release of plasmid,resulting in long-lasting transfection and increased growth promotion through the expressed growth factors.

Despite the fact that these scaffolds successfully promoted the generation of blood vessels in vivo,low cell viability,pronounced in fl ammation and necrosis lead to the development of a system using calcium phosphate/DNA(CP/DNA)nanoparticles instead of PEI/DNA polyplexes[110].It was assumed that the transfection ef fi ciency of plain CP/DNA nanoparticles decreases over time due to aggregation and growth of nanoparticles, fi nally resulting in particles too large for cellularuptake[112].Li et al.hypothesized that encapsulation of CP/ DNA nanoparticles into electrospun f i bers might alleviate this phenomenon.CP/DNAnanoparticlesindeedsignif i cantly reduced cytotoxicity and inf l ammation reaction compared to PEI/DNA polyplexes.Furthermore,the combined encapsulation of pVEGF and pFGF was shown to be more effective with regards to the density of newly formed blood vessels in vivo than delivery of individual plasmids[110].

3.3.Physical methods

Besides polymer-and liposome-based non-viral systems, several physical methods have been applied for the localized administration of NA in an attempt to improve safety,eff i cacy and targeting.Physical approaches to NA delivery include hydrodynamic methods,iontophoresis,microneedles,ultrasound and electroporation.The latter two methods are most frequently applied and are described in more detail in the following sections.

3.3.1.Ultrasound

Ultrasound facilitated delivery(sonoporation)of nucleic acids is probably the physical method,which is most often applied for localized delivery.Sonoporation relies on the transient formation of pores in the cell membrane in the presence of microbubbles,resulting in eff i cient uptake of various molecules into the cells[113].Microbubbles have been used as ultrasound contrast agents for several decades and various formulations are commercially available and FDA approved for human use(e.g.SonoVue®from Bracco International, Optison®from Mallinckrodt and Def i nity®from Lantheus Medical Imaging).However,besides use as a contrast agent, these microbubbles have also been recognized as drug delivery vehicles for non-permeable molecules such as macromolecular drugs and NA[114,115].Deshpande and Prausnitz showed in vitro that the combination of sonoporation using Optison microbubbles and PEI/DNA complexes signif i cantly improved transfection eff i ciency over each of the treatments alone[116].Similarly,improved cytoplasmatic uptake of lipoplexes through cell membrane pores was observed[117]. Ultrasound facilitated delivery was also successfully used for siRNA transfection with naked siRNA[118],siRNA-liposome complexes[119]andsiRNA-polycationcomplexes[120]. These studies point towards potential improvements of NA delivery eff i ciency in vivo using ultrasound.Already in 2003, Pislaru et al.found that ultrasound exposure enhanced gene delivery eff i ciency in rat skeletal muscle after intramuscular injection of microbubbles mixed with naked plasmid DNA coding for luciferase compared to the same formulation without ultrasound application[121].A dose-related increase of ultrasound facilitated transfection eff i ciency was observed and transfections through sonoporation were spatially highly restricted compared to control experiments with adenoviral gene transfer(Fig.8).Interestingly,signif i cant differences in transfection eff i ciency in different muscles were observed, which were unexpected and attributed to f l aws inherent to IM injection and varying doses per volume in the different muscles[121].

Anotherapproachtoutilizingsonoporationforthe spatially restricted transfection of tissues was presented by Kowalczuk et al.[122].In this study,transfection of the ocular ciliary muscle with naked DNA in combination with microbubbles and ultrasound was investigated.Similar to the study by Pislaru et al.,signif i cantly improved gene expression was observed after ultrasound treatment.Despitea rise oflens and ciliary muscle temperature during the treatment,no damage to the tissue was observed.Unfortunately,the study was performed using reporter genes only(luciferase,green f l uorescent protein(GFP)and β-galactosidase)but nevertheless shows that expression of therapeutic proteins in the ciliary muscle is potentially feasible.

Ziadloo,Xie and Frenkel recently presented an alternative approach to gene delivery through sonoporation[123].In contrast to many studies in the f i eld,no microbubbles were usedtoimproveultrasoundeff i ciency.Instead,pulsed, focused ultrasound(pFUS)was applied immediately prior to intratumoral injection of naked tumor necrosis factor alpha (TNF-α)plasmidDNA.Exposure to pFUS alone had noeffect on tumor growth compared to the control group.Injection of TNF-α plasmid DNA alone(without pFUS treatment)resulted in signif i cant tumor growth inhibition,which was further signif i cantly improved after pFUS treatment in combination with TNF-α plasmid.Histological evaluation of the tumors revealed larger necrotic regions and improved distribution and penetration of f l uorescent surrogate nanoparticles after pFUS treatment.The authors therefore conclude that besides the structural effects of ultrasound treatment that resulted in an increase of the pore size of the tissue and enhanced distribution of the plasmid,the enlarged space between cells could have also improved the hydraulic conductivity.This would result in increased f l uid f l ow from the tumor core to its periphery and further contribute to improved penetration and distribution of the injected solution(Fig.9).

3.3.2.Electroporation

The permeability of the cell membrane for non-permeable, macromolecular drugs can also be increased through application of an external electrical f i eld.Electroporation has several advantages compared to polymeric and liposomal transfection vectors:a)high transfection eff i ciency in primary cells is achievable,b)fewer safety concerns are associated with this method,c)electroporation is easy to perform, and d)eff i ciency is only marginally inf l uenced by the cell line used[124].Over 30 years of research on electroporation[125] have led to technologically quite advanced methods with several systems being commercially available.Electroporation has been tested in numerous clinical trials,providing evidence that the method can be successfully applied in clinical therapy[126].Due to the wealth of reports available and decades of research,we herein focus on selected recent studies and otherwise refer to the reviews by Gotthelf and Gehl[127]and Wells[128]for detailed information on electroporation.

ThebasicmechanismofsiRNAelectroporationwas recently investigated on single-cell level by Paganin-Gioanni et al.[129].In this study,electrotransfer of NA was directly observed by confocal microscopy,providing evidence for the direct transfer of siRNA across the cell membrane.Electropermeabilization occurred on the two opposite sides of the cell facing the electrodes,whereas siRNA entry was found to occur only on the side facing the cathode,suggesting that electrophoresis aids in electrotransfer.Interestingly,the mechanism of siRNA electrotransfer was found to be somewhat different from plasmid DNA transfer.While siRNA appeared to rapidly and freely penetrate the cell membrane during electroporation,plasmid DNA f i rst formed long-lived spots on the plasma membrane and translocation into the cytoplasm occurred in a second step several minutes after electroporation took place.

Electroporation is well established for the transfer of DNA and extensive studies provided both optimized electroporation devices as well as-parameters.However,fewer studies haveinvestigated methodsfor optimal RNA delivery.Knowing that the basic mechanisms of electroporation-enhanced NA transfer on a cellular level differ between DNA and RNA,it might be hypothesized that optimal transdermal transfer conditions might be widely disparate.Broderick et al.studied in vivo transdermal electroporation for both DNA and siRNA [130].It was shown in this study that a)low voltage electroporation(10 V range)elicited the most robust transfection for plasmid DNA and b)the same parameters applied for plasmid DNA resulted in successful electrotransfer of siRNA.Overall, no site inf l ammation or tissue damage was observed using this procedure conf i rming the good tolerability of low voltage electroporation.

Fromapatientperspective,topicalelectroporationenhanced NA delivery might appear inconvenient due to the necessity of intradermal injection followed by more or less invasive electroporation.Lee et al.therefore developed a monolithic hybrid system consisting of a dissolving microneedle and anelectrode to combine bothsteps within the same device(Fig.10)[131].In contrast to NA-coated metal microneedles,this system allows the transfer of signif i cantly larger amounts of NA into the skin,leading to improved therapeutic eff i ciency.Thedissolvingmicroneedleswereproduced through antidromic isolation by drawing lithography from maltose using the aluminum electrode as drawing pillar and were loaded with plasmid DNA coding for luciferase.Insertion of this device into the skin bypasses the stratum corneum and releases DNA into the interstitial space of the skin.Electroporation can be performed directly through the electrode.Using these hybrid microneedles,subcutaneous tumors were treated with plasmid DNA containing the proinf l ammatory cytokine interleukin 12 in a mouse model.Hybrid electro-microneedles were most eff i cient at suppressing tumor growth and with regards to survival after tumor inoculation.

4.Conclusions and outlook

Numerous approaches to local delivery of NA have been published in recent years.Besides drug delivery systems intended for topical or localized therapy,tissue engineering has been a main driver for research and development. Without reiterating the basic considerations as described in the introduction,it should be emphasized here that for applications where the drug delivery system is in direct contactwith the body for an extended duration,NA delivery might be a promising alternative to protein or peptide delivery due to higher stability of NA and the ability to employ resident cells as bioreactors.Signif i cant progress has been made in the past decades in the development of non-viral vectors for gene delivery,which forms the basis for scaffold-based NA delivery. However,systemic administration of non-viral vectors is still associated with problems such as insuff i cient blood half-life, ineff i cient targeting and cytotoxicity,all of which can be avoided or alleviated by localized delivery.

The discontinuation of the clinical development of naked siRNAfortreatmentofchoroidalandretinalneovascularization was a setback for localized NA delivery in the eye and the discovery of non-specif i c,TLR3-mediated effects of siRNA has questioned the therapeutic suitability of naked siRNA.Nevertheless,both the indication as well as the delivery route are still highly interesting.To improve siRNA specif i c effects,further research into drug delivery systems allowing protecting siRNA from degradation and unspecif i c recognition and with the ability to facilitate eff i cient cellular uptake is necessary.

The development of bioresponsive,enzyme sensitive delivery systems might result in a major improvement of drug therapy as these systems add a new dimension to controlled drug release.Current systems mainly focus on improved cell migration within hydrogels facilitated by MMP-sensitive crosslinks.However,somestudiesshowedthatMMP-sensitive peptides may also be used to control drug release, e.g.resulting in release of MMP inhibitors specif i cally at elevated MMP levels.Advanced electrospun drug delivery system might in the future use this concept for the selfregulated control and normalization of the wound milieu, while at the same time providing mechanical support and protection from contamination by bacteria.

Finally,physical methods for NA delivery such as electroporation have been continuously developed for decades and are highly versatile with regards to the type of NA used.The combination of advanced electroporation techniques with specialized microneedle arraysrepresentsa clinicallyfeasible, relatively simple and potentially safe approach to dermal delivery of NA.While the range of applications of such systems is limited compared to hydrogels or electrospun nonwovens,their simplicity,the improved control over device structure and the well-def i ned location of NA delivery might represent signif i cant advantages with regards to clinical evaluation and regulatory acceptance.

Localized NA delivery therefore is a promising strategy for the safe and eff i cacious treatment of numerous diseases. Further development of non-viral vectors,nano-and microstructuredscaffoldsandNAderivativesisexpectedto resultin signif i cant improvements with regards to controlled NA release,biocompatibilityofscaffoldsandtransfection eff i ciency.

REFERENCES

[1]Xu L,Anchordoquy T.Drug delivery trends in clinical trials and translational medicine:challenges and opportunities in the delivery of nucleic acid-based therapeutics.J Pharm Sci 2011;100:38-52.

[2]Hickerson RP,Vlassov AV,Wang Q,et al.Stability study of unmodif i ed sirna and relevance to clinical use. Oligonucleotides 2008;18:345-354.

[3]Rohland N,Reich D,Mallick S,et al.Genomic DNA sequences from mastodon and woolly mammoth reveal deep speciation of forest and savanna elephants.Plos Biol 2010:8.

[4]Ewe A,Schaper A,Barnert S,et al.Storage stability of optimal liposome-polyethylenimine complexes (lipopolyplexes)for DNA or siRNA delivery.Acta Biomater 2014;10:2663-2673.

[5]Kundu AK,Chandra PK,Hazari S,et al.Stability of lyophilized siRNA nanosome formulations.Int J Pharm 2012;423:525-534.

[6]Kasper JC,Troiber C,Kuchler S,et al.Formulation development of lyophilized,long-term stable siRNA/ oligoaminoamide polyplexes.Eur J Pharm Biopharm 2013;85:294-305.

[7]Liemann S,Glockshuber R.Inf l uence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein. Biochemistry 1999;38:3258-3267.

[8]Newton DL,Boque L,Wlodawer A,et al.Single amino acid substitutions at the N-terminus of a recombinant cytotoxic ribonuclease markedly inf l uence biochemical and biological properties.Biochemistry 1998;37:5173-5183.

[9]Sah DWY.Therapeutic potential of RNA interference for neurological disorders.Life Sci 2006;79:1773-1780.

[10]Uprichard SL.The therapeutic potential of RNA interference.Febs Lett 2005;579:5996-6007.

[11]Guo X,Huang L.Recent advances in nonviral vectors for gene delivery.Acc Chem Res 2012;45:971-979.

[12]Buyens K,De Smedt SC,Braeckmans K,et al.Liposome based systems for systemic siRNA delivery:stability in blood sets the requirements for optimal carrier design. J Control Release 2012;158:362-370.

[13]Bauhuber S,Hozsa C,Breunig M,et al.Delivery of nucleic acids via disulf i de-based carrier systems.Adv Mater 2009;21:3286-3306.

[14]Gullotti E,Yeo Y.Extracellularly activated nanocarriers:a new paradigm of tumor targeted drug delivery.Mol Pharm 2009;6:1041-1051.

[15]Ganta S,Devalapally H,Shahiwala A,et al.A review of stimuli-responsive nanocarriers for drug and gene delivery. J Control Release 2008;126:187-204.

[16]Bertin A.Polyelectrolyte complexes of DNA and polycations as gene delivery vectors.Polyelectrolyte complexes in the dispersed and solid state ii:application aspects,vol.256; 2014.p.103-195.

[17]Germershaus O,Mao SR,Sitterberg J,et al.Gene delivery using chitosan,trimethyl chitosan or polyethylenglycolgraft-trimethyl chitosan block copolymers:establishment of structure-activity relationships in vitro.J Control Release 2008;125:145-154.

[18]Boussif O,Lezoualch F,Zanta MA,et al.A versatile vector for gene and oligonucleotide transfer into cells in culture and in-vivo-polyethylenimine.Proc Natl Acad Sci U S A 1995;92:7297-7301.

[19]Mao SR,Sun W,Kissel T.Chitosan-based formulations for delivery of DNA and siRNA.Adv Drug Deliv Rev 2010;62:12-27.

[20]Dufes C,Uchegbu IF,Schatzlein AG.Dendrimers in gene delivery.Adv Drug Deliv Rev 2005;57:2177-2202.

[21]Benjaminsen RV,Mattebjerg MA,Henriksen JR,et al.The possible“proton sponge”effect of polyethylenimine(PEI) does not include change in lysosomal pH.Mol Ther 2013;21:149-157.

[22]Rehman ZU,Hoekstra D,Zuhorn IS.Mechanism of polyplex-and lipoplex-mediated delivery of nucleic acids: real-time visualization of transient membrane destabilization without endosomal lysis.ACS Nano 2013;7:3767-3777.

[23]Petersen H,Fechner PM,Martin AL,et al.Polyethyleniminegraft-poly(ethylene glycol)copolymers:inf l uence of copolymer block structure on DNA complexation and biological activities as gene delivery system.Bioconjug Chem 2002;13:845-854.

[24]Ogris M,Brunner S,Schuller S,et al.PEGylated DNA/ transferrin-PEI complexes:reduced interaction with blood components,extended circulation in blood and potential for systemic gene delivery.Gene Ther 1999;6:595-605.

[25]Germershaus O,Merdan T,Bakowsky U,et al. Trastuzumab-polyethylenimine-polyethylene glycol conjugates for targeting Her2-expressing tumors.Bioconjug Chem 2006;17:1190-1199.

[26]Germershaus O,Neu M,Behe M,et al.HER2 targeted polyplexes:the effect of polyplex composition and conjugation chemistry on in vitro and in vivo characteristics.Bioconjug Chem 2008;19:244-253.

[27]Neu M,Germershaus O,Mao S,et al.Crosslinked nanocarriers based upon poly(ethylene imine)for systemic plasmid delivery:in vitro characterization and in vivo studies in mice.J Control Release 2007;118:370-380.

[28]Neu M,Germershaus O,Behe M,et al.Bioreversibly crosslinked polyplexes of PEI and high molecular weight PEG show extended circulation times in vivo.J Control Release 2007;124:69-80.

[29]Florea BI,Meaney C,Junginger HE,et al.Transfection eff i ciency and toxicity of polyethylenimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures.AAPS PharmSci 2002:4.

[30]vandeWetering P,Cherng JY,Talsma H,et al.Relation between transfection eff i ciency and cytotoxicity of poly(2-(dimethylamino)ethyl methacrylate)/plasmid complexes. J Control Release 1997;49:59-69.

[31]Breunig M,Lungwitz U,Liebl R,et al.Breaking up the correlation between eff i cacy and toxicity for nonviral gene delivery.Proc Natl Acad Sci U S A 2007;104:14454-14459.

[32]Peng Q,Zhong ZL,Zhuo RX.Disulf i de cross-linked polyethylenimines(PEI)prepared via thiolation of low molecular weight PEI as highly eff i cient gene vectors. Bioconjug Chem 2008;19:499-506.

[33]Yin H,Kanasty RL,Eltoukhy AA,et al.Non-viral vectors for gene-based therapy.Nat Rev Genet 2014;15:541-555.

[34]Felgner PL,Gadek TR,Holm M,et al.Lipofection-a highly eff i cient,lipid-mediated DNA-transfection procedure.Proc Natl Acad Sci U S A 1987;84:7413-7417.

[35]Konopka K,Stamatatos L,Larsen CE,et al.Enhancement of human-immunodef i ciency-virus type-1 infection by cationic liposomes-the role of cd4,serum and liposome cell-interactions.J Gen Virol 1991;72:2685-2696.

[36]Felgner JH,Kumar R,Sridhar CN,et al.Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations.J Biol Chem 1994;269:2550-2561.

[37]Zuhorn IS,Kalicharan R,Hoekstra D.Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis.J Biol Chem 2002;277:18021-18028.

[38]Dass CR.Cytotoxicity issues pertinent to lipoplex-mediated gene therapy in-vivo.J Pharm Pharmacol 2002;54:593-601.

[39]de Ilarduya CT,Sun Y,Duezguenes N.Gene delivery by lipoplexes and polyplexes.Eur J Pharm Sci 2010;40:159-170.

[40]Ozpolat B,Sood AK,Lopez-Berestein G.Liposomal siRNA nanocarriers for cancer therapy.Adv Drug Deliv Rev 2014;66:110-116.

[41]Li SD,Huang L.Targeted delivery of antisense oligodeoxynucleotide and small interference RNA into lung cancer cells.Mol Pharm 2006;3:579-588.

[42]Garcia L,Urbiola K,Duzgunes N,et al.Lipopolyplexes as nanomedicines for therapeutic gene delivery. Nanomedicine:infectious diseases,immunotherapy, diagnostics,antif i brotics,toxicology and gene medicine, vol.509;2012.p.327-338.

[43]Chono S,Li SD,Conwell CC,et al.An eff i cient and low immunostimulatory nanoparticle formulation for systemic siRNA delivery to the tumor.J Control Release 2008;131:64-69.

[44]Jain S,Kumar S,Agrawal AK,et al.Enhanced transfection eff i ciency and reduced cytotoxicity of novel lipid-polymer hybrid nanoplexes.Mol Pharm 2013;10:2416-2425.

[45]Shen J,Samul R,Silva RL,et al.Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1. Gene Ther 2006;13:225-234.

[46]Kaiser PK,Symons RCA,Shah SM,et al.RNAi-based treatment for neovascular age-related macular degeneration by Sirna-027.Am J Ophthalmol 2010;150:33-39.

[47]Nguyen QD,Schachar RA,Nduaka CI,et al.Phase 1 doseescalation study of a siRNA targeting the RTP801 gene in age-related macular degeneration patients.Eye 2012;26:1099-1105.

[48]Nguyen QD,Schachar RA,Nduaka CI,et al.Dose-ranging evaluation of intravitreal siRNA PF-04523655 for diabetic macular edema(the DEGAS Study).Invest Ophthalmol Vis Sci 2012;53:7666-7674.

[49]Kleinman ME,Yamada K,Takeda A,et al.Sequence-and target-independent angiogenesis suppression by siRNA via TLR3.Nature 2008;452:591-597.

[50]Meuli M,Liu Y,Liggitt D,et al.Eff i cient gene expression in skin wound sites following local plasmid injection.J Invest Dermatol 2001;116:131-135.

[51]Hengge UR,Chan EF,Foster RA,et al.Cytokine geneexpression in epidermis with biological effects following injection of naked DNA.Nat Genet 1995;10:161-166.

[52]Sun LY,Xu L,Chang H,et al.Transfection with aFGF cDNA improves wound healing.J Invest Dermatol 1997;108:313-318.

[53]Sawamura D,Yasukawa K,Kodama K,et al.The majority of keratinocytes incorporate intradermally injected plasmid DNA regardless of size but only a small proportion of cells can express the gene product.J Invest Dermatol 2002;118:967-971.

[54]Ciernik IF,Krayenbuhl BH,Carbone DP.Puncturemediated gene transfer to the skin.Hum Gene Ther 1996;7:893-899.

[55]Eriksson E,Yao F,Svensjo T,et al.In vivo gene transfer to skin and wound by microseeding.J Surg Res 1998;78:85-91.

[56]Germershaus O,Luehmann T,Rybak J-C,et al.Application of natural and semi-synthetic polymers for the delivery of sensitive drugs.Int Mater Rev 2014;60:101-131.

[57]Cohen-Sacks H,Elazar V,Gao JC,et al.Delivery and expression of pDNA embedded in collagen matrices. J Control Release 2004;95:309-320.

[58]Capito RM,Spector M.Collagen scaffolds for nonviral IGF-1 gene delivery in articular cartilage tissue engineering.Gene Ther 2007;14:721-732.

[59]Wang WW,Li WZ,Ong LL,et al.Localized SDF-1alpha gene release mediated by collagen substrate induces CD117+ stem cells homing.J Cell Mol Med 2010;14:392-402.

[60]des Rieux A,Shikanov A,Shea LD.Fibrin hydrogels for nonviral vector delivery in vitro.J Control Release 2009;136:148-154.

[61]Lei YG,Rahim M,Ng Q,et al.Hyaluronic acid and fi brin hydrogels with concentrated DNA/PEI polyplexes for local gene delivery.J Control Release 2011;153:255-261.

[62]Kong HJ,Kim ES,Huang YC,et al.Design of biodegradable hydrogel for the local and sustained delivery of angiogenic plasmid DNA.Pharm Res 2008;25:1230-1238.

[63]Krebs MD,Salter E,Chen E,et al.Calcium alginate phosphate-DNA nanoparticle gene delivery from hydrogels induces in vivo osteogenesis.J Biomed Mater Res Part A 2010;92A:1131-1138.

[64]Han HD,Mora EM,Roh JW,et al.Chitosan hydrogel for localized gene silencing.Cancer Biol Ther 2011;11:839-845.

[65]Segovia N,Pont M,Oliva N,et al.Hydrogel doped with nanoparticles for local sustained release of siRNA in breast cancer.Adv Healthc Mater 2014[Epub ahead of print].

[66]Shepard JA,Wesson PJ,Wang CE,et al.Gene therapy vectors with enhanced transfection based on hydrogels modi fi ed with af fi nity peptides.Biomaterials 2011;32:5092-5099.

[67]Li ZH,Ning W,Wang JM,et al.Controlled gene delivery system based on thermosensitive biodegradable hydrogel. Pharm Res 2003;20:884-888.

[68]Nguyen K,Dang PN,Alsberg E.Functionalized, biodegradable hydrogels for control over sustained and localized siRNA delivery to incorporated and surrounding cells.Acta Biomater 2013;9:4487-4495.

[69]Lei YG,Huang SX,Sharif-Kashani P,et al.Incorporation of active DNA/cationic polymer polyplexes into hydrogel scaffolds.Biomaterials 2010;31:9106-9116.

[70]Skiles M,Blanchette J.Polymeric drug delivery systems in tissue engineering.Engineering polymer systems for improved drug delivery.2014.

[71]Takahashi H,Wang YW,Grainger DW.Device-based local delivery of siRNA against mammalian target of rapamycin (mTOR)in a murine subcutaneous implant model to inhibit fi brous encapsulation.J Control Release 2010;147:400-407.

[72]Manaka T,Suzuki A,Takayama K,et al.Local delivery of siRNA using a biodegradable polymer application to enhance BMP-induced bone formation.Biomaterials 2011;32:9642-9648.

[73]Kim YM,Park MR,Song SC.Injectable polyplex hydrogel for localized and long-term delivery of siRNA.ACS Nano 2012;6:5757-5766.

[74]Li ZB,Yin H,Zhang ZX,et al.Supramolecular anchoring of DNA polyplexes in cyclodextrin-based polypseudorotaxane hydrogels for sustained gene delivery.Biomacromolecules 2012;13:3162-3172.

[75]Kim YM,Park MR,Song SC.An injectable cell penetrable nano-polyplex hydrogel for localized siRNA delivery. Biomaterials 2013;34:4493-4500.

[76]Nguyen MK,Jeon O,Krebs MD,et al.Sustained localized presentation of RNA interfering molecules from in situ forming hydrogels to guide stem cell osteogenic differentiation.Biomaterials 2014;35:6278-6286.

[77]Kwon JS,Park IK,Cho AS,et al.Enhanced angiogenesis mediated by vascular endothelial growth factor plasmidloaded thermo-responsive amphiphilic polymer in a rat myocardial infarction model.J Control Release 2009;138:168-176.

[78]Lei YG,Ng QKT,Segura T.Two and three-dimensional gene transfer from enzymatically degradable hydrogel scaffolds. Microsc Res Tech 2010;73:910-917.

[79]Orsi S,Guarnieri D,Netti PA.Design of novel 3D gene activated PEG scaffolds with ordered pore structure.J Mater Sci Mater Med 2010;21:1013-1020.

[80]Gojgini S,Tokatlian T,Segura T.Utilizing cell-matrix interactions to modulate gene transfer to stem cells inside hyaluronic acid hydrogels.Mol Pharm 2011;8:1582-1591.

[81]Shepard JA,Huang A,Shikanov A,et al.Balancing cell migration with matrix degradation enhances gene delivery to cells cultured three-dimensionally within hydrogels. J Control Release 2010;146:128-135.

[82]Keeney M,Onyiah S,Zhang Z,et al.Modulating polymer chemistry to enhance non-viral gene delivery inside hydrogels with tunable matrix stiffness.Biomaterials 2013;34:9657-9665.

[83]You JO,Almeda D,Ye GJ,et al.Bioresponsive matrices in drug delivery.J Biol Eng 2010;4:15.

[84]Nagase H,Visse R,Murphy G.Structure and function of matrix metalloproteinases and TIMPs.Cardiovasc Res 2006;69:562-573.

[85]Lutolf MP,Lauer-Fields JL,Schmoekel HG,et al.Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration:engineering cellinvasion characteristics.Proc Natl Acad Sci U S A 2003;100:5413-5418.

[86]Lee SH,Moon JJ,Miller JS,et al.Poly(ethylene glycol) hydrogels conjugated with a collagenase-sensitive fl uorogenic substrate to visualize collagenase activity during three-dimensional cell migration.Biomaterials 2007;28:3163-3170.

[87]Raeber GP,Lutolf MP,Hubbell JA.Molecularly engineered PEG hydrogels:a novel model system for proteolytically mediated cell migration.Biophys J 2005;89:1374-1388.

[88]Purcell BP,Lobb D,Charati MB,et al.Injectable and bioresponsive hydrogels for on-demand matrix metalloproteinase inhibition.Nat Mater 2014;13:653-661.

[89]Lei Y,Segura T.DNA delivery from matrix metalloproteinase degradable poly(ethylene glycol) hydrogels to mouse cloned mesenchymal stem cells. Biomaterials 2009;30:254-265.

[90]Guelcher SA.Biodegradable polyurethanes:synthesis and applications in regenerative medicine.Tissue Eng Part B Rev 2008;14:3-17.

[91]Nelson DM,Baraniak PR,Ma Z,et al.Controlled release of IGF-1 and HGF from a biodegradable polyurethane scaffold. Pharm Res 2011;28:1282-1293.

[92]Li B,Yoshii T,Hafeman AE,et al.The effects of rhBMP-2 released from biodegradable polyurethane/microsphere composite scaffolds on new bone formation in rat femora. Biomaterials 2009;30:6768-6779.

[93]Li B,Davidson JM,Guelcher SA.The effect of the local delivery of platelet-derived growth factor from reactive two-component polyurethane scaffolds on the healing in rat skin excisional wounds.Biomaterials 2009;30:3486-3494.

[94]Li B,Brown KV,Wenke JC,et al.Sustained release of vancomycin from polyurethane scaffolds inhibits infection of bone wounds in a rat femoral segmental defect model. J Control Release 2010;145:221-230.

[95]Nelson CE,Gupta MK,Adolph EJ,et al.Sustained local delivery of siRNA from an injectable scaffold.Biomaterials 2012;33:1154-1161.

[96]Nelson CE,Kim AJ,Adolph EJ,et al.Tunable delivery of siRNA from a biodegradable scaffold to promote angiogenesis in vivo.Adv Mater 2014;26:607-614.

[97]Rives CB,des Rieux A,Zelivyanskaya M,et al.Layered PLG scaffolds for in vivo plasmid delivery.Biomaterials 2009;30:394-401.

[98]Andersen MO,Le DQS,Chen MW,et al.Spatially controlled delivery of siRNAs to stem cells in implants generated by multi-component additive manufacturing.Adv Funct Mater 2013;23:5599-5607.

[99]Meinel AJ,Germershaus O,Luhmann T,et al.Electrospun matrices for localized drug delivery:current technologiesand selected biomedical applications.Eur J Pharm Biopharm 2012;81:1-13.

[100]Luu YK,Kim K,Hsiao BS,et al.Development of a nanostructured DNA delivery scaffold via electrospinning of PLGA and PLA-PEG block copolymers.J Control Release 2003;89:341-353.

[101]Liang DH,Luu YK,Kim KS,et al.In vitro non-viral gene delivery with nano fi brous scaffolds.Nucleic Acids Res 2005:33.

[102]Nie HM,Wang CH.Fabrication and characterization of PLGA/HAp scaffolds for delivery of BMP-2 plasmid composite DNA.J Control Release 2007;120:111-121.

[103]Saraf A,Baggett LS,Raphael RM,et al.Regulated non-viral gene delivery from coaxial electrospun fi ber mesh scaffolds. J Control Release 2010;143:95-103.

[104]Sakai S,Yamada Y,Yamaguchi T,et al.Surface immobilization of poly(ethyleneimine)and plasmid DNA on electrospun poly(L-lactic acid) fi brous mats using a layerby-layer approach for gene delivery.J Biomed Mater Res Part A 2009;88A:281-287.

[105]Li L,Puhl S,Meinel L,et al.Silk fi broin layer-by-layer microcapsules for localized gene delivery.Biomaterials 2014;35:7929-7939.

[106]Kim HS,Yoo HS.MMPs-responsive release of DNA from electrospun nano fi brous matrix for local gene therapy: in vitro and in vivo evaluation.J Control Release 2010;145:264-271.

[107]Blocker KM,Kiick KL,Sullivan MO.Surface immobilization of plasmid DNA with a cell-responsive tether for substratemediated gene delivery.Langmuir 2011;27:2739-2746.

[108]Kim HS,Yoo HS.In vitro and in vivo epidermal growth factor gene therapy for diabetic ulcers with electrospun fi brous meshes.Acta Biomater 2013;9:7371-7380.

[109]Kim HS,Yoo HS.Matrix metalloproteinase-inspired suicidal treatments of diabetic ulcers with siRNA-decorated nano fi brous meshes.Gene Ther 2013;20:378-385.

[110]Chen F,Wan HY,Xia T,et al.Promoted regeneration of mature blood vessels by electrospun fi bers with loaded multiple pDNA-calcium phosphate nanoparticles.Eur J Pharm Biopharm 2013;85:699-710.

[111]He SH,Xia T,Wang H,et al.Multiple release of polyplexes of plasmids VEGF and bFGF from electrospun fi brous scaffolds towards regeneration of mature blood vessels.Acta Biomater 2012;8:2659-2669.

[112]Sokolova VV,Radtke I,Heumann R,et al.Effective transfection of cells with multi-shell calcium phosphate-DNA nanoparticles.Biomaterials 2006;27:3147-3153.

[113]Miller DL,Pislaru SV,Greenleaf JE.Sonoporation: mechanical DNA delivery by ultrasonic cavitation.Somat Cell Mol Genet 2002;27:115-134.

[114]Bao SP,Thrall BD,Miller DL.Transfection of a reporter plasmid into cultured cells by sonoporation in vitro. Ultrasound Med Biol 1997;23:953-959.

[115]Duvshani-Eshet M,Machluf M.Therapeutic ultrasound optimization for gene delivery:a key factor achieving nuclear DNA localization.J Control Release 2005;108:513-528.

[116]Deshpande MC,Prausnitz MR.Synergistic effect of ultrasound and PEI on DNA transfection in vitro.J Control Release 2007;118:126-135.

[117]Lentacker I,Wang N,Vandenbroucke RE,et al.Ultrasound exposure of lipoplex loaded microbubbles facilitates direct cytoplasmic entry of the lipoplexes.Mol Pharm 2009;6:457-467.

[118]Otani K,Yamahara K,Ohnishi S,et al.Nonviral delivery of siRNA into mesenchymal stem cells by a combination of ultrasound and microbubbles.J Control Release 2009;133:146-153.

[119]Vandenbroucke RE,Lentacker I,Demeester J,et al. Ultrasound assisted siRNA delivery using PEG-siPlex loaded microbubbles.J Control Release 2008;126:265-273.

[120]Florinas S,Kim J,Nam K,et al.Ultrasound-assisted siRNA delivery via arginine-grafted bioreducible polymer and microbubbles targeting VEGF for ovarian cancer treatment. J Control Release 2014;183:1-8.

[121]Pislaru SV,Pislaru C,Kinnick RR,et al.Optimization of ultrasound-mediated gene transfer:comparison of contrast agents and ultrasound modalities.Eur Heart J 2003;24:1690-1698.

[122]Kowalczuk L,Boudinet M,El Sanharawi M,et al.In vivo gene transfer into the ocular ciliary muscle mediated by ultrasound and microbubbles.Ultrasound Med Biol 2011;37:1814-1827.

[123]Ziadloo A,Xie JW,Frenkel V.Pulsed focused ultrasound exposures enhance locally administered gene therapy in a murine solid tumor model.J Acoust Soc Am 2013;133:1827-1834.

[124]Geng T,Zhan YH,Wang HY,et al.Flow-through electroporation based on constant voltage for large-volume transfection of cells.J Control Release 2010;144:91-100.

[125]Neumann E,Schaeferridder M,Wang Y,et al.Gene-transfer into mouse lyoma cells by electroporation in high electricfi elds.EMBO J 1982;1:841-845.

[126]Bodles-Brakhop AM,Heller R,Draghia-Akli R. Electroporation for the delivery of dna-based vaccines and immunotherapeutics:current clinical developments.Mol Ther 2009;17:585-592.

[127]Gothelf A,Gehl J.What you always needed to know about electroporation based DNA vaccines.Hum Vaccin Immunother 2012;8:1694-1702.

[128]Wells DJ.Gene therapy progress and prospects: electroporation and other physical methods.Gene Ther 2004;11:1363-1369.

[129]Paganin-Gioanni A,Bellard E,Escoffre JM,et al.Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells.Proc Natl Acad Sci U S A 2011;108:10443-10447.

[130]Broderick KE,Chan A,Lin F,et al.Optimized in vivo transfer of small interfering rna targeting dermal tissue using in vivo surface electroporation.Mol Ther Nucleic Acids 2012;1.

[131]Lee K,Kim JD,Lee CY,et al.A high-capacity,hybrid electromicroneedle for in-situ cutaneous gene transfer. Biomaterials 2011;32:7705-7710.

*Corresponding author.Institute for Pharma Technology,School of Life Sciences,University of Applied Sciences and Arts Northwestern Switzerland,Gru¨ndenstrasse 40,CH 4132,Muttenz,Switzerland.Tel.:+41 61 467 44 48.

E-mail address:oliver.germershaus@fhnw.ch(O.Germershaus).

Peer review under responsibility of Shenyang Pharmaceutical University.

http://dx.doi.org/10.1016/j.ajps.2014.10.001

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