Nanoparticulate systems for the delivery of antisense oligonucleotides

Advanced Drug Delivery Reviews 47 (2001) 99–112www.elsevier.com/ locate /drugdeliv

Nanoparticulate systems for the delivery of antisenseoligonucleotides

*Gregory Lambert, Elias Fattal, Patrick Couvreur

´Laboratoire de Physico-Chimie, Pharmacotechnie et Biopharmacie, URA CNRS 8612, Faculte de Pharmacie, 5,´ ˆRue Jean-Baptiste Clement, 92296 Chatenay-Malabry, France

Accepted 9 October 2000

Abstract

Antisense oligonucleotides are molecules that are able to inhibit gene expression being therefore potentially active for thetreatment of viral infections or cancer. However, because of their poor stability in biological medium and their weakintracellular penetration, colloidal drugs carriers such as nanoparticles were developed for the delivery of oligonucleotides(ODN). ODN associated to nanoparticles were shown to be protected against degradation and to penetrate more easily intodifferent types of cells. As a consequence, nanoparticles were shown to improve the efficiency of ODNs for the inhibition ofthe proliferation of cells expressing the point mutated Ha-ras gene. In vivo, polyalkylcyanoacrylate (PACA) nanoparticleswere able to efficiently distribute the ODNs to the liver whereas the alginate nanosponges could concentrate the ODNs in thelungs. Finally, ODN loaded to PACA nanoparticles were able to improve in mice, the treatment of RAS cells expressing thepoint mutated Ha-ras gene. 2001 Elsevier Science B.V. All rights reserved.

Keywords: Nanoparticles; Nanocapsules; Nanospheres; Oligonucleotide; Antisense

Contents

1. Introduction: antisense strategy and the need for particulate formulations .................................................................................... 1002. Nanospheres ........................................................................................................................................................................... 103

2.1. Preparation of nanospheres by polymerisation of a monomer............................................................................................... 1032.2. Nanospheres obtained from preformed polymers ................................................................................................................ 1042.3. Association of ODNs to nanospheres ................................................................................................................................. 105

3. In vitro stability of ODNs adsorbed onto nanospheres ................................................................................................................ 1074. Cell interactions with ODNs loaded nanospheres ....................................................................................................................... 1085. In vitro pharmacological activity of oligonucleotides-loaded nanospheres.................................................................................... 1086. In vivo studies with oligonucleotide nanospheres ....................................................................................................................... 1097. Conclusion ............................................................................................................................................................................. 109References .................................................................................................................................................................................. 110

*Corresponding author. Tel.: 1 33-146-83-5396; fax: 1 33-146-61-9634.E-mail address: [email protected] (P. Couvreur).

0169-409X/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0169-409X( 00 )00116-2

100 G. Lambert et al. / Advanced Drug Delivery Reviews 47 (2001) 99 –112

1. Introduction: antisense strategy and the need TA base pairs and between protonated cytosine andfor particulate formulations CG base pairs [3]. A second motif for triple helix

recognition of double-stranded DNA is comprised byThe aim of antisense strategy is to interfere with a homopurine motif in which a purine-rich oligo-

gene expression by preventing the translation of nucleotide binds to DNA antiparallel to the Watson–proteins from mRNA. Theoretically, an antisense Crick purine strand [4]. Pyrimidine unmodifiedoligonucleotide is a short fragment (from 15 to 20 oligodeoxynucleotides or backbone-modified oligo-sequence bases) of deoxynucleotides that have a nucleotides are able to block gene transcription in asequence complementary to a portion of the targeted sequence specific manner [3,5,6]. Another mecha-mRNA. The antisense oligonucleotides then hybrid- nism of RNA inactivation is mediated by ribozymes.ize with the mRNA by Watson–Crick base pairing The 59 and 39 ends of these ribonucleotides areand blocks sterically the translation of this transcript complementary to the target RNA and contain aninto a protein [1] (Fig. 1). This mechanism is called intramolecular hairpin loop, which induces the cleav-translational arrest (Fig. 1). Another mechanism that age of the target RNA [7]. However, the limitationswas widely described is the destruction of antisense- of ribozymes as potential antisense therapeutic aremRNA hybrids by an enzyme which get activated: similar to those described below for antisense RNA.the RNaase H [2] (Fig. 1). Inactivation of gene Finally, it has been recently described that a hairpinexpression by oligonucleotides might also be exerted structure in the target mRNA can be cleaved by anby triple helix formation between genomic double- antisense oligodeoxynucleotide bound to both sides

21stranded DNA and oligonucleotides (Fig. 1). This of the hairpin in the presence of Cu ions and asequence specific binding is achieved through reducing agent [8].Hoogsteen hydrogen bonds between thymidine and Antisense oligonucleotides consist in natural phos-

Fig. 1. Main mechanisms of the antisense strategy.

G. Lambert et al. / Advanced Drug Delivery Reviews 47 (2001) 99 –112 101

phodiester compounds (Fig. 2). However, crucial 29 position with a hydroxymethyl group converts theproblems such as the poor stability of these oligo- sugar to a modified ribose. The resultant RNA/RNAnucleotides versus nuclease activity in vitro and in duplex formed with the target RNA is more stable,vivo and their low intracellular penetration have as indicated by the elevated Tm. One modificationlimited their use in therapeutics [9,10]. As a result, can also be provided by replacement of the deoxy-practical application of antisense oligonucleotides ribose phosphate backbone as in peptide nucleichas required modifications with the aim of retaining acids [11]. Thus, by these modifications one canthe hybridization capacity while increasing stability synthesize tailored antisense oligonucleotides with aand cellular penetration. The chemical modifications balance of characteristics of hybridization affinity,have mainly focused on the phosphodiester backbone hydrophobicity and capacity to recruit RNase H-and/or the sugar moiety (Fig. 2). Replacement of the mediated hydrolysis of the target RNA.non-bridging oxygen of the phosphodiester backbone Although these chemical modifications have pro-by sulfur results in a phosphorothioate (PS) with vided an improvement of stability and cell penetra-enhanced stability to enzymatic degradation (Fig. 2). tion, they have also resulted in a variety of non-Although the duplex formed with the target RNA has antisense activities. Thus, when evaluating an oligo-a lower melting temperature (Tm) (i.e. lower affini- nucleotide, which has been designed as, antisense toty) than the phosphodiester compound, it remains a a target RNA, assaying the pharmacological activitysubstrate for RNase H. Another approach is the alone is insufficient and may be misleading. Thereplacement of the non-bridging oxygen by a methyl so-called aptameric effect of oligonucleotide is basedgroup which results in a greater hydrophobicity due on the fact that oligonucleotides can fold into three-to the loss of the negative charge. However in this dimensional structures [12]. Oligonucleotide aptam-case, RNase H activation is also reduced. Alter- ers are able to bind to receptors, enzymes and othernatively, replacing the hydrogen at the deoxyribose proteins and affect their function; this can occur in a

Fig. 2. Different types of chemically modified oligonucleotides.


non-specific fashion. In this view, oligonucleotides process used for the preparation of nanoparticles,containing a four repeated G bases interacts spe- nanospheres or nanocapsules can be obtained.cifically with proteins [13,14]. In addition, phosphor- Nanocapsules are vesicular systems in which theothiate oligonucleotides were described to bind to a drug is confined to a cavity (an oily or aqueous core)wide number of proteins in a sequence-independent surrounded by a unique polymeric membrane; nanos-manner [15]. They can also affect clotting and pheres are matrix systems in which the drug iscomplement systems [16]. Finally, oligonucleotides dispersed throughout the particles. Nanoparticlescontaining unmethylated CpG dinucleotides were were first developed in the mid-1970s by Birrenbachshown to display immunomodulation properties and Speiser [20]. Later on, their application for the[17,18]. design of drug delivery systems was made available

Thus, in order to increase ODN stability, to by the use of biodegradable polymers that wereimprove cell penetration and to avoid non specific considered to be highly suitable for human applica-aptameric effect, the use of particulate carriers such tions [21]. At that time, the research on colloidalas liposomes or nanoparticles, may be considered as carriers was mainly focusing on liposomes but nobeing the more realistic approach to deliver ODNs. one was able to produce stable lipid vesicles suitableIndeed colloidal carriers were found to be able to for clinical applications. In some cases, nanoparticlesprotect natural unmodified phosphodiester ODNs have been shown to be more efficient drug carriersagainst degradation and since they are taken up by than liposomes due to their better stability [22]. Thisendocytosis they could also increase cell penetration is the reason why many drugs were associated toof ODNs (Fig. 3). Among drug carriers, nanoparti- nanoparticles in the last decades (e.g.; antibiotics,cles biodegradable or not have shown interesting antiviral and antiparasitic drugs, cytostatics, proteinpotentialities to bind and deliver ODNs in an effi- and peptides). The main interest of nanoparticles iscient manner [19]. their ability to achieve tissue targeting and enhance

Nanoparticles are defined as being submicronic the intracellular penetration of drugs. Indeed,( , 1 mm) colloidal systems generally made of nanoparticles are mainly taken up by the cells of thepolymers (biodegradable or not). According to the Mononuclear Phagocyte System (MPS) in the liver,

Fig. 3. Mechanism of internalization and hypothesized intracellular release of ODNs associated to biodegradable nanoparticles.


the spleen, lungs and bone marrow [23]. The uptakeoccurs through an endocytosis process after whichthe particles end up in the lysosomal compartment[23] where they are degraded producing low molecu-lar weight soluble compounds that are eliminatedfrom the body by renal excretion [24]. As a result ofthe MPS site specific targeting, avoidance of someorgans was made possible thus, reducing the sideeffects and toxicity of some active compounds. Dueto their lysosomal localization, it may be consideredthat nanoparticles are not suitable to address ODNsefficiently to the cell cytoplasm. Therefore, and inorder to avoid trapping of ODN loaded nanoparticleswithin the lysosomal compartment, several com-pounds able to destabilize the lysosomal membranewere associated with the nanoparticles (e.g.; cationicsurfactant or cationic hydrophobic peptides) [25–27].Recently, in order to avoid MPS uptake, severalgroups have developed a strategy consisting in thelinkage to the nanoparticles of polyethylene glycolderivatives [28–32]. This approach has resulted in alower uptake of nanoparticles by the MPS and in alonger circulation time [29,32]. As a consequence,

these so-called stealth nanoparticles would be ableto extravasate in a selective manner across endo-thelium that becomes permeable due to the presenceof solid tumors [33,34] or inflamed tissues [35].However, this technology although available has notbeen applied so far for the delivery of ODNs.

2. Nanospheres

Several methods have been developed for prepar-ing nanospheres. They can be classified in two maincategories according to whether the formation ofnanospheres requires a polymerization reaction, orwhether it is achieved directly from a macromoleculeor a preformed polymer (Fig. 4).

2.1. Preparation of nanospheres by polymerisationof a monomer

Nanospheres preparation methods based on thepolymerization of monomers generally consist in

Fig. 4. Examples of preparation methods of nanoparticles.introducing a monomer into an aqueous phase or indissolving the monomer in a non solvent of thepolymer. The polymerization reaction in these sys-


tems generally occurs in two steps: a nucleation the overall polymerization rate and particle numberphase followed by a growth phase. Couvreur et al. increased dramatically upon increasing the functional[21] developed nanospheres consisting of poly- monomer concentration and the molecular weight of(alkylcyanoacrylates) (PACA). These polymers, polymer samples decreased with the functionalwhich have been used for several years as surgical monomer concentration revealing the strong activityglues are bioerodible [24] which is the most signifi- of VBAH and AEMH in the chain transfer. Thecant advantage of alkylcyanoacrylates over other colloidal and surface properties of both copolymeracrylic derivatives previously used. In contrast to were investigated and shown to induce rather similarother acrylic derivatives, requiring an energy input properties [44]. It was found that the final particlefor the polymerization, alkylcyanoacrylates can be size was decreasing with increasing the functionalpolymerized easily without such a contribution monomer concentration. In addition, on both types ofwhich is another advantage regarding the stability of latexes, increasing the functional monomer concen-the associated drug. These nanospheres are prepared tration caused the surface amino groups density to

2by emulsion polymerization of cyanoacrylic mono- increase from 0 to a plateau value at 8.2 mC/cmmers dispersed in an acidic aqueous phase (Fig. 4a). [44]. Furthermore, Ganachaud et al. [45] have com-The size of the nanospheres obtained is approximate- pared the synthesis of these polystyrene latex par-ly 200 nm, but it can be reduced to 30–40 nm using ticles by different polymerization method: either bya non ionic surfactant in the polymerization medium seed particle functionalization or by the shot-growth[36] or by adding SO to the monomer [37]. Freeze procedure using 2,29-azobis(2-amidino-propane)2

fracture studies have revealed that the internal struc- dihydrichloride as an initiator and VBAH as ature of these cyanoacrylic nanospheres consisted well cationic monomer. The seed particle functionaliza-in a matrix made up of a dense polymeric network tion is based on the functionalization of preformed[38]. Molecular weight determinations made by gel spheres. The seeding material (styrene and VBAH)permeation chromatography suggested that nanos- were added according to a batch process (one shot)pheres are built up from an entanglement of numer- or in a semi continuous manner. The shot growthous small oligomeric subunits rather than from the approach requires as a first step the synthesis ofrolling up of one or a few long polymer chains [39]. particles with low amounts of functional monomer inThe anionic emulsion polymerization has been order to set the particle size. The remaining amountsturned to good account for preparing nanospheres of of styrene and VBAH were added in a second step atpoly(dialkylmethilidene malonate), a biomaterial dis- about 80% conversion which is high enough to avoidplaying a great potential as alternative drug delivery secondary nucleation process [45]. It was found thatsystem [40–42]. functionalizing seed particles gave poor function-

Although non biodegradable, cationic polystyrene alization yields whereas the shot growth methodnanospheres were proposed also for the adsorption of allows to synthesize spherical monodispersion par-ODNs. ODNs that are bound to such solid support ticles with differing charge densities and fairly goodcan be useful as hybridization probes and affinity yields [45]. In this process, ‘‘core-shell’’ like struc-matrices for binding specifically to complementary tures took place, the shell containing most of thetarget DNA or RNA sequences. These systems might functional monomer added in the second processbe used in clinical diagnostic for detecting patho- step. Most amine and amidine groups resulting fromgenic microorganisms. For this purpose, copoly- the first step were partially buried [46].merisation in batch conditions of styrene in thepresence of two amino-containing monomers: amino- 2.2. Nanospheres obtained from preformedethyl methacrylate hydrocholoride (AEMH) and polymersvinylbenzylamine hydrdochloride) (VBAH) using2,29-azobis(2-amidino-propane) dihydrichloride as With the exception of alkylcyanoacrylates andcationic initiator was achieved [43]. In this study, it dialkylmethylidene malonate, most of the monomerswas shown that the two monomers affected similarly suitable for a micellar polymerisation process in anthe kinetics of emulsion polymerisation of styrene: aqueous phase lead to slowly biodegradable or non


biodegradable polymers. In addition, residual mole- et al. [55]. It is based on the precipitation of acules in the polymerisation medium (monomer, polymer in solution following the addition of aoligomer, surfactant . . . ) can be more or less toxic non-solvent of the polymer (Fig. 4b). This method,which needs tedious purification of the colloidal thus, allows the formation of nanospheres withoutmaterial. In order to avoid those limitations, methods prior emulsification. Of course, the choice of thehave been proposed using preformed polymers in- polymer /solvent /non-solvent system is extremelystead of monomers. important since it governs the production of nanos-

Two important methodologies have been proposed pheres [55]. This is because the solvent and thefor the preparation of nanospheres from synthetic non-solvent of the polymer must be mutually misc-polymers. The first is based on the emulsification of ible. The progressive addition of the polymer solu-water–non miscible organic solutions of preformed tion to the non-solvent generally leads to the forma-polymers in aqueous phases containing surfactants, tion of nanospheres close to 200 nm in size. Thefollowed by the removal of solvents under reduced nanospheres appear to be formed, in this technique,pressure (Fig. 4b). This method which relates to the by a mechanism comparable to the ‘diffusion andmethods for preparing pseudolatex or artificial latex standing’ process found in spontaneous emulsifica-developed by Vanderhoff et al. [47] in polymer tion. This phenomenon, beyond the scope of thischemistry, has been applied to polylactic acid [47– review, has been explained by local variations of the49]. The main advantage is that polylactic polymers interfacial tension between the two non miscibleare generally considered as biocompatible and well liquids due to the reciprocal diffusion of the thirdtolerated. Poly(b-hydroxybutyrate) is also a very liquid. This method has been successfully applied topromising biodegradable polymer which has been various polymers such as polylactide and polylac-used as material for producing nanospheres by tide-co-glycolide, polyecaprolactone, ethylcellulose,solvent evaporation [50,51]. Using this polymer, it polyalkylcyanoacrylate and polystyrene.was possible by high pressure emulsification to More recently Aynie et al. [56] have developed areduce the particle sizes to 170 nm. Biodegradable process which leads to the preparation of spongelikenanospheres of polylactic acid were also prepared by alginate and polylysine nanospheres (Fig. 4c). Thisemulsion, microfluidization and solvent evaporation process involves the formation of a calcium alginatemethod using human serum albumin as emulsifying pregel on which a polylysine solution is added underagent [52]. The molecular conformation of albumin magnetic stirring. The resulting so-called nanos-at the surface of the polymer has been investigated ponges display a size of 320 nm and a Zeta potentialand the matrix structure of the system evidenced by of –34 mV.freeze–fracture [52]. Another interesting technology,applicable to a wide range of polymers, is based on

2.3. Association of ODNs to nanospheresthe selection of salts producing the salting-out ofacetone from water [53,54]. In fact, the saturated

Due to their hydrophilic and polyanionic charac-aqueous solution prevents acetone from mixing withter, ODNs are poorly interacting with polymericwater. After the preparation of an oil-in-water emul-materials. Therefore, the association of ODNs tosion, water is added in a sufficient amount to allownanospheres is a challenge which is particularlycomplete diffusion of acetone into the aqueousdifficult to achieve.phase, inducing the formation of nanospheres. The

Three main strategies have been considered for amain advantage is that this process does not requiresuccessful binding of ODNs to nanospheres:an increase in temperature and therefore may be

useful when heat-sensitive substances have to beprocessed [53]. This method has allowed to prepare 1. ODNs may be covalently linked to a hydrophobicpolylactic, polymethylmetacrylate as well as molecule allowing the hydrophobic interactionethylcellulose nanospheres [54]. with polymer surface (Fig. 5a).

The second principle for obtaining nanospheres 2. Cationic polymers may be used to coat thefrom synthetic polymers has been proposed by Fessi particles (Fig. 5b). These cations are used to


Fig. 5. Different possibilities of association of ODNs to polymeric nanoparticles: (a) by the intermediate of a hydrophobic derivative ofODN, (b) by by the association of ODN to a cationic amphiphilic molecule and (c) by incorporation in alginate nanosponges.

obtain successful interactions with the negatively medium [25]. More recently, Zobel et al. [57] havecharged ODNs molecules. replaced CTAB by DEAE-dextran that was intro-

3. Nanosponges may be used, the loading of ODNs duced into the polymerisation medium before nanos-being performed through a diffusion / reptation phere formation. Although the amount of DEAE-process (Fig. 5c). dextran covalently linked to the polymer was not

precisely measured, the unloaded nanospheres dis-Initially, ODNs were successfully associated to played a positive charge showing that the DEAE-

polyalkylcyanoacrylate (PACA) nanospheres pre- dextran has been associated with the nanospheres.coated with a hydrophobic cation such as Cetyl- With this formulation, the highest loading of ODNstrimethylammonium bromide (CTAB) [25] (Fig. 5b). (35 mmol of ODNs per gram of polymer) wasOligonucleotide adsorption onto nanospheres was achieved at pH 5.5 using a 10 mM phosphate buffer.therefore mediated by the formation of ion pair Other hydrophobic compounds such as cationicbetween the negatively charged phosphate groups of lipids were also employed as adjuvants for loadingthe nucleic acid chain and the positively charged ODNs to nanospheres [58]. However, using com-compound preadsorbed onto nanospheres [25]. The pounds such as polyamines (DOGS), the absorptionadsorption efficacy of oligonucleotide–cation com- was lower than with CTAB in similar conditions ofplexes on PACA nanospheres was found to be highly adsorption [58]. Another approach to load ODN ontodependent on several parameters: the oligonucleotide PACA nanospheres was to use a hydrophobic conju-chain length, the nature of the cyanoacrylic polymer, gate of ODNs like ODNs coupled to cholesterol (Fig.the hydrophobicity of the cation used as ion-pairing 5a) [59]. However, because this conjugate wasagent and the ionic strength of the dispersing negatively charged, it was partially repulsed by the


negative charges displayed by the polymer which forces could be generated between the bases ofexplains the lower loading efficacy in the absence of ODNs and the particle surface [62,64].CTAB.

ODNs were also associated to poly(lactic acid)-PEG nanospheres [27] using two types of oligopep-tides with alternating hydrophobic and hydrophilic 3. In vitro stability of ODNs adsorbed ontoamino acids and containing lysines residues. It was nanospheresfound that ODNs interacted with the lysine residuesof the peptides forming non-water-soluble complexes When adsorbed onto PIHCA nanospheres throughthat were able to coprecipitate with PEG-PLA co- binding to CTAB, ODNs were efficiently protectedpolymers when nanospheres were prepared using against enzymatic degradation even after 5 h incuba-nanoprecipitation technology [27]. tion with phosphodiesterase or in cell culture media

ODNs were also successfully associated to cat- [25]. In addition, about 90% of the oligonucleotideionic polystyrene nanospheres [60–63]. Owing to the still remained intact after overnight incubation withpresence of positive surface charge onto the par- the enzyme (0.1 mg/ml). Similar results were ob-ticles, it was found in a preliminary study that tained by Zobel et al. [57] with ODNs adsorbed ontoelectrostatic forces played a major role in the ad- DEAE containing nanospheres incubated withsorption process [62]. It was also observed that the DNAase.kinetic of adsorption of ODNs to the particles was ODNs-DEAE-Nanospheres complexes were foundvery rapid. About 70% of the maximum of ad- to be more stable in cell culture medium containingsorption occurred within a few minutes after which a 10 percent of fetal calf serum than ODNs associatedslow second phase took place which was explained nanospheres containing CTAB. It was assumed thatby reduced diffusion of ODNs to the particles adsorption of plasma protein could turn to a protec-surfaces caused by the electrostatic repulsion be- tive coating in DEAE containing nanospheres pre-tween free and adsorbed ODNs [62]. The influence venting the action of esterases [57]. On the contrary,of the pH on the adsorption was also found to be the lower stability of the ODNs in CTAB containingvery important: raising the pH above the pK of the nanospheres was explained by a competition betweena

amino groups decreased the adsorption [61]. On the CTAB and plasma protein inducing a partial desorp-contrary, the lower the pH the more ODNs was tion of the ODNs from the nanospheres [26]. Inadsorbed [62]. The higher adsorption at low pH was addition, PACA nanospheres were shown to beexpected by the increase of the cationic surface bioerodible, degradation by plasma esterases startingcharge at acidic pH. This confirmed that electrostatic from the surface [24]. Therefore the differenceforces played a major role in the adsorption process. between both nanospheres (containing DEAE orGanachaud et al. [60] have also shown that the CTAB) can be provided from the difference ofmaximal amount of ODNs adsorbed was near to the stability of the coating layer, DEAE being muchtheoretical value of a close-packed monolayer of flat strongly attached to the nanospheres than CTAB.conformation assuming that the ODNs was in a The displacement of CTAB-ODNs complexes bycylindrical conformation [60]. It is important to note plasma proteins was confirmed in the presence ofthat this conformation is not at all favorable for more concentrated mice plasma (70%) at 378C. Thefurther hybridization of the ODN with complemen- half-lives of pdT16 incubated free or bound to thetary sequence. On the other hand, the amount of nanospheres were short: 6.0 and 12.5 min, respec-ODNs adsorbed at basic pH was still not negligible tively [65]. However, thirty minutes after incubation[62]. It is also worth mentioning that adsorption in plasma, the percentage of non degraded 33P-pdToccurred also in the case of negatively charged 16 was only 2.9% for free ODNs whereas 28.9% ofparticles [61,62]. It is therefore likely that the 33P-pdT 16 associated with nanospheres were stilladsorption was not only governed by electrostatic intact [65]. This suggested that at least part of theinteractions [60] but that hydrophobic or hydrogen phosphodiester linkages were not available for nu-bonding may contribute to the adsorption. Such cleases degradation even if in concentrated plasma


medium the localization of ODN at the surface of the nuclear and extranuclear fraction. The increasednanospheres still remains not favorable. stability of the oligonucleotide associated to the

On the contrary, alginate nanosponges lead to an nanospheres which is observed in the extranuclearimportant protection of the ODNs, 80% of the ODNs fraction could be explained by the fact that nanos-remained undegraded after 1 h incubation in fetal pheres protect them against digestion by lysosomalcalf serum. This could be explained by the fact that enzymes. How they escape from the lysosomes stillthe ODNs are localized into the solid core of the remains unclear, but it is supposed that CTAB whichnanosponges rather than at their surface. is a quaternary ammonium could destabilize the

lysosomal membrane after a certain period of time,thus allowing the release of the oligonucleotide into

4. Cell interactions with ODNs loaded the cell cytoplasm. Escape from the lysosomes wasnanospheres however not observed on Vero cells when ODNs

were delivered by nanospheres containing DEAE.Cell uptake studies of a 15mer oligothymidilate Indeed, this type of nanospheres remained localized

adsorbed onto PIHCA nanospheres was performed in in vesicular structure in the cytoplasm suggestingnon toxic conditions using U937 cells. It was shown that in this case no destabilization of the lysosomalthat the uptake of the ODNs was dramatically membrane occurred which might be a strong limita-increased when associated with nanospheres. After tion of these type of nanospheres for ODNs delivery.24 h incubation, uptake of oligonucleotide was 8-times higher when adsorbed to nanospheres thanwhen incubated as an ODN free solution [26], and it 5. In vitro pharmacological activity ofwas markedly reduced (95%) at 48C as compared to oligonucleotides-loaded nanospheres378C. These results clearly show that ODNs ad-sorbed onto nanospheres were internalized by U937 The in vitro activity of oligonucleotide loadedcells through an endocytic /phagocytic process and PIHCA nanospheres was demonstrated in a fewnot simply adsorbed at the membrane surface. This studies. Schwab et al. [66,67] have shown thathas been confirmed by confocal microscopy with nanosphere-adsorbed antisense ODNs directed to afluorescently labeled nanospheres: after internaliza- point mutation (G → U) in codon 12 of the Ha-rastion, nanospheres accumulate into phagosomes or mRNA selectively inhibited the proliferation of cellslysosomes. Such an intracellular distribution profile expressing the point mutated Ha-ras gene. Withis supposed to lead to the rapid degradation of the nanospheres, the efficient concentration was 100-ODNs after their intracellular release from nanos- times lower than with ODN free. A sequence specificpheres. This is the reason why the intracellular inhibition of the profileration of T24 human bladderstability of 59 labeled 15-mer free or adsorbed onto carcinoma cells (containing the human Ha-ras on-PIHCA nanospheres was investigated [26]: no unde- cogene mRNA) was evidenced using the same ODNgraded ODNs were detected in cell lysate after 1.5 h conjugated to cholesterol and loaded onto PIHCAincubation with free ODNs whereas the 15-mer nanospheres [59]. The efficiency was comparable toadsorbed onto PIHCA nanospheres remained intact that of CTAB containing nanospheres. More recent-even after a 6.5 h incubation period. After 24 h, ly, Lambert et al. [68] in an attempt to inhibit PKCa

some degradation products appeared, but the fraction expression in Hep G2 cells have observed thatof intact ODNs remained considerable with nanos- PIBCA nanospheres in subtoxic concentration werepheres. The intracellular distribution of ODNs was inducing by themselves a depletion of PKCa. Thealso measured after lysis in the presence of Nonidet- same observation was done for lipofectin, a cationicP40, a nonionic detergent which protects the nuclear lipid transfecting agent [69]. The main conclusion ofstructure. It should be stressed out that the cyto- this study is that the commonly used strategy ofplasmic fraction contains also endocellular vesicles ODNs targeting with cationic non viral vectors maylike lysosomes or phagosomes. When adsorbed onto display non-specific effects which can lead to arti-nanospheres, intact ODNs were detected in both factual results.


6. In vivo studies with oligonucleotide or the sense sequence adsorbed onto PIHCA nanos-nanospheres pheres was injected 1 day before and 48 h after

implantation of the tumor in the same area whereThe pharmacokinetic studies carried out with cells were inoculated. Twenty three days after cell

PIBCA nanospheres [65] have shown that although inoculation, animals were sacrificed and the tumorsnanospheres did not markedly increase the blood were excised and weighed [67]. It was shown that

33half-life of a P-16 mer oligothymidylate, its tissue nanospheres containing the antisense markedly in-distribution was significantly modified. Indeed, when hibited Ha-ras- dependent tumor in a highly specifictransported by PIBCA nanospheres, oligothymidylate manner. Indeed, only the antisense was able towas importantly taken up by the liver whereas a reduce significantly tumors weight and volume assubsequent reduced distribution in the other organs compared to the sense sequence adsorbed onto thewas observed, especially in the kidneys. These data same nanospheres [67].suggest that with the aid of nanospheres, the oligo-nucleotide could be delivered to the liver with acertain specificity and the urinary excretion de- 7. Conclusioncreased. To address the crucial problem of oligo-nucleotide degradation, the state in which (degraded The results presented in this review show that theor not) the oligonucleotide was in the plasma and association of ODNs with biodegradable nanospheresdelivered to the liver was investigated [65]. In this is possible if using a hydrophobic counter-ion or inview, an original assay method was proposed that the case of modified ODNs to become more hydro-consists in the use of a TLC analyser allowing, in phobic or finally when oligonucleotides are entrap-polyacrylamide gels, to quantify the amount of ped in an hydrophilic system such as alginateundegraded 16 mer oligothymidylate in tissues such nanosponges. These systems have been proved to beas liver and plasma [70]. This method was able to efficient not only for protecting the oligonucleotidedistinguish between ODNs differing by only one from the degradation by 39-exonucleases but also fornucleotide in length. Using that methodology, it was increasing the intracellular capture of these mole-found that a significant amount of oligothymidylate cules. Tissue distribution profile of ODNs could bewas kept intact in the liver and the plasma when dramatically modified after association with nanos-administered under the form of nanospheres [65]. pheres and organs such as the liver or the lungs mayWhen intravenously administered with alginate be targeted. Thus, new antisense treatment of livernanosponges, oligonucleotide accumulation in the diseases such as viral hepatitis or liver cancers orlungs was 10 fold greater than with poly- metastasis may be considered with this type ofisobutylcyanoacylate [56]. This massive accumula- carrier.tion may be the result of microembolizations in the One important drawback to associate ODNs at thepulmonary capillary bed due to aggregation of the surface of nanospheres is that ODNs are available forparticles after injection. Another realistic explanation degradation or desorption in the presence of proteinof this lung pulmonary distribution is the presence of rich biological medium. Moreover, when associatedspecific interactions of the particles with the alveolar through electrostatic forces, ODNs are adopting amacrophages due to the polysaccharidic nature of conformation which make them unlikely to hybridizethese particles [56]. with their biological target. Thus, conceptually, the

In one study performed in vivo, nanospheres has ideal case for optimal activity would be to entrap thebeen proved to be efficient after intratumoral ad- ODNs within the internal core of polymeric nanocap-ministration [67]. In a nude mice model HBL100ras1 sules in order to mask them and to prevent themcells (expressing the point mutated Ha-ras genes) from any interaction with proteins. In the state of thewere inoculated by subcutaneous administration. art, all the methodologies available to prepareThese clones are able to induce tumors since the nanocapsules involve the preparation of emulsions,mutated ras gene are directly involved in cell prolif- none of them were applied until now for the en-eration and tumorigenicity. Anti-ras oligonucleotide capsulation of oligonucleotides. O/W emulsions lead


properties of peptide nucleic acids, Science 258 (1992)to nanocapsules with an oily core in which oligo-1481–1485.nucleotides may be solubilized by linkage to an

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Nanoparticulate systems for the delivery of antisense oligonucleotides