Hindawi Publishing Corporation · 2019. 7. 31. · mdx mice were killed following one week ( =3 )orone month ( =3 ) from administration of the last dose. e analysis of cryosections

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 527418 13 pageshttpdxdoiorg1011552013527418

Research ArticleBiodistribution and Molecular Studies onOrally Administered Nanoparticle-AON ComplexesEncapsulated with Alginate Aiming at InducingDystrophin Rescue in mdx Mice

Maria Sofia Falzarano1 Chiara Passarelli2 Elena Bassi1 Marina Fabris1

Daniela Perrone3 Patrizia Sabatelli4 Nadir M Maraldi4 Silvia Donagrave5

Rita Selvatici1 Paolo Bonaldo5 Katia Sparnacci6 Michele Laus6 Paola Braghetta5

Paola Rimessi1 and Alessandra Ferlini17

1 Department of Medical Sciences University of Ferrara 44121 Ferrara Italy2 Unit of Molecular Medicine for Neuromuscular and Neurodegenerative Diseases Bambino Gesu Childrenrsquos HospitalIRCCS 00146 Rome Italy

3 Department of Biology and Evolution University of Ferrara 44121 Ferrara Italy4 IGM-CNR Unit of Bologna co IOR 40136 Bologna Italy5 Department of Biomedical Sciences University of Padua 35121 Padua Italy6Department of Environmental and Life Sciences INSTM University of Eastern Piedmont 15121 Alessandria Italy7 Department of Medical Sciences Section of Medical Genetics University of Ferrara via Fossato di Mortara 74 44121 Ferrara Italy

Correspondence should be addressed to Alessandra Ferlini flaunifeit

Received 1 July 2013 Revised 10 October 2013 Accepted 14 October 2013

Academic Editor Akinori Nakamura

Copyright copy 2013 Maria Sofia Falzarano et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

We have previously demonstrated that intraperitoneal injections of 21015840-O-methyl-phosphorothioate (21015840OMePS) antisense oligori-bonucleotides adsorbed onto a cationic core-shell nanoparticles (NPs) termed ZM2 provoke dystrophin restoration in themusclesofmdxmice The aim of the present work was to evaluate the oral route as an alternative way of administration for ZM2-antisenseoligoribonucleotides complexes The biodistribution and elimination of nanoparticles were evaluated after single and multiple oraldoses of IR-dye conjugated nanoparticles Labeled nanoparticles were tracked in vivo as well as in tissue cryosections urines andfeces byOdyssey infrared imaging system and revealed a permanence in the intestine and abdominal lymph nodes for 72 hours to 7days before being eliminated We subsequently tested alginate-free and alginate-encapsulated ZM2-antisense oligoribonucleotides(AON) complexes orally administered 2 and 3 times per week respectively in mdx mice for a total of 12 weeks Treatment withalginate ZM2-AON induced a slight dystrophin rescue in diaphragm and intestine smooth muscles while no dystrophin wasdetected in alginate-free ZM2-AON treated mice These data encourage further experiments on oral administration testing of NPand AON complexes possibly translatable in oligoribonucleotides-mediated molecular therapies

1 Introduction

The X-linked recessive Duchenne muscular dystrophy(DMD) affects 1 in 3500 newborn boys [1] and it is causedby the loss of dystrophin expression The antisense mediatedexon skipping approach represents a promising therapy for

DMD It is based on the possibility to convert a severephenotype (DMD) into a milder form (Becker musculardystrophy) acting on dystrophin pre-mRNA [2 3]

Two AON chemistries 21015840OMePS and phosphorodiami-date morpholino oligomer (PMO) have already been thesubject of clinical trials in humans [1 4ndash8] Phosphorothioate

2 BioMed Research International

PMMA PMMA

Quaternary ammoniumgroups

AON

Biocompatible core Fast release region

Slow release regiondepot effect

(a)

PMMAPMMA

Quaternary ammonium

AON



groups + NIPAM

(b)

Figure 1 Nanoparticles characteristics Representation of the interactions between antisense oligoribonucleotide (M23D AON) andquaternary ammonium groups on the surface of T1 (a) and ZM2 (b) nanoparticles

(PS) oligonucleotides are the most widely studied AONs dueto their stability to nucleolytic degradation and relative easeof synthesis In PS oligonucleotides the nonbridging oxygenis replaced with a sulfur atom in the oligonucleotide chainThis substitution confers sufficient stability in plasma tissuesand cells to avoid degradation before binding to target RNAPS-modified AONs are water-soluble and have high protein-binding capacity which prevents rapid renal excretion andfacilitate uptake into tissues PMOconsists of the replacementof the phosphodiester bond by a phosphorodiamidate linkagewith the ribose replaced by a morpholino moiety PMOs arecharge neutral refractory to biological degradation and stablein serum and plasma [9ndash11]

In order to increase stability and half-life in biologicalfluids thereby improving AON efficacy different polymericnanoparticles have been developed their subcellular andsubmicron size allows them to penetrate deeply into tissuesthrough the fine capillaries crossing the fenestration presentin the epithelial lining We have previously demonstratedthe effectiveness of polymethylmethacrylate (PMMA) T1(Figure 1(a)) nanoparticles conjugated with AON to inducedystrophin restoration in body-wide muscles in themdx ani-mal model Although we obtained functional effect with one-eightieth of the normal AON dose regimen the efficiencyof this compound was relatively unsatisfactory in terms ofsize AON loading capacity and efficiency of treatmentTherefore to obtain a more efficient compound we designedand prepared a novel type of cationic core-shell NP (termed

ZM2) made up of a predominantly PMMA core and arandom copolymer shell consisting of units derived from N-isopropyl-acrylamide+ (NIPAM) and reactive methacrylate-bearing cationic groups (Figure 1(b)) ZM2 nanoparticleswere able to bind and deliver 21015840OMePS M23D AON in mdxmice inducing dystrophin restoration in the skeletal musclesarrector pili smooth muscle and heart after intraperitoneal(ip) treatment [12ndash15]

The mdx mouse is the most widely used animal modelfor exon skipping experiments since it is a natural mutantwith a stop mutation within exon 23 of the dystrophin geneThis causes a complete absence of dystrophin protein as wellas a moderate muscle weakness This stop mutation can becorrected using the exon skipping approach [16]

Indeed treatment of mdx mice with 21015840OMePS M23DAON induces the exclusion of the mutated exon 23 frommRNA resulting in an in-frame mRNA transcript and sub-sequent expression of a slightly shorter dystrophin protein inmdxmuscle [12 17ndash19]

The objective of this work was to study the biodistribu-tion elimination and efficacy of orally administered NPs-AON in mdx mice The oral route is considered the mostattractive for small molecules and macromolecular drugdelivery encompassing the advantages of easy administra-tion high patient compliance and cost-effectiveness [20 21]The PS oligonucleotides molecules are poorly bioavailablewhen administered orally for their low mucosal permeabilityand poor transcytosis across the gut due to both unfavorable

BioMed Research International 3

Table 1 Nanoparticles characteristics

SampleDiameter (SEM) nm(plusmnstandard error of

the mean)

Diameter (PCS) nm(plusmnpolydispersity

index)

Quaternaryammonium

groups 120583molg

Primaryamino groups120583molg

NIPAM Studies

ZM2 137 plusmn 9 156 plusmn 0015 202 mdash yesFunctional analysis (dystrophinrescue) with ZM2-AON andZM2-AON alginate

ZM4 138 plusmn 7 143 plusmn 0012 107 102 yes Biodistribution and eliminationusing dye NIR-797

physicochemical properties (hydrophilicity high molecularweight high negative charge density) and to the depurinationin the acidic gastric environment [22ndash25]

In order to protect molecules and facilitate drug perme-ation different kinds of delivery systems are under study inorder to inhibit hydrolysis prolong intestinal retention andthereby enhance drug bioavailability [26ndash30]

Alginate is one of themost common and suitable biopoly-mers It is nontoxic biodegradable and employed to makeacidic pH-resistant hydrogel [31] The most important prop-erty of alginates is their ability to form a reticulated structurein the presence of calcium ions allowing the entrapmentof molecules making them good mucoadhesive agents [3233] Alginate is a family of polysaccharides composed of120572-L-guluronic acid (G) and 120573-D-mannuronic acid residues(M) arranged in homopolymeric blocks of each type and inheteropolymeric blocks [31]

Swelling of alginate is minimal in the stomach while itincreasesmoving toward the intestine due to the pH increase[34] In the present paper we describe the biodistributionand elimination of ZM4 NPs conjugated with the dye NIR-797 after single and multiple oral administrations using theNear-Infrared fluorescent imaging System Odyssey (LI-CORBiosciences) The biodistribution studies indicate that NPspersist in the intestinal lumen for at least 72 hours after asingle administration and are concentrated in the intestineand abdominal lymph nodes after multiple administrationsbefore being completely eliminated at day 7 from the lasttreatment Successively we report on the results obtainedusing alginate as encapsulating agent for ZM2-AON com-plexes orally administered in mdx mice mdx mice orallytreated with alginate-free ZM2-M23D or alginate-coatedZM2-M23D revealed a slight rescue of dystrophin protein inthe intestinal smooth muscles and a mild positivity in thediaphragm only when in the presence of alginate The lowrescue was associated with the absence of AON in tissues asassayed by AON specific ELISA

These results encourage further research into the oraladministration route for antisense molecules

2 Results

21 Biodistribution and Elimination of Nanoparticle OralTreatment To assess the biodistribution and eliminationpathways a novel NP sample was prepared nominated ZM4ZM4 features the same size size distribution and surfacehydrophilicity as ZM2 but it also exposes at the surface or

in the swelled shell primary amino groups These groupsare necessary to interact with the isothiocyanate groups ofthe NIR-797 fluorescent dye (Sigma-Aldrich) giving riseto ZM4-IR nanoparticles in which the dye is covalentlyincorporated into the shell (see Table 1 reporting the NPscharacteristics) The in vivo biodistribution was evaluatedafter a single or multiple oral administrations employing theOdyssey Imaging System (Li-Cor Biosciences) that allows thespecific distribution and clearance of the infrared dye-labelednanoparticles to be tracked in live animals over time SeeTable 2 for the schedule of treatments

The Odyssey in vivo imaging time course of ZM4-IRafter single or multiple administrations demonstrated thepersistence of ZM4-IR in the intestinal lumen for at least 72hours (Figure 2) and the absence of any NP accumulationThe positivity observed in the mouth (masseter) is due toresidues of NP IR-dye formulations administered by gavage

In the multiple administration schedule ZM4-IR treatedmdx mice were killed following one week (119899 = 3) or onemonth (119899 = 3) from administration of the last dose Theanalysis of cryosections from different organs shows thatZM4-IR NPs are still present in intestine and abdominallymph nodes (Figure 3(a)) one week after treatment while noNPs were detected in the organs of mice sacrificed 1 monthafter the last administration (Figure 3(b)) The positivity ofthe masseter muscle is probably due to local adsorption as aresult of oral administration by gavage

In the nanoparticles clearance studies feces and urinesamples were collected and analyzed with Odyssey The fecesof 3 untreated mdx mice were used as negative controlsNeither the feces nor urine samples of the untreated miceshowed any fluorescence while the feces of treated miceshowed an intense signal NP clearance occurred almostexclusively through the feces and mainly in the first 48 hoursafter administration In fact by semiquantitative analysis itresulted that 90 of administered nanoparticles was detectedin the feces samples collected during the first 12ndash36 hoursafter treatment while the remaining 10 was graduallyeliminated in the next few days being completely removedafter 7 days (data not shown)

22 Dystrophin Restoration Studies ZM2-M23D Complexes

221mdxMice Treatment Table 3 summarizes the oral treat-ments and the sacrifice of mice analyzed in this work 3 mdxmice were administered for 12 weeks (2 dosesweek) withZM2-M23D complexes 3mdxmice were treated for 12 weeks


(a) (b)

(c) (d)

(e) (f)

Figure 2 Biodistribution of ZM4-IR-dye Three mice (1 2 3) were analyzed at 12 (a) 24 (b) 48 (c) and 72 (d) hours post ZM4-IR singleadministration Fluorescence was visualized using the Odyssey Infrared Imaging System Green fluorescence represents the IR signal (sim800 nm) associated to the nanoparticle The signal appears localized to the abdominal region (intestine) of the mouse (e) Mice located onthe mouse pod of the Odyssey scanner (f) The image shows the absence of fluorescence signal inmdx untreated mice

(a) (b)

Figure 3 Odyssey analysis of organmuscle cryosections (20120583m) from ZM4-IR multiple-dose administered mice 7 days (a) and 1 month(b) after last treatment Green fluorescence represents IR-dye signal (sim800 nm) associated to the nanoparticle red represents tissueautofluorescence at sim700 nm The signal is evident in abdominal lymph nodes ileum and masseter muscle at 7 days and absent at 1 monthHematoxylin-Eosin (HE) staining of the same cryosections is presented next to IR positive sections to clarify the position on the slide


Table 2 Schedule of IR-dye conjugate nanoparticle treatments for biodistribution and elimination analysis

No ofmice Oral formulations Frequency No of administration Time of analysis

119899 = 3 ZM4-IR (25mg) 1week 1 Elimination (metabolic cages) 12 hours after administrationTime of collection of Odyssey images 12ndash24ndash48ndash72 hours

119899 = 3 ZM4-IR (25mg) 2week 15Time of collection of Odyssey images before the new

administrationSacrifice 1 week 1 month after last administration

Table 3 Schedule ofmdx treatments for dystrophin restoration studies

Oral formulations Frequency No ofadministrations Total dose Time of analysis

Group 1Alginate-free(119899 = 6)

ZM2 (25mg)-AON (225 120583g)(119899 = 3) 2week 32 240mgKg 1 week after last administration

ZM2 (25mg)(119899 = 3) 2week 32 mdash 1 week after last administration

Group IIAlginate-coated(119899 = 6)



Group IIIUntreated(119899 = 3)

mdash mdash mdash mdash 1 week after last administration

with AON-free ZM2 3 mdx mice were treated for 12 weeks(3 dosesweek) with ZM2-M23D complexes coated withalginate 3 mdx mice were treated for 12 weeks with alginatecoated AON-free ZM2 3 untreated mice were included asnegative controls

The amount of AON loaded onto alginate-coated ZM2nanoparticles was lower with respect to the one used forZM2 uncoated nanoparticles 200120583g versus 225120583g in orderto facilitate the interaction of the free surface positive chargesof ZM2-AON complexes with alginate For this reason thenumber of injectionsweek (2 versus 3) are different Howeverthe total amount of M23D oligonucleotide received in eachgroup of treatment was the same represented by 240mgkgAll groups of mice were sacrificed 1 week after the lastadministration

The only side effect we observed was a mild laxative effectafter the treatment with alginate formulations

222 RNA Analysis In order to test the effect of ZM2-AON treatments on the dystrophin transcript we assessedexon 23 skipping levels using a Real-Time PCR exon-specificassay (ESRA) as previously reported [13 35] ESRAs wereperformed using the RNA pool for each treatment groupand skipping percentages were calculated for the treatedcompared to the untreated mice After 12 weeks of treatmentwith alginate-ZM2-AON we only detected a low level of exon23 skipping in the diaphragm of mice treated with alginate-coated ZM2-AON (mean skipping value for 3 mice 8)

Exon-23 skipping was undetectable in the cardiac musclegastrocnemius quadriceps and intestine for both ZM2-AONand alginate-ZM2-AON treatedmdxmice

223 AON Hybridization Ligation Assay To evaluate thepresence and amount of AON in tissues an AON sequence-specific hybridization ligation assay was performed Weanalyzed the diaphragm from treated and untreated micegiven that the diaphragm from alginate-ZM2-AON treatedmice resulted positive in ESRA analysis (8 of skipping) Tocompare the data with amuscle that was negative in the ESRAanalysis we also analyzed the quadriceps from untreatedand treated mice The AON hybridization assay revealed theabsence of AON both in diaphragm and quadriceps fromall the treatments probably due to the low amount of AONadministered in our experiments (240mgkg) and M23Dcould be too low to be detectable by this assay (data notshown)

224 Immunofluorescence Analysis In order to evaluate thepresence and the correct localization of dystrophin wildtype (WT) treated and untreated mdx sections were doublelabeled with a polyclonal antidystrophin antibody followedby an antilaminin alpha2 chain antibody for skeletal andcardiac muscles [36] and an antidesmin antibody for theintestine

After 12 weeks of treatment with alginate ZM2-AONimmunofluorescence analysis revealed a slight rescue ofdystrophin only in intestinal smooth muscle (Figure 4) and


WT

Dystrophin Desmin

mdx

Treated

Treated

Figure 4 Immunofluorescence analysis of intestinal smooth muscle of wild type (WT) untreated (mdx) and alginate-ZM2-M23D orallytreated (Treated)mdxmiceThe sections of small intestine were labeled with antidystrophin antibody Serial sections of intestine were labeledwith a polyclonal antibody for desmin (green) All samples were observedwith aNikon Eclipse 80i fluorescencemicroscope Dystrophin (red)is clearly visible in the intestinal smooth muscle of WTmice absent in untreatedmdx and rescued in treatedmdxmice (Scale bar = 50120583m)


diaphragm (Figure 5) the latter result being consistent withthe RNA data

In mice treated with ZM2-AON alginate-free complexesno dystrophin was detected in any of the muscles analyzed

225 Western Blotting Western blot analysis performedas previously described [13] confirmed the presence ofhigh molecular-weight dystrophin protein only in the intes-tine smooth muscle from alginate ZM2-AON-treated mice(Figure 6)

For WT samples the total protein loaded was 110 (15120583g)of the quantity in the other lanes (150 120583g) Quantitationperformed by densitometric analysis of autoradiographicbands followed by normalization with the quantity of totalprotein loaded on the gels showed 31 plusmn 15 (119875 = 00003) ofdystrophin recovery in the intestine from alginate ZM2-AONtreatedmicewith respect toWTmice controlsThe results arerepresentative of three separate experiments Data are givenas means plusmn SEM statistical significance was calculatedwith the Studentrsquos t-test for unpaired data No protein wasdetected in skeletal muscles (gastrocnemius quadriceps anddiaphragm) or the heart of alginate ZM2-AON treated miceNo dystrophin was detected in tissuesmuscles from all theother treated mice analyzed The high (31) percentageof dystrophin in the smooth muscle of intestine togetherwith the positivity immunostaining might be not surprisingconsidering the oral administration route

3 Discussion

In this work we demonstrate that the oral route is veryappealing as administration for AON and more in generaldrugs The promising fact is the very low but measurabledystrophin rescue on the diaphragm that needs to be fur-ther studied to improve efficiency reaching a remarkabledystrophin restoration We believed that our data althoughpreliminary might represent the first step for further studiesaiming at delivering nanoparticle-AON orally

Here we reveal the biodistribution and elimination oforally administered NIR-dye marked NPs as well as the exonskipping efficacy of NPs combined with AON molecules inmdxmice

Preclinical studies and clinical trials [1 4 5 7 37] havedescribed the use of AONs limited to parenteral intraperi-toneal (ip) intravenous (iv) and subcutaneous (sc) routes[13ndash15 19 38ndash40] To date the oral route has received limitedattention because it is known that AON-RNA molecules aresubjected to the depurination process in the acidic gastricenvironment [22ndash25]

Here we use alginate as an encapsulating agent for theoral formulation in order to protect the AON further in addi-tion to its combination with nanoparticles against the gastricpH and to increase the intestinal adhesiveness of ZM2-AONcomplexes Sodium alginate is biocompatible biodegrad-able nontoxic and approved by the US Food and DrugAdministration for oral use [41ndash46] It is also extensivelyused in food industry as a thickener emulsifier and as astabilizer [33] Until now alginates have been used for oral

administration in preclinical models to encapsulate antitu-bercular drugs [43] hepatitis B antigen [47] pDNA codingfor fish lymphocystis disease virus (LCDV) [48] insulin [49]the anti-inflammatory tripeptide Lys-Pro-Val (KPV) to treatinflammatory bowel disease (IBD) [50] oral DNA vaccineagainst infectious pancreatic necrosis virus [51] and antisenseDNA oligonucleotides [46]

Alginate has never been used as a protective agent forexon-skipping inducing RNA molecules systemic delivery

After a single oral administration ZM4 remains in theintestine for about 72 hours before being adsorbed andoreliminated The imaging in live animals before each dose ofthe multiple treatment shows the absence of NP accumula-tion Adsorption through the intestinal wall is demonstratedby Odyssey cryosection analysis showing ZM4 in abdominallymph nodes one week after treatment no ZM4 was detectedin the organs ofmice sacrificed 1month after the last adminis-tration ZM4 clearance studies reveal that they are eliminatedalmost exclusively through feces and mainly in the first 48hours after administration This result suggests that the posi-tivity observed in the intestine 72 hours after administrationis represented by a low percentage (10) of residual ZM4 andthat the biodistribution evaluation could be affected by thisearly elimination of the majority of the administered NPsFurthermore in this context the potential functional effectof NP-AON complexes in terms of dystrophin protein rescuecould be reduced by the low amount of systemically availabletherapeutic molecules

Following the verification of the intestinal absorption ofthe ZM4 we continued to demonstrate that oral treatmentwith NP-AON complexes is able to restore dystrophin syn-thesis though at very low level in mdx mice The compar-ison between two different treatments one with ZM2-AONcomplexes encapsulated with alginate and the other withalginate-free ZM2-AON complexes reveals that the alginateis necessary to protect the AONmolecules maintaining theirability to recover the dystrophin protein

Our data demonstrate that only in mdx mice orallytreated with alginate-coated ZM2-AON complexes there is aslight rescue of dystrophin protein in the intestinal smoothmuscles and a weak positivity in the diaphragm No dys-trophinwas detected on the other hand in any of themusclesof mice treated with alginate-free ZM2-AON complexesTable 4 summarizes the dystrophin restoration results

It is known that dystrophin is present in several celltypes in the gastrointestinal wall in smooth muscle cells(SMCs) of the muscularis externa and muscularis mucosaein the myoid cells located in the mucosa in the perivascularSMCs and all the submucous and myenteric neurons [52]On the contrary full-length dystrophin is lacking in thegastrointestinal tract of DMD patients and mdx mice whichshow several alterations in gastrointestinal motility

Notably our results give the first evidence that the oraldelivery of an antisense oligoribonucleotide co-formulatedwith ZM2 and alginate can produce a functional effect onRNA splicing We observe that the protective action of thealginate is essential for preventing damage to the AONmolecules by gastric acid since only with alginate-coatedZM2-AON we observe dystrophin synthesis showing that


WT

Dystrophin Desmin

mdx

Treated

Figure 5 Immunofluorescence analysis of dystrophin protein in the diaphragm of wild type (WT) untreated (mdx) and alginate-ZM2-M23D orally treated (treated) mdx mice The sections were labeled with antidystrophin antibody (red signal) and double-labeled with a ratmonoclonal antibody for laminin-2 (green) Dystrophin traces are visible in diaphragm from alginate ZM2-M23D oral treated mice Nodystrophin labeling was detected at the sarcolemma in mice untreated or treated with ZM2-AONmdx (Scale bar = 50 120583m)

ZM2 alone are not sufficient for AON protection The lowefficiency on the restoration of dystrophin protein might bedue to the inability of ZM2 to efficiently overcome the intesti-nal barrier as documented by the biodistribution studiesby Odyssey Concomitantly the observed laxative effect ofalginate may result in a reduced amount of available ZM2-AON complexes and a consequent decrease in the contacttime between particles and the intestinal epithelium leadingto a lower uptake of the molecules AON specific ELISA con-cordantly demonstrated the absence of AON in mice tissues

Nonetheless some AONs are protected enough to pass thebody barriers and reach at least the diaphragm with measur-able functional effect

Smaller size orand modified hydrophilicity of NPsand AON chemical modifications may also improve thein vivo biodistribution and stability of the binding withNPs-AON and therefore deserve to be addressed In con-clusion the challenge for future studies will be to improveboth the AON structure (backbone) and molecule pro-tection by NPs to improve the AON bioavailability and


Table 4 Summary of results of dystrophin restoration studies

Oral formulations Tissues Exon 23 skipping IHC WB

Group INo alginate(119899 = 6)

ZM2 (25mg)-AON (225 120583g)(119899 = 3)

Diaphragm 0 minus minus

Intestine 0 minus minus

Gastrocnemius 0 minus minus

Quadriceps 0 minus minus

Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus

Group II+alginate(119899 = 6)

ZM2 (25mg)-AON (200 120583g)(119899 = 3)

Diaphragm 8 + minus

Intestine 0 + +Gastrocnemius 0 minus minus


Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


mdash





Heart 0 minus minus

WT NT ZM2-AONZM2-AON

+

alginate

Figure 6 Immunoblotting of dystrophin in the intestine ofuntreated mdx mice (NT) ZM2-AON treated mice and alginate-coated ZM2-AON treated mice Intestine fromWT mice were usedas positive controls For WT samples the total protein loaded was110 (15120583g) of the quantity used in the other lanes (150 120583g) Quantita-tion performed by densitometric analysis of autoradiographic bandsfollowed by normalization with the quantity of total protein loadedon the gels showed 31 plusmn 15 (119875 = 00003) of dystrophin recoveryin the intestine from alginate ZM2-AON-treated mice compared toWT mice The arrows show the bands of dystrophin (427 kD)

to enhance the intestinal cellsrsquo uptake of orally delivereddrugs

4 Materials and Methods

41 Animals All experiments were performed on malemdx mice (C57BL10ScSn-DmdmdxJ) All procedures wereapproved by the Animal Experimentation Ethics CommitteeMice were housed in temperature-controlled rooms (22∘C) ata humidity level of 50 and a 1212 hour light-dark cycleMicewere purchased from the Jackson Laboratory (Bar HarborME)

42 ZM4 Nanoparticles Synthesis ZM4 nanoparticle samplewere prepared by emulsion polymerization of methyl metha-crylate employing as emulsion stabilizers different functionalcomonomers (N-isopropyl acrylamide (NIPAM) (NN-dimethyl N-octyl ammonium) ethyl methacrylate bromide(MOAEMA) and 2-aminoethyl methacrylate hydrochloride(AEMA))

In particular 300mL of methyl methacrylate were intro-duced in a flask containing 500mL of an aqueous solution


of 234 g (67mmol) of MOAEMA 055 g (33mmol) ofAEMA and 113 g (10mmol) NIPAM The flask was fluxedwith nitrogen under constant stirring for 30 minutes then85mg (0313mmol) of the cationic free radical initiator AIBAdissolved in water were added The polymerization wasperformed at 80 plusmn 10∘C for 4 hours under constant stirringAt the end of the reaction the samples were recovered andpurified by repeated dialysis

Particle sizes and size distributions were measured bydynamic light scattering and scanning electron microscopy(SEM) analysis Dynamic light scattering analysis was per-formed at 25∘C with a Malvern Zetasizer Nano ZS systemat a fixed scattering angle of 90∘ using a He-Ne laser and aPCS software (Malvern UK version 611) Five individualmeasurements were performed for each sample The valuesthat we report are the average of these 5 measurements SEManalysis were performed with a Field Emission Gun InspectF microscope from FEI Company The SEM micrographswere elaborated by the Scion Image processing (NIH publicdomain) program From 200 to 250 individual nanosphereswere measured for each sample The characteristics of theobtained nanoparticles are reported in Table 1

43 ZM4Conjugation with NIR-797 Dye 12mL of a solution3mgmL of dye NIR-797 Isothiocyanate (Sigma-Aldrich 120582ex795 nm 120582em 817 nm) in anhydrous DMSO were added to15mL of a suspension of nanoparticles (22120583g NIR-797mgnanoparticle) in phosphate buffer (10mM pH 8) undermagnetic stirring for 2 hours protected from light Aftercentrifugation (10 minutes at 11000 g) the pellet was washedtwice then resuspended inwater obtainingZM4-IRnanopar-ticles

44 Oral Administration Schedule for Biodistribution andElimination Studies 25mg of ZM4-IR NPs dissolved in200120583L of water were administered to mice by oral gavage insingle or multiple treatments 8ndash6 weeks old mdx mice weredivided into 2 groups (119899 = 2) of treatment the first groupreceived a single dose of ZM4-IR the second group received2 administrationsweek for a total of 15 administrations ofZM4-IR Table 2 summarizes the administration scheduleTwo untreated mice were used as negative controls

45 Biodistribution and Elimination Studies Prior to theacquisition of in vivo images the mice were fed with a specialdiet (Research Diets AIN-76A or AIN-93G) in order tominimize autofluorescence (red signal) due to chlorophyllcontained in plant-based ingredients The animals wereanesthetized with 1 Avertin shaved in abdominal and chestregions and located on MousePod of Odyssey for infraredin vivo imaging The scanning was performed 12 24 48 72hours after a single administration of ZM4-IR and beforeeach dose for multiple administrations For nanoparticlesclearance studies mice were placed into metabolic cages forcollecting feces and urine for 24 hours starting after 12 hoursand 6 days after ZM4-IR administration The feces wereweighed and then dissolved in 4mL of PBS by vigorousshaking while the urine samples were spin for 10 minutesat 11000 g and solubilized in 100 120583L The total urine sample

(100 120583L) 100 120583L of feces suspension and progressive dilutionsof both samples were put in a 96 well-dish and analyzed byOdyssey Along with samples a dilution of the fluorescentnanoparticles were prepared as a standard To perform quan-tification of signals regions of interest (ROIs) of equal sizewere drawn and placed over each well A calibration curvewas obtained from the dilution of the fluorescent nanoparti-cles after normalization of each ROI to the sum of the totalintensity of all the standard ROIs The same normalizationprocedure was applied to the feces and urine samples andnormalized values were used

At the end of treatments mice were sacrificed their tis-sues and organs excised frozen in liquid N2-cooled isopen-tane and stored at minus80∘C Twenty-micrometer-thick frozentransverse sections were cut from different specimens ana-lyzed by Odyssey and stained with hematoxylin and eosin(HampE) (Sigma-Aldrich Milan Italy)

46 AON Synthesis M23D(+07-18) (51015840-GGCCAAACC-UCGGCUUACCUGAAAU-31015840) AON against the boundarysequences of the exon and intron 23 of mouse dystrophingene contains 21015840-O-methyl modified RNA and full-lengthphosphorothioate backbone Oligonucleotide synthesis wascarried out on an AKTA oligopilot plus 10DNARNA synthe-sizer (GE Healthcare Milano Italy) using its trityl-on modeThe sequence was synthesized on a 2120583mol scale using PrimerSupport 200 loaded at 80 120583molg (Amersham BiosciencesMilano Italy) Commercial 21015840-O-methyl phosphoramidites(Proligo Boulder CO) were dissolved to a nominal concen-tration of 50mmolL in anhydrous acetonitrile (CH

3CN) and

activated with a 025molL solution of 5-(bis-35-trifluoro-methylphenyl)-1H-tetrazole (Proligo) in CH

3CN The final

detritylation was achieved using a 01molL aqueous solutionofNaOAc (pH30) Crude dimethyltryptamine protected anddetritylated oligonucleotide were purified by an AKTAbasicultraphysical contact liquid chromatography system using anAmersham Biosciences Resource RPC 3mL column elutedunder a gradient of CH

3CN in 01molL triethylammonium

acetate (pH 8) The final oligonucleotide was dissolved inwater and filtered through a short column of Dowex 50WX8(Na+ form 100ndash200mesh) to obtain after lyophilization08 120583mol (40) of target compound The purity of the full-length desired product was evaluated by MALDI-TOF MS31P-NMR and RP-HPLC analyses

47 ZM2-M23D Loading ZM2 nanoparticles were preparedby emulsion polymerization of methyl methacrylate employ-ing as emulsion stabilizers two functional comonomers(N-isopropyl acrylamide (NIPAM) and (NN-dimethyl N-octyl ammonium) ethyl methacrylate bromide) as previouslyreported [14] They have a particle diameter of 137 nm (asdetermined by scanning electron microscopy) and a surfacecharge density of 202120583mol ammonium groups per gram ofnanoparticles M23DAON contains 21015840-O-methyl RNA phos-phorothioate backbone and its synthesis was previouslydescribed [13] Loading experiments have shown that ZM2nanoparticles (1mgmL) adsorbed 21015840OMePS M23D oligori-bonucleotide onto their surface in the concentration rangeof 10ndash100 120583gmL M23D adsorption onto ZM2 nanoparticles


is a highly reproducible process with a loading value of90120583gmg [14]

48 Naked ZM2 or ZM2-AON Coating with Alginate Sus-pension of naked ZM2 or ZM2-AON complexes in waterwere mixed with a solution of sodium alginate (052mgalginatemg of ZM2) under magnetic stirring for 10 minutesCaCl2(3mM) was added to the solution and kept under

agitation for 10 additional minutes After centrifugation at11000 g for 15 minutes the supernatant was removed and col-lected in new tubes while alginate-ZM2 or alginate ZM2-AON (200120583g AON25mg ZM2) pellets were loaded ininsulin syringes for oral administration by gavage The col-lected supernatantswere drawn filtered on aMillexGV

4filter

and the absorbance (260ndash280 nm) measured by Nanodrop1000 (Thermo Scientific)

49 Oral Administration Schedule for Dystrophin RestorationStudies Two groups ofmdxmice (6 weeks old) were treatedby oral gavage The I group received 200120583L of suspensioncontaining 225120583g of M23D loaded onto 25mg of ZM2nanoparticles (3 mice) or 25mg of AON-free ZM2 nanopar-ticles as negative control (3 mice) for a total of 32 admin-istrations (240mgKg total) The II group was treated with200120583L of suspension containing 200120583g ofM23D loaded onto25mg of ZM2 nanoparticles or 25mg of AON-free ZM2nanoparticles but in the presence of alginate (3 mice foreach different treatment) for a total of 36 administrations(240mgKg total) Three untreated age-matched mdx micewere included as negative controls All groups of mice weresacrificed 1 week after last administration

Table 3 summarizes the administration schedule

410 Dystrophin RNA Studies Total RNAwas extracted fromfrozen sections of muscle biopsies using TRIzol (InvitrogenMilano Italy) and reverse-transcribed into cDNA using theHigh-Capacity cDNA Reverse Transcription kit (AppliedBiosystems Frankfurt Germany) We performed Exon-Specific Real Time Assays (ESRAs) using RNA pool for eachtreatment group ESRAs were used on exons 8 23 and 25to quantify the percentage of exon-23 skipping in treatedwith respect to untreated mice (ΔCt method) using 120573-actinas endogenous control All these ESRAs are based on Taq-Man technology and have been designed by PrimerExpressApplied Biosystems software (Applied Biosystems) Primersand probes sequences are available upon requestThe amountof target sequences compared to appropriate endogenouscontrol (120573-actin gene) was evaluated by the comparativeCT method with respect to the endogenous 120573-actin control(ΔΔCt Method) (Applied Biosystems User Bulletin no 2)[13 53]

411 AON Hybridization Ligation Assay The assay for mea-suring the concentration of 2OMePS oligoribonucleotide23AON in tissue samples is based on a hybridization ligationassay [38] A template probe (51015840-gaatagacgaggtaagccgaggtt-tggcc-biotin-31015840 29-merDNAphosphate oligonucleotide) anda ligation probe (51015840-P-cgtctattc-DIG-31015840 9-mer DNA phos-phate oligonucleotide) were used The sample was incubated

with the template probe (50 nmolL) at 37∘C for 1 hourand the hybridized samples were transferred to streptavidin-coated 96-well plates Subsequently the digoxigenin-labeledligation probe (2 nmolL) was added Detection was per-formed with anti-DIG-POD (1 4000 Roche DiagnosticsMilan Italy) 331015840551015840-tetramethylbenzidine substrate (SigmaAldrich Srl Milan Italy) and stop solution (SigmaAldrich)Absorption at 450 nm was measured in a SpectraFluorPlus(Tecan Italia Srl Cernusco sul Naviglio Milan Italy) Thetissue samples were homogenized to a concentration of60mgmL in proteinase K buffer (100mmolL Tris-HClpH 85 200mmolL NaCl 5mmolL EDTA 02 sodiumdodecylsulfate) containing 2mgmL of proteinase K (LifeTechnologies Monza Italy) followed by incubation for 4hours rotating at 55∘C in a hybridization oven Next thesamples were centrifuged for 15 minutes at maximum speedand the supernatant was used in the assay We analyzeddiaphragm and quadriceps from untreated and oral treatedmdx mice Muscle samples were first diluted 60-fold in PBSsubsequent dilutions and the calibration curves were done in60-fold control muscle in PBS

412 Dystrophin Protein Immunofluorescence Analysis Oneweek after the last injection mdx mice were killed and theirdiaphragm quadriceps gastrocnemius cardiac muscles andintestine isolated snap-frozen in liquid N2-cooled isopen-tane and stored at minus80∘C until further processing

Seven-micrometer-thick frozen transverse sections werecut from at least two-thirds of the length of heart diaphragmgastrocnemius quadriceps muscles and intestine for eachmuscle at least 5 slices were cut at 150120583m intervals To analyzedystrophin restoration freshly-cut muscle and intestine sec-tions were labeled with a polyclonal antidystrophin antibody(H-300) (epitope corresponding to amino acids 801ndash1100mapping within an internal region of dystrophin SantaCruz Biotechnology Santa Cruz CA) diluted 1 100 revealedwith anti-rabbit Cy3-conjugated secondary antibody (Jack-son Immunoresearch Suffolk UK) Muscles samples weredouble-labeled with a rat monoclonal antibody for laminin-2(1205722-chain 4H8-2 diluted 1 500 Alexis Biochemical Farm-ingdale NY USA) revealed with anti-rat Cy2-conjugatedsecondary antibody (Jackson Immunoresearch Suffolk UK)Seriate sections of intestine were labeled with a polyclonalantibody for desmin (diluted 1 100 Abcam Cambridge UK)followed by anti-rabbit FITC-conjugated secondary antibodyfor desmin (DAKO Glostrup Denmark) All images wereobserved with a Nikon Eclipse 80i fluorescence microscope(Nikon Instruments Firenze Italy) connected to at a high-resolution CCD camera (Nikon Instruments Firenze Italy)at 20x magnification

413 Dystrophin Analysis by Western Blotting Western blotanalysis was performed as previously described [13] Twenty-micrometer-thick frozen muscle sections were homogenizedwith a lysis buffer (7molL urea 2molL thiourea 1amidosulfobetaine-14 and 03dithioerythritol) Aliquots ofproteins from wild type mice (15120583g) and from the muscles oftreated or untreatedmdxmice (150 120583g)were loaded onto a 6sodium dodecyl sulphate-polyacrylamide gel and separated


by electrophoresis Samples were transferred to a nitrocel-lulose membrane at 75V The membrane was incubatedovernight at 4∘C with the specific antibody DYS2 (NovoCas-tra Newcastle UK)

To quantify the restoration of dystrophin protein intreated versus wild type mice a densitometric analysis ofautoradiographic bands was performed with a Bio-Rad Den-sitometer GS 700 (Bio-Rad Milan Italy) followed by nor-malization with the quantity of total protein loaded onto thegels

Conflict of Interests

The authors declared no conflict of interests

Acknowledgments

The Telethon Italy Grant GGP09093 (to A Ferlini Depart-ment of Medical Science Section of Medical GeneticsUniversity of Ferrara Ferrara Italy) is acknowledged Theauthors are grateful to Judith CT van Deutekom (ProsensaTherapeutics BV Leiden) for her help and suggestions withAON hybridization ligation assay

References

[1] J C van Deutekom A A Janson I B Ginjaar et al ldquoLocaldystrophin restoration with antisense oligonucleotide PRO051rdquoTheNew England Journal of Medicine vol 357 no 26 pp 2677ndash2686 2007

[2] S D Wilton and S Fletcher ldquoExon skipping and Duchennemuscular dystrophy hope hype and how feasiblerdquo NeurologyIndia vol 56 no 3 pp 254ndash262 2008

[3] G J van Ommen J van Deutekom and A Aartsma-Rus ldquoThetherapeutic potential of antisense-mediated exon skippingrdquoCurrent Opinion in Molecular Therapeutics vol 10 no 2 pp140ndash149 2008

[4] N M Goemans M Tulinius J T van den Akker et al ldquoSys-temic administration of PRO051 in Duchennersquos muscular dys-trophyrdquo The New England Journal of Medicine vol 364 no 16pp 1513ndash1522 2011

[5] S Cirak V Arechavala-Gomeza M Guglieri et al ldquoExonskipping and dystrophin restoration in patients with Duchennemuscular dystrophy after systemic phosphorodiamidate mor-pholino oligomer treatment an open-label phase 2 dose-escalation studyrdquo The Lancet vol 378 no 9791 pp 595ndash6052011

[6] httpwwwclinicaltrialsgovct2resultsterm=NCT01396239[7] M Kinali V Arechavala-Gomeza L Feng et al ldquoLocal restora-

tion of dystrophin expression with the morpholino oligomerAVI-4658 in Duchenne muscular dystrophy a single-blindplacebo-controlled dose-escalation proof-of-concept studyrdquoThe Lancet Neurology vol 8 no 10 pp 918ndash928 2009

[8] httpwwwclinicaltrialsgovct2resultsterm=DMDamppg=2[9] W-H Pan and G A Clawson ldquoAntisense applications for bio-

logical controlrdquo Journal of Cellular Biochemistry vol 98 no 1pp 14ndash35 2006

[10] C F Bennett and E E Swayze ldquoRNA targeting therapeuticsmolecular mechanisms of antisense oligonucleotides as a ther-apeutic platformrdquo Annual Review of Pharmacology and Toxicol-ogy vol 50 pp 259ndash293 2010

[11] V Arora D C Knapp M T Reddy D D Weller and P LIversen ldquoBioavailability and efficacy of antisense Morpholinooligomers targeted to c-myc and cytochrome P-450 3A2 fol-lowing oral administration in ratsrdquo Journal of PharmaceuticalSciences vol 91 no 4 pp 1009ndash1018 2002

[12] C JMann KHoneyman A J Cheng et al ldquoAntisense-inducedexon skipping and synthesis of dystrophin in the mdx mouserdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 1 pp 42ndash47 2001

[13] P Rimessi P Sabatelli M Fabris et al ldquoCationic PMMAnanoparticles bind and deliver antisense oligoribonucleotidesallowing restoration of dystrophin expression in the mdxmouserdquoMolecular Therapy vol 17 no 5 pp 820ndash827 2009

[14] A Ferlini P Sabatelli M Fabris et al ldquoDystrophin restorationin skeletal heart and skin arrector pili smooth muscle of mdxmice by ZM2 NP-AON complexesrdquo GeneTherapy vol 17 no 3pp 432ndash438 2010

[15] E Bassi S Falzarano M Fabris et al ldquoPersistent dystrophinprotein restoration 90 days after a course of intraperitoneallyadministered naked 21015840 OMePS AON and ZM2 NP-AON com-plexes in mdx micerdquo Journal of Biomedicine and Biotechnologyvol 2012 Article ID 897076 8 pages 2012

[16] P Sicinski Y Geng A S Ryder-Cook E A Barnard M GDarlison and P J Barnard ldquoThe molecular basis of musculardystrophy in the mdx mouse a point mutationrdquo Science vol244 no 4912 pp 1578ndash1580 1989

[17] E P Hoffman R H Brown Jr and L M Kunkel ldquoDystrophinthe protein product of the Duchenne muscular dystrophylocusrdquo Cell vol 51 no 6 pp 919ndash928 1987

[18] E P Hoffman J E Morgan S C Watkins and T A PartridgeldquoSomatic reversionsuppression of the mouse mdx phenotypein vivordquo Journal of the Neurological Sciences vol 99 no 1 pp9ndash25 1990

[19] L L Qi A Rabinowitz Y C Chen et al ldquoSystemic delivery ofantisense oligoribonucleotide restorers dystrophin expressionin body-wide skeletal musclesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no1 pp 198ndash203 2005

[20] J Xu S Ganesh and M Amiji ldquoNon-condensing polymericnanoparticles for targeted gene and siRNA deliveryrdquo Interna-tional Journal of Pharmaceutics vol 427 no 1 pp 21ndash34 2012

[21] S Akhtar ldquoOral delivery of siRNA and antisense oligonu-cleotidesrdquo Journal of Drug Targeting vol 17 no 7 pp 491ndash4952009

[22] A A Raoof P Chiu Z Ramtoola et al ldquoOral bioavailability andmultiple dose tolerability of an antisense oligonucleotide tabletformulated with sodium capraterdquo Journal of PharmaceuticalSciences vol 93 no 6 pp 1431ndash1439 2004

[23] R S Geary O Khatsenko K Bunker et al ldquoAbsolute bioavail-ability of 21015840-O-(2-methoxyethyl)-modified antisense oligonu-cleotides following intraduodenal instillation in ratsrdquo Journalof Pharmacology and Experimental Therapeutics vol 296 no3 pp 898ndash904 2001

[24] A des Rieux V FievezMGarinot Y-J Schneider andV PreatldquoNanoparticles as potential oral delivery systems of proteins andvaccines amechanistic approachrdquo Journal of Controlled Releasevol 116 no 1 pp 1ndash27 2006


[25] S Maher T W Leonard J Jacobsen and D J Brayden ldquoSafetyand efficacy of sodium caprate in promoting oral drug absorp-tion from in vitro to the clinicrdquo Advanced Drug DeliveryReviews vol 61 no 15 pp 1427ndash1449 2009

[26] L G Tillman R S Geary and G E Hardee ldquoOral delivery ofantisense oligonucleotides in manrdquo Journal of PharmaceuticalSciences vol 97 no 1 pp 225ndash236 2008

[27] L Yin J Ding J Zhang C He C Tang and C Yin ldquoPolymerintegrity related absorption mechanism of superporous hydro-gel containing interpenetrating polymer networks for oraldelivery of insulinrdquo Biomaterials vol 31 no 12 pp 3347ndash33562010

[28] R M Samstein K Perica F Balderrama M Look and T MFahmy ldquoThe use of deoxycholic acid to enhance the oral bio-availability of biodegradable nanoparticlesrdquo Biomaterials vol29 no 6 pp 703ndash708 2008

[29] N J Kavimandan E Losi and N A Peppas ldquoNovel deliverysystem based on complexation hydrogels as delivery vehicles forinsulin-transferrin conjugatesrdquo Biomaterials vol 27 no 20 pp3846ndash3854 2006

[30] Z S Haidar R C Hamdy and M Tabrizian ldquoProtein releasekinetics for core-shell hybrid nanoparticles based on the layer-by-layer assembly of alginate and chitosan on liposomesrdquo Bio-materials vol 29 no 9 pp 1207ndash1215 2008

[31] F Tugcu-Demiroz F Acarturk S Takka and O Konus-Boyunaga ldquoEvaluation of alginate based mesalazine tablets forintestinal drug deliveryrdquoEuropean Journal of Pharmaceutics andBiopharmaceutics vol 67 pp 491ndash497 2007

[32] P V Finotelli D da Silva M Sola-Penna et al ldquoMicrocapsulesof alginatechitosan containingmagnetic nanoparticles for con-trolled release of insulinrdquo Colloids and Surfaces B vol 81 no 1pp 206ndash211 2010

[33] M George and T E Abraham ldquoPolyionic hydrocolloids for theintestinal delivery of protein drugs alginate and chitosanmdashareviewrdquo Journal of Controlled Release vol 114 no 1 pp 1ndash142006

[34] Y-H Lin H-F Liang C-K Chung M-C Chen and H-W Sung ldquoPhysically crosslinked alginateNO-carboxymethylchitosan hydrogels with calcium for oral delivery of proteindrugsrdquo Biomaterials vol 26 no 14 pp 2105ndash2113 2005

[35] P Spitali P Rimessi M Fabris et al ldquoExon skipping-mediateddystrophin reading frame restoration for small mutationsrdquoHuman Mutation vol 30 no 11 pp 1527ndash1534 2009

[36] C A Sewry ldquoImmunocytochemical analysis of human muscu-lar dystrophyrdquo Microscopy Research and Technique vol 48 no3-4 pp 142ndash154 2000

[37] Y Takeshima M Yagi H Wada et al ldquoIntravenous infusion ofan antisense oligonucleotide results in exon skipping in muscledystrophin mRNA of Duchenne muscular dystrophyrdquo PediatricResearch vol 59 no 5 pp 690ndash694 2006

[38] H Heemskerk C de Winter P van Kuik et al ldquoPreclinicalPK and PD studies on 21015840-O-methyl-phosphorothioate RNAantisense oligonucleotides in themdxmouse modelrdquoMolecularTherapy vol 18 no 6 pp 1210ndash1217 2010

[39] H A Heemskerk C L de Winter S J de Kimpe et al ldquoIn vivocomparison of 21015840-O-methyl phosphorothioate and morpholinoantisense oligonucleotides for Duchenne muscular dystrophyexon skippingrdquo Journal of Gene Medicine vol 11 no 3 pp 257ndash266 2009

[40] T Yokota Q-L Lu T Partridge et al ldquoEfficacy of systemicmor-pholino exon-skipping in duchenne dystrophy dogsrdquo Annals ofNeurology vol 65 no 6 pp 667ndash676 2009

[41] H H Toslashnnesen and J Karlsen ldquoAlginate in drug delivery sys-temsrdquo Drug Development and Industrial Pharmacy vol 28 pp621ndash630 2002

[42] R Pandey and G K Khuller ldquoChemotherapeutic potential ofalginate-chitosan microspheres as anti-tubercular drug carri-ersrdquo Journal of Antimicrobial Chemotherapy vol 53 no 4 pp635ndash640 2004

[43] Z Ahmad R Pandey S Sharma and G K Khuller ldquoPharma-cokinetic and pharmacodynamic behaviour of antituberculardrugs encapsulated in alginate nanoparticles at two dosesrdquoInternational Journal of Antimicrobial Agents vol 27 no 5 pp409ndash416 2006

[44] B Thu P Bruheim T Espevik O Smidsroslashd P Soon-Shiongand G Skjak-Braeligk ldquoAlginate polycation microcapsules IInteraction between alginate and polycationrdquo Biomaterials vol17 no 10 pp 1031ndash1040 1996

[45] W R Gombotz and S F Wee ldquoProtein release from alginatematricesrdquo Advanced Drug Delivery Reviews vol 31 no 3 pp267ndash285 1998

[46] M Gonzalez Ferreiro L G Tillman G Hardee and R Bod-meier ldquoAlginatepoly-L-lysine microparticles for the intesti-nal delivery of antisense oligonucleotidesrdquo PharmaceuticalResearch vol 19 no 6 pp 755ndash764 2002

[47] O Borges J Tavares A de Sousa G Borchard H E Jungingerand A Cordeiro-da-Silva ldquoEvaluation of the immune responsefollowing a short oral vaccination schedulewith hepatitis B anti-gen encapsulated into alginate-coated chitosan nanoparticlesrdquoEuropean Journal of Pharmaceutical Sciences vol 32 no 4-5 pp278ndash290 2007

[48] J-Y Tian X-Q Sun and X-G Chen ldquoFormation and oraladministration of alginate microspheres loaded with pDNAcoding for lymphocystis disease virus (LCDV) to Japaneseflounderrdquo Fish and Shellfish Immunology vol 24 no 5 pp 592ndash599 2008

[49] Y Zhang W Wei P Lv L Wang and G Ma ldquoPreparation andevaluation of alginate-chitosan microspheres for oral deliveryof insulinrdquo European Journal of Pharmaceutics and Biopharma-ceutics vol 77 no 1 pp 11ndash19 2011

[50] H Laroui GDalmassoH T TNguyen Y Yan S V Sitaramanand D Merlin ldquoDrug-loaded nanoparticles targeted to thecolon with polysaccharide hydrogel reduce colitis in a mousemodelrdquo Gastroenterology vol 138 no 3 pp 843ndash853 2010

[51] A I de lasHeras S Rodrıguez Saint-Jean and S I Perez-PrietoldquoImmunogenic and protective effects of an oral DNA vaccineagainst infectious pancreatic necrosis virus in fishrdquo Fish andShellfish Immunology vol 28 no 4 pp 562ndash570 2010

[52] M-G Vannucchi C Zardo L Corsani and M-S Faussone-Pellegrini ldquoInterstitial cells of Cajal enteric neurons andsmooth muscle and myoid cells of the murine gastrointestinaltract express full-length dystrophinrdquo Histochemistry and CellBiology vol 118 no 6 pp 449ndash457 2002

[53] A Ferlini and P Rimessi ldquoExon skipping quantification by real-time PCRrdquoMethods in Molecular Biology vol 867 pp 189ndash1992012

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


PMMA PMMA

Quaternary ammoniumgroups

AON



(a)

PMMAPMMA

Quaternary ammonium

AON



groups + NIPAM

(b)

Figure 1 Nanoparticles characteristics Representation of the interactions between antisense oligoribonucleotide (M23D AON) andquaternary ammonium groups on the surface of T1 (a) and ZM2 (b) nanoparticles

(PS) oligonucleotides are the most widely studied AONs dueto their stability to nucleolytic degradation and relative easeof synthesis In PS oligonucleotides the nonbridging oxygenis replaced with a sulfur atom in the oligonucleotide chainThis substitution confers sufficient stability in plasma tissuesand cells to avoid degradation before binding to target RNAPS-modified AONs are water-soluble and have high protein-binding capacity which prevents rapid renal excretion andfacilitate uptake into tissues PMOconsists of the replacementof the phosphodiester bond by a phosphorodiamidate linkagewith the ribose replaced by a morpholino moiety PMOs arecharge neutral refractory to biological degradation and stablein serum and plasma [9ndash11]

In order to increase stability and half-life in biologicalfluids thereby improving AON efficacy different polymericnanoparticles have been developed their subcellular andsubmicron size allows them to penetrate deeply into tissuesthrough the fine capillaries crossing the fenestration presentin the epithelial lining We have previously demonstratedthe effectiveness of polymethylmethacrylate (PMMA) T1(Figure 1(a)) nanoparticles conjugated with AON to inducedystrophin restoration in body-wide muscles in themdx ani-mal model Although we obtained functional effect with one-eightieth of the normal AON dose regimen the efficiencyof this compound was relatively unsatisfactory in terms ofsize AON loading capacity and efficiency of treatmentTherefore to obtain a more efficient compound we designedand prepared a novel type of cationic core-shell NP (termed

ZM2) made up of a predominantly PMMA core and arandom copolymer shell consisting of units derived from N-isopropyl-acrylamide+ (NIPAM) and reactive methacrylate-bearing cationic groups (Figure 1(b)) ZM2 nanoparticleswere able to bind and deliver 21015840OMePS M23D AON in mdxmice inducing dystrophin restoration in the skeletal musclesarrector pili smooth muscle and heart after intraperitoneal(ip) treatment [12ndash15]

The mdx mouse is the most widely used animal modelfor exon skipping experiments since it is a natural mutantwith a stop mutation within exon 23 of the dystrophin geneThis causes a complete absence of dystrophin protein as wellas a moderate muscle weakness This stop mutation can becorrected using the exon skipping approach [16]

Indeed treatment of mdx mice with 21015840OMePS M23DAON induces the exclusion of the mutated exon 23 frommRNA resulting in an in-frame mRNA transcript and sub-sequent expression of a slightly shorter dystrophin protein inmdxmuscle [12 17ndash19]

The objective of this work was to study the biodistribu-tion elimination and efficacy of orally administered NPs-AON in mdx mice The oral route is considered the mostattractive for small molecules and macromolecular drugdelivery encompassing the advantages of easy administra-tion high patient compliance and cost-effectiveness [20 21]The PS oligonucleotides molecules are poorly bioavailablewhen administered orally for their low mucosal permeabilityand poor transcytosis across the gut due to both unfavorable




the mean)


index)

Quaternaryammonium

groups 120583molg


NIPAM Studies








2 Results









(a) (b)

(c) (d)

(e) (f)


(a) (b)



























WT

Dystrophin Desmin

mdx

Treated

Treated







3 Discussion













WT

Dystrophin Desmin

mdx

Treated









ZM2 (25mg)-AON (225 120583g)(119899 = 3)





Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


ZM2 (25mg)-AON (200 120583g)(119899 = 3)

Diaphragm 8 + minus



Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


mdash





Heart 0 minus minus


+

alginate
















3CN) and


3CN The final









4filter















Acknowledgments


References






























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






the mean)


index)

Quaternaryammonium

groups 120583molg


NIPAM Studies








2 Results









(a) (b)

(c) (d)

(e) (f)


(a) (b)



























WT

Dystrophin Desmin

mdx

Treated

Treated







3 Discussion













WT

Dystrophin Desmin

mdx

Treated









ZM2 (25mg)-AON (225 120583g)(119899 = 3)





Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


ZM2 (25mg)-AON (200 120583g)(119899 = 3)

Diaphragm 8 + minus



Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


mdash





Heart 0 minus minus


+

alginate
















3CN) and


3CN The final









4filter















Acknowledgments


References






























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b)

(c) (d)

(e) (f)


(a) (b)



























WT

Dystrophin Desmin

mdx

Treated

Treated







3 Discussion













WT

Dystrophin Desmin

mdx

Treated









ZM2 (25mg)-AON (225 120583g)(119899 = 3)





Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


ZM2 (25mg)-AON (200 120583g)(119899 = 3)

Diaphragm 8 + minus



Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


mdash





Heart 0 minus minus


+

alginate
















3CN) and


3CN The final









4filter















Acknowledgments


References






























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




























WT

Dystrophin Desmin

mdx

Treated

Treated







3 Discussion













WT

Dystrophin Desmin

mdx

Treated









ZM2 (25mg)-AON (225 120583g)(119899 = 3)





Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


ZM2 (25mg)-AON (200 120583g)(119899 = 3)

Diaphragm 8 + minus



Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


mdash





Heart 0 minus minus


+

alginate
















3CN) and


3CN The final









4filter















Acknowledgments


References






























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




WT

Dystrophin Desmin

mdx

Treated

Treated







3 Discussion













WT

Dystrophin Desmin

mdx

Treated









ZM2 (25mg)-AON (225 120583g)(119899 = 3)





Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


ZM2 (25mg)-AON (200 120583g)(119899 = 3)

Diaphragm 8 + minus



Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


mdash





Heart 0 minus minus


+

alginate
















3CN) and


3CN The final









4filter















Acknowledgments


References






























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials








3 Discussion













WT

Dystrophin Desmin

mdx

Treated









ZM2 (25mg)-AON (225 120583g)(119899 = 3)





Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


ZM2 (25mg)-AON (200 120583g)(119899 = 3)

Diaphragm 8 + minus



Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


mdash





Heart 0 minus minus


+

alginate
















3CN) and


3CN The final









4filter















Acknowledgments


References






























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




WT

Dystrophin Desmin

mdx

Treated









ZM2 (25mg)-AON (225 120583g)(119899 = 3)





Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


ZM2 (25mg)-AON (200 120583g)(119899 = 3)

Diaphragm 8 + minus



Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


mdash





Heart 0 minus minus


+

alginate
















3CN) and


3CN The final









4filter















Acknowledgments


References






























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







ZM2 (25mg)-AON (225 120583g)(119899 = 3)





Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


ZM2 (25mg)-AON (200 120583g)(119899 = 3)

Diaphragm 8 + minus



Heart 0 minus minus

ZM2 (25mg)(119899 = 3)





Heart 0 minus minus


mdash





Heart 0 minus minus


+

alginate
















3CN) and


3CN The final









4filter















Acknowledgments


References






























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials












3CN) and


3CN The final









4filter















Acknowledgments


References






























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







4filter















Acknowledgments


References






























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials








Acknowledgments


References






























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials








































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Documents

Hindawi Publishing Corporation · 2019. 7. 31. · mdx mice were killed following one week ( =3 )orone month ( =3 ) from administration of the last dose. e analysis of cryosections