Developments of mucus penetrating nanoparticles

Developments of mucus penetrating nanoparticles

ARTICLEINFO

Article history∶

Received 6 December 2014

Accepted 31 December 2014

Available online 16 February 2015

∶

Mucus can effectively protect the exposed mucosal surfaces due to its adhesive and viscoelastic properties.Most foreign particulates are ef fi ciently trapped in mucus layers via steric obstruction and adhesion.Trapped particles are typically removed from the mucosal tissue within seconds to a few hours depending on their location sites.This article focuses on describing the tenacious mucus barrier properties,the strategies to investigate the interaction of nanoparticles with the mucus as well as the novel developments of mucus penetrating nanoparticles.

?2015 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

1. Introduction

Nanocarriers have emerged as an effective strategy for mucosa delivery of drugs,which possess a series of desirable properties,including small steric obstruction due to their nanometer size,and protection of cargo therapeutics at both the extracellular and intracellular levels[1].However,one of the greatest challenges that limit the success of nanoparticles (NPs)is their ability to penetrate quickly through mucus to reach the underlying cells.

Mucus is a viscoelastic and adhesive hydrogel that covers in the surface of lung airways,gastrointestinal(GI)tract,female reproductive tracts,eye and other mucosa[2].Mucus protects underlying epithelium by ef fi ciently trapping pathogens and foreign particulates,then rapidly removing them.Therefore, mucus is not only vital for human health,but also represents a substantial barrier to mucosal drug delivery.Mucus forms adhesive interactions with particulates via electrostaticinteractions,van derWaalsforces,hydrophobicforces, hydrogen bonding,and chain entanglement[3,4].Mucoadhesive NPs is to prolong the retention timeof particles in mucosal surface by maximize these interactions[5],which would undergo either direct transit or elimination.Different mucoadhesive systems have been well reviewed previously[6,7].

Another strategy to overcome the mucus barrier and achieve longer retention time in cell surface is to develop a nanocarrier which can effectively penetrate the mucus layer and accumulate in epithelial surface.Justin Hanes and coworkers fi rst proposed mucus penetrating particles(MPP)by mimicking the essential surface properties of viruses that allow them to avoid mucoadhesion[5],showing great promise in mucosal drug delivery.Thereby,the aim of present study is to summarize the properties of mucus,approaches for designing NPs to conquer the mucus barrier as well as the strategies used to investigate the interactions between mucus and NPs.

2. Barrier of mucus layer

Mucus is a viscoelastic,adhesive gel that coats and protects most epithelial surfaces,which ef fi ciently trap most foreign particles and pathogens through adhesive and steric interactions,followed by rapid clearance.Fig.1 illustrated the fate of foreign particulates,including penetrating through mucus(A),trapping in mucus(B)and excluding by mucus(C) [8].The following will elaborate the reasons resulting in various fates of foreign particulates.

2.1. Composition of mucus

Mucus is a hydrogel complex composed of carbohydrates, protein,lipids,antibody,cellular debris bacteria and inorganic salts[9].The barrier properties of mucus are rooted in its dense network of mucin fi bers,which contain highly glycosylated(negatively charged)segments[5],thus show high affi nity with positively charged particles.For example,Laf fl eur F et al.reported that the diffusion rate of neutral polyacrylic acid(PAA)-polypropylene amide(PAM)nanoparticles(NPs)is 2.5-fold higher than positively charged PAM NPs[8].Similar phenomenon also has been reported by other groups that NPs with positively charged surface can be trapped in mucus effectively own to strong electrostatic interactions[10].

Additionally,there exist periodic hydrophobic domains along the mucin strains[11],which can bind hydrophobic particles with high avidity.Although hydrophobic interactions effectively limit the transport of some harmful agents such as bacteria[12,13],italso represents a challenge for the delivery of drug carriers,since the commonly used biomaterials are hydrophobic,like poly(lactic-co-glycolic acid)(PLGA)[14,15]and polystyrene(PS)[16].However,after coating PLGA NPs with hydrophilic DNA,the average transport rates can be improved 10-fold in reconstituted pig gastric mucus[17].

2.2. Viscoelasticity

The viscoelasticity of mucus are essential for its protective properties.As reported,a rather moderate decrease of viscoelasticity can signi fi cantly promote bacterialand sperm motility[11].Mucin,as the main component of mucus,directly affects the viscoelastic properties of mucus. A series studies have shown that the mucins can be changed in amount,type and size in disease[18-20].For instance,compared with healthy secretions,the mucin concentration increased approximately 7-fold for patient with asthma,further increasing the dif fi culty of mucosal drug delivery[21].Apart from mucin,other factors also play a key role in regulating mucus viscoelasticity,including lipids,inorganic salts,pH and cellular debris.The cell debris DNA can further increase the viscoelasticity of mucus due to its fi bers are even longer than mucin fi bers[18].Besides, highly acidic environments(pH＜4)would cause the aggregation of mucin fi bers and greatly increase the mucus viscoelasticity[22].Therefore,to ensure free diffusion of NPs in mucus,large amounts of mucus mucolytic agents can be adopted to reduce the viscoelasticity of mucus (Fig.2),including papain[23,24],recombinant human DNase (rhDNase)[10,25],N-acetyl-L-cysteine(NAC)[26]and guluronate oligomers[27].

2.3. Steric obstruction

Mucus gel is composed of highly cross-linked mucin fi bers by hydrophobic interactions and disul fi des link,creating a dense porous structure.However,mucus displays different pore size depending on its location on the body,such as,the average pore size of human cervicovaginal mucus(CVM)is 340±70 nm[28]and 550±50 nm for fresh bovine vitreous [29],while smaller mesh spacing inherent to cystic fi brosis (CF)sputum(140±50 nm)own to the higher concentrations of mucins,DNA,and actin[30].Thereby,to penetrate mucus,nanocarriersmustbesmallenoughtoavoid steric obstruction in spite of NPs with larger size are preferred to improve drug loading and release kinetics[5]. As reported,a 2-fold increase in particle size,from 510 nm to 1190 nm for PS-PEG NP,would led to a 30-fold decrease in the ensemble-averaged mean squared displacement[29]. Italsohasbeen reported thatthenanospheressize approaching 560 nm were almost completely blocked by the sputum[25].Similarly,Norris and Sinko studied the diffusion of variously sized PS particles in reconstituted porcine gastric mucin gel,and observed a sharp decrease in translocation permeability when particle sizes reach 300 nm[31].

2.4. Dynamic properties

Mucus is constantly secreted,subsequently shed and discarded or digested and recycled.Its turnover time is short, especiallyforthelooselyadherentmucuslayer,often measured in minutes to hours.For oral drug delivery,the intestinal mucus turnover time is 50-270 min[32],resulting in ef fi cient clearance of administered particulates.

In conclusion,the understanding of mucus compositions and properties is important to design nanocarriers which can avoid the blockagein mucus,meanwhile penetratemucusat a rate signi fi cantly higher than mucus turnover cycle.

3. MPP by modi fi cation their surface physicochemical properties

NPs properties including charge and hydrophobicity have a great in fl uence on their behavior of penetrating through mucus.As a consequence,to prepare NPs with a suf fi ciently hydrophilic and uncharged surface to effectively minimize the adhesive interactions betweenmucin and NPs by reducing hydrophobicorelectrostatic interactions,show promise prospect on mucus penetrating.

3.1. PEG-modi fi ed NPs

Coating NPs with low molecular weight(MW)PEG is the most widely studied mucus penetrating strategy(Fig.3A)[33].There are various PEG modi fi ed MPP described in the literature (Table 1).PEG is an uncharged hydrophilic polymer which was used to increase the mucus adhesion in earlier studies[34,35]. While later researches have shown that coating NPs with a high density of low MW PEG can reduce the interactions between particle and mucus.The possible reasons are as followings:the MW of PEG was too low to support adhesion via polymer chains interpenetration and the PEG density is suffi cient to shield the hydrophobic core effectively[36].To determine the effect of PEG MW on the interactions of coated particles with mucus,Hanes et al.studied the diffusion rate of PS NPs modi fi ed by different MW(2,5 and 10 kDa)and densities(42±3%,65±1%and 69±1%)of PEG in CVM.The experimental results showed that low MW(e.g.2 kDa)and high-density(e.g.65-70%)PEG coating can facilitate the NPs to pass through mucus[36].Apart from modi fi ed PS NPs,PEG also can be used to conjugate with other polymeric materials to prepare MPP,like PLGA[37],poly sebacic acid(PSA)[38], polyethylenimine (PEI)[39]and poly-L-lysine(PLL)[40]. Furthermore,densely PEGylated particles are able to readily penetrate chronic rhinosinusitis mucus(CRSM)samples and sputum expectorated from the cystic fi brosis(CF)patients with higher viscosity[37].

In addition to covalent conjugation of PEG to the particles core,PEG also can be physically absorbing on the particle surface by hydrophobic or electrostatic interactions[41,42]. This kind of NPs has similar mucus penetrating properties as long as their surface is densely coated by low MW PEG.

3.2. Pluronic F-127 modi fi ed NPs

Triblock copolymer of poly(ethylene glycol)-poly(propylene oxide)-poly(ethylene glycol)(PEG-PPO-PEG;known as Pluronics)has a long application history in oral,intravenous and ophthalmic administration,which can coat hydrophobic particle surfaces by adsorption of the hydrophobic PPO segments,leaving a dense brush of uncharged,hydrophilic PEG segments protruding from the particle surface[43](Fig.3B). Studies have shown that Pluronics containing PPO segments with MW＞3 kDa,such as F-127,can produce MPP.Coating PLGA nanoparticles with Pluronic F127 can effectively block adhesive interactions between the PLGA core and mucus constituents,which are similar to covalent conjugation of PEG toPLGA,simultaneously without changingthestructureofthe PLGA carrier materials[44].Other studies also indicated that the average speed for PLGA/F127 particles was only 20-fold reduced compared to their theoretical speed in water(uncoated PLGA particles were slowed by＞1000-fold)[37].Similarly,X.Li et al.designed a core shell corona nanolipoparticles (CSC)which contains chitosan NPs as a core component and pluronic F127-lipid vesicles as a shell with hydrophilic chain and polyethylene oxide PEO as a corona for oral protein delivery.CSC can further improve the absorption of drug through enhanced intestinal mucus penetration[45].Additionally,otherstudycomparedthemucuspenetrating behavior of Pluronic F127-modi fi ed liposomes(PF127-Lip)and chitosan-modi fi ed liposomes(CS-Lip).Pharmacokinetic analysis in rats shows that the Cmaxand AUC0-tof F127-Lip were 1.73-fold and 1.84-fold higher than those of CS-Lip,respectively.This indicated F127-Lip more suitable for drug nanocarriers[46].Besides,it is interesting to found that PS NPs can rapidly penetrate CVM if the CVM is pretreated with suf fi cient concentrations of F127[47].

However,it needs to be noticing that not all hydrophilic and neutral modi fi cation can facilitate mucus penetration. Like hydrophilic and uncharged polyvinyl alcohol coated PS NPs were immobilized,with speeds at least 4000-fold lower in mucus than in water,regardless of the MW or incubation concentration of PVA[48].

4. MPP by disrupting the mucus barrier

Due to the tenacious and sticky network of mucin fi bers,the diffusion of foreign particles is restricted by trapping and steric hindrance.According to the nature of mucus,a technique to reach the underlying cell layer is presented by disrupting the mucoglycoprotein substructures using mucolytic agents[49] (Fig.3C).For example,Mu¨ller C and coworkers prepared NPs composed of polyacrylic acid(PAA)and papain.The presence of papain on the surface and inside of the particles could strongly decrease viscosity of the mucus thus leading to particle transition across the mucus layer quickly[23,24].

In addition,recombinant human DNase(rhDNase)is the most commonly used mucolytic in CF,which can hydrolyzes the DNA that forms dense entanglements with mucin glycoproteins and other mucus constituents,thus reducing the number of viscoelasticity and crosslinks of mucus[50]. Sanders et al.observed rhDNase moderately facilitated the transport of nanospheres through CF sputum[25].However, Dawson et al.found that treatment with rhDNase dramatically narrowed the distribution of individual particle diffusion rates and reduced macroviscoelastic properties of CF sputum by up to 50%,but did not signi fi cantly alter the ensembleaverage particle diffusion rate.This might attribute to the hydrolytic cleavage of DNA into smaller fragments which diffuse freely into micropores and then increased the microviscosity within the pores[10].

N-acetyl-L-cysteine(NAC)is another common mucolytic for its ability to facilitate the penetration of NPs across CF sputum.NAC disrupts the structure of the mucus polymer by substituting free thiol(sulfhydryl)groups for the disul fi de bonds connecting with mucin proteins[26].As a result,both the elasticity and viscosity of the mucus are lowered.Alton et al.found that the mucus barrier to non-viral gene vectors can be overcome partially by treatment with NAC in an ex vivo model of sheep tracheal epithelium[51].However,it is still need further investigation to clarify if the combination of polymeric NPs with mucolytic agents will promote more endotoxins and other toxic substances absorption by decreasing the local viscosity of mucus layer.

5. Research strategies of mucus penetration

5.1. Diffusion experiments

Methods used to study particle diffusion in mucus include multiple particle tracking(MPT),Ussing chamber or Transwell-Snapwelldiffusion chamberand so on.Transwell-Snapwelldiffusion chamberwas fi rstused for quantitatively measuring the diffusivity of particles in mucus, which consists of two chambers with the placement of the mucus layer in the middle.Although this method is conceptually straightforward,it is also sensitive to parameters that are dif fi cult to control,including the thickness of the mucussample,alterations in mucus properties and blockage of fi lter pores by mucus[52].

In order to avoid the defects in diffusion chamber experiments,many studies recorded the NPs dynamic transit in the mucus using fl uorescence microscopy,such as fl uorescence recovery after photobleaching(FRAP)and multiple particle tracking(MPT).FRAP is the fl uorescently labeled NPs exposure to a laser beam to form a fl oating white spot.The diffusion coef fi cient is obtained by the recovery of the fl uorescence intensity,which results following diffusion of the fl uorescently labeled molecules into this area with the fl ow of NPs[53].Shen etal.applied FRAPtoexamine the diffusion ofplasmid DNAsin mucus[54].Additionally,FRAP also has been used to investigate the effect of guluronate oligomers on mobility of NPs in mucous matrices,and results showed that guluronate oligomers can improve NPs mobility in native pig gastric mucus (Fig.4A and B)[27].FRAPcan beused to investigate the mobility of labeled molecules in mucus and biogels,but it provides only ensemble-averaged diffusion rates and cannot be used to quantify the transport rates of individual particles.A lack of information at the individual particle level may limit its application in complex mucus[55].For this purpose,Hanes et al.have pioneered MPT to measure the transport of NPs in mucus.As shown in Fig.4C and D[56],it can record each particletrajectoriesbyinverted fl uorescencemicroscope meanwhile MPT can be used for analysis of NPs in some complex biological fl uids,such as sputum[57].Apart from this, the diffusion behavior of NPs in the mucus also can be studied by rotating diffusion tubes[58]and mucus slices[59]etc.

5.2. Cell models

5.2.1. HT29-MTX cell model

HT29 cells belong to human colonic adenocarcinoma cell line. Under the in fl uence of methotrexate(MTX)[60],HT29 differentiated into mature goblet cells,such as E12 cells,which can secrete mucus.Therefore,HT29-MTX cells can be used to study thein fl uenceofmucuslayeronNPstransport[61].However,this modelhassomedrawbackscomparedtotheinvivosituationdue to it is based on only one cell type of the intestinal epithelium.

5.2.2. Caco-2/HT29-MTX co-cultures cell model

Human colon carcinoma Caco-2 and HT29 cells were established to represent the two most abundant cell populations in the intestinal epithelium,absorptive cells and goblet cells. Therefore,co-cultures of Caco-2 cells and mucus-producing goblet cells HT29-MTX would provide a drug absorption model incorporating the mucus barrier[62].Wilkman-Larhead fi rst characterized co-cultures of Caco-2 and goblet-like HT29-MTX cells as in vitro drug and peptide absorption models[63]. In general,a higher amount of Caco-2 cells leads to higher transepithelial electrical resistance(TEER)values probably due to more intensely formed tight junctions.When HT29 cell ratio is 25%,the change of TEER value is 0-790 Ω·cm2in 0-23 days. whentheratiois75%,theTEERvaluedowngradeto 0-310 Ω·cm2.Goblet cells comprise a quantitatively signi fi cant component of the GI tract,comprising approximately 10%of the duodenal epithelium and increasing to 24%in the distal colon.Thus,to maintain better in vivo/in vitro correlationrelevance,the co-cultures proportion of Caco-2/HT29-MTX is 90%/10%or 75%/25%mostly[64,65].

5.2.3. Caco-2/HT29-MTX/Raji B triple culture model

Apart from enterocytes and goblet cells,M cells located in the epithelium that overlay the Peyer's patches also play a dominant role.Several reportssuggest that NPs are capable to enter intestinal epithelia via M cells while uptake by absorptive enterocytes only plays a minor role[66,67].In order to design a model which can mimic the small intestinal epithelial more accurately,some studies establish an in vitro cellular model based on Caco-2,mucus-producing HT29-MTX,and human Burkitt's lymphoma Raji B cells which represent M cells.The model was set up by seeding co-cultures of Caco-2 and HT29 cells into Transwell fi lters and maintained under identical conditions following the addition of Raji B to the basolateral chamber[68,69].

In addition,there were also studies designed and characterized biosimilar mucus compatible with Caco-2 cell monolayers cultured in vitro to establish a more representative in vitro model for the intestinal mucosa to predict the in fl uence of mucus on intestinal drug absorption[70].

5.3. Animal models

To better understand the fate of the particles and how the results might translate in humans,many studies adopt animal models to investigate the therapeutic effects or pharmacokinetics of NPs,which mainly include isolated intestinal experiments,in situ experiments and in vivo experiments.

5.3.1. Isolated intestinal model

To avoid the shortcomings of cell monolayer model such as lack of three-dimensional macrostructure and cells of varying degrees of differentiation,some studies adopted isolated intestinal experiments(including everted intestinal sac and permeability study by Ussing chamber[71])to measure the mucoadhesive properties of NPs.However,in such model,the intestinalneed toberemoved,opened,washed and segmented,which may change the intestinal property and failed to predict what occurred in vivo[72].

5.3.2. In situ model

Intestinal loop models have been used for decades to investigate systemic absorption of drugs.In this model,a portion of the small intestine is excised from the abdominal cavity, subsequently ligated at both ends to make an isolated“l(fā)oop”, and the test NPs is directly injected into the loop.After a chosen time period,the animal is sacri fi ced and the intestinal loop is removed from the body cavity for further morphology or quantitative analysis[73].Several studies have used this modelto investigate thein fl uenceofmucusonNPsabsorption and the amounts of NPs trapped in mucus[45,46].

5.3.3. In vivo model

No matter how sophisticated an in vitro model,eventually in vivo evaluation is necessary to validate the true performance of a drug delivery system.For example，the signi fi cant difference existed in mucus composition and thickness with the position of the GI tract,it is dif fi cult to simulate these in vitro experiments.However,a shortcoming existed in all models so far is their non-human nature,which shows great difference in human studies[72].

6. Conclusions

Mucus layer covering in exposed epithelial surfaces of the body has vital protective and lubrication effect.However,the adhesive and rapidly update properties of mucus is one of the main barriers for mucosal drug delivery.A promising strategy to tacklethisproblemisuse ofMPPwhichcanreadilyin fi ltrate into the mucus layer before turnover occurring.While some problems for MPP including the epithelial barrier,the security of MPP and suitable mucus models still need to be further investigated.

Acknowledgement

We gratefully acknowledge fi nancial support from the National Natural Science Foundation of China(81173010).

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Min Liu,Jian Zhang,Wei Shan,Yuan Huang*

Key Laboratory of Drug Targeting and Drug Delivery System,Ministry of Education,West China School of Pharmacy, Sichuan University,No.17,Block 3,Southern Renmin Road,Chengdu 610041,China

*Corresponding author.West China School of Pharmacy,Sichuan University,Chengdu,Sichuan 610041,China.Tel./fax:+86 028 85501617. E-mail address:huangyuan0@163.com(Y.Huang).

Peer review under responsibility of Shenyang Pharmaceutical University.

http://dx.doi.org/10.1016/j.ajps.2014.12.007

1818-0876/?2015 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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