The particle in the spider's web: transport through biological ...mitmrsec.mit.edu/sites/default/files/images/The particle...Nanoscale FEATURE ARTICLE Cite this: Nanoscale, 2017, 9,

Nanoscale

FEATURE ARTICLE

Cite this: Nanoscale, 2017, 9, 8080

Received 18th December 2016,Accepted 12th May 2017

DOI: 10.1039/c6nr09736g

rsc.li/nanoscale

The particle in the spider’s web: transport throughbiological hydrogels

Jacob Witten a,b and Katharina Ribbeck*a

Biological hydrogels such as mucus, extracellular matrix, biofilms, and the nuclear pore have diverse func-

tions and compositions, but all act as selectively permeable barriers to the diffusion of particles. Each

barrier has a crosslinked polymeric mesh that blocks penetration of large particles such as pathogens,

nanotherapeutics, or macromolecules. These polymeric meshes also employ interactive filtering, in which

affinity between solutes and the gel matrix controls permeability. Interactive filtering affects the transport

of particles of all sizes including peptides, antibiotics, and nanoparticles and in many cases this filtering

can be described in terms of the effects of charge and hydrophobicity. The concepts described in this

review can guide strategies to exploit or overcome gel barriers, particularly for applications in diagnostics,

pharmacology, biomaterials, and drug delivery.

1. Introduction: hydrogels areubiquitous selective barriers in biology

Biological hydrogels, which are composed of hydrated polymernetworks, are found throughout every domain of life. For

example, both archaea1 and bacteria can form biofilms, ormicrobial aggregates surrounded by secreted extracellular poly-meric substances (EPS) that act as a protective barrier andcreate microenvironments within which microbes thrive andadapt to harsh conditions.2,3 In eukaryotes, hydrogels haveevolved to fulfill an astonishingly diverse set of functions. Forexample, the nuclear pore is a barrier formed from crosslinkedintrinsically disordered proteins called nucleoporins, whichcontrol the passage of macromolecules between the nucleusand the cytoplasm.4,5 One of the largest hydrogels in the body

Jacob Witten

Jacob Witten received his BA inMathematics and Biophysicsfrom Amherst College in 2014,where he researched compu-tational methods for hydrogenexchange mass spectrometry. In2015, he joined ProfessorKatharina Ribbeck’s group in thedepartment of BiologicalEngineering at MassachusettsInstitute of Technology as a doc-toral student. His currentresearch combines compu-tational and experimental tech-

niques to study molecular transport in mucus, focusing on appli-cations to drug delivery for treating diseases such as cysticfibrosis.

Katharina Ribbeck

Katharina Ribbeck is Eugene BellCareer Development Professor ofTissue Engineering in theDepartment of BiologicalEngineering at the MassachusettsInstitute of Technology. Shereceived her Ph.D. in Biology atthe University of Heidelberg inGermany. Her lab studies thebasic mechanisms by which bio-logical hydrogels barriersexclude, or allow passage ofdifferent molecules and patho-gens, and the mechanisms patho-

gens have evolved to penetrate mucus barriers. Her goal is todevelop the foundation for a theoretical framework that capturesgeneral principles governing selectivity biological hydrogels, and forthe reconstitution of synthetic gels that mimic the basic selectiveproperties of biological gels.

aDepartment of Biological Engineering, Massachusetts Institute of Technology,

Cambridge, MA 02139, USA. E-mail: [email protected]; Tel: +1 (617) 715-4575bComputational and Systems Biology Initiative, Massachusetts Institute of

Technology, Cambridge, MA 02139, USA

8080 | Nanoscale, 2017, 9, 8080–8095 This journal is © The Royal Society of Chemistry 2017

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is the mucus gel (Fig. 1a) that lines all wet epithelia and pro-tects the underlying cells against toxins, pollutants, and invad-ing pathogens.6–10 Another example of a hydrogel-basedbarrier is the extracellular matrix (ECM) surrounding cellswithin tissues. ECM provides mechanical stability but alsoforms a selective barrier that regulates transport of signalingmolecules secreted by the cells (Fig. 1b).11–16

The permeability of biological hydrogels poses a challengefor biomedical development. For example, biofilms areinvolved in the majority of infections in developed countries;they form on medical implants and wounds, cause middle-earinfections and gingivitis, and more.17 One driver of antibioticresistance in biofilms is binding of antibiotics to EPS, whichsequesters them and/or reduces their penetration into thebiofilm. In humans, the ECM is an obstacle to drug delivery,as its mesh prevents large nanoparticles from penetratingdeep into tissue or tumors.18–24 Mucus similarly controls thepermeability of nanoparticles,25,26 but can also limit diffusionof small molecules such as antibiotics.

Biological hydrogels are complex molecular assemblieswith context-dependent properties. For biofilm EPS, the struc-ture of the matrix varies widely across species, strains, andenvironments, complicating the characterization of EPS com-

ponents. However, biofilm EPS commonly contains extracellu-lar DNA and various types of polysaccharides.2 One well-studied biofilm is that formed by Pseudomonas aeruginosa, anopportunistic pathogen that can cause biofilm-associatedinfection in wounds27 or in the lungs of patients withcystic fibrosis (CF).28 Interestingly, even this single species pro-duces anionic (alginate), cationic (Pel),29 and neutral (Psl)30

polysaccharides in differing amounts depending on contextand bacterial strain.31

Fibrous structural proteins such as collagens, elastin, fibro-nectin, and laminins, together provide much of the structuralintegrity of ECM. ECM also contains high levels of polyanionicglycosaminoglycans (GAGs) and proteoglycans (proteins withdensely grafted GAGs) that give ECM a high net negativecharge density. Structural components of ECM are covalentlycrosslinked to varying degrees, regulating matrix stiffness.11

In human mucus, the main gel-forming componentsconsist of a family of large, intrinsically disordered secretedglycoproteins called mucins. The main secreted mucins areMUC2, MUC5B, and MUC5AC, although other secreted mucinscan be present in smaller amounts as well.7 Mucins have alter-nating regions of small, globular hydrophobic domains andhighly glycosylated, primarily anionic (due to sialic acid andsulfation) unstructured regions.7,32,33 Other components ofmucus include lipids, soluble proteins and peptides, andnucleic acids.32 Mucus composition can vary depending ondisease state, and in this review we highlight CF, a geneticdisease in which improper ion balance results in pathologi-cally thick, dehydrated mucus.34 The presence of necroticneutrophils in CF airways results in high levels of free DNAand actin filaments in lung mucus,35 increasing the mucus’viscoelasticity32 and playing important roles in binding tocations. More detailed reviews can be found on composition ofbiofilms,2,36 of the ECM,11,24,37 and of mucus.6,7,32,38,39

While biological hydrogels are distinct in terms of theirlocations and molecular compositions, they share certaincommon principles that govern selective filtration. The goal ofthis review is to address the general principles that apply tothe permeability of many types of biological hydrogels.

Broadly speaking, the transport of a solute through a gel iscontrolled by the solute’s size, its interactions with the com-ponents of the gel, or a combination of the two (Fig. 2).40 Size,or steric, filtering is a universal feature of biological gels,which have a polymeric mesh size that constrains thediffusion of large particles (Fig. 2a). Pure steric filtering is animportant component of hydrogel selectivity, but it is alsocrude because it only selects based on one parameter. Filteringbased on chemical interactions between solutes and gel com-ponents is also a common feature of biological gels.Depending on their chemistry, the gel components interactwith solutes across gradients of chemical properties includingcharge and hydrophobicity, thereby differentially affectingdiffusion.40 A lack of interactions enables unhindereddiffusion (Fig. 2b), while certain weak and diffuse solute–gelinteractions can facilitate penetration of the solute into the gelwithout slowing transport (Fig. 2c). Binding to hydrogels, on

Fig. 1 Scanning electron microscopy of biological hydrogels. (a)Cervical mucus samples from pregnant patients at low or high risk forpreterm birth. Scale bar: 200 nm. Reprinted with permission fromCritchfield et al. (2013).196 Copyright (2013) PLOS. Published under CCBY license. https://creativecommons.org/licenses/by/4.0/legalcode. (b)Reconstituted basal lamina ECM gels. Scale bar: 25 μm. Reprinted withpermission from Arends et al. (2015).197 Copyright (2015) PLOS.Published under CC BY license. https://creativecommons.org/licenses/by/4.0/legalcode.

Nanoscale Feature article

This journal is © The Royal Society of Chemistry 2017 Nanoscale, 2017, 9, 8080–8095 | 8081

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http://dx.doi.org/10.1039/C6NR09736G

the other hand, reduces effective solute diffusivity and hinderspenetration (Fig. 2d). In drug delivery applications, facilitatedtransport and reduced effective diffusivity from binding can becombined to optimize drug delivery.21,41

2. Size-dependent filtration2.1 Length scales and hydrogel mesh

Size effects are important for the transport of viruses, bacteria,eukaryotic cells, particulate pollutants, nanoparticles, and anyother particle on the same length scale as the gel mesh. In thissection, we discuss size filtering while assuming that all par-ticles are inert to (do not interact with) gel components. To afirst approximation, steric interactions are quite simple,corresponding in the macroscopic world to the fact that an ele-phant, but not a fly, can be stopped by a chain-link fence,while a tennis ball can pass through with some prodding.

In biological hydrogels, the microscopic mesh is formed byentangled and crosslinked polymers. The distance betweenadjacent links in the chain-link fence corresponds to the meshsize; objects much smaller than the mesh size diffuse at a ratecorresponding to the viscosity of the interstitial fluid (the fluidbetween fibers; Fig. 3a), objects on the order of the mesh sizeare obstructed but not completely stopped (Fig. 3b), andobjects much larger than the mesh size are trapped (Fig. 3c).This steric barrier is important for mucus because it blocksand/or traps large pollutants and potential pathogens, thusallowing some mechanism, such as mucociliary clearance, toclear the invading particle before it can reach the epithelium.8

Mesh size is also an important consideration for nanoparticledesign, as it sets a maximum possible size for a nanoparticlethat must penetrate a hydrogel barrier. The mesh sizes ofmucus, biofilm EPS, and ECM vary and are on the order of10–1000 nm.19,20,23,24,42–46 Note that measurements of meshsize may be incorrect if the particles used to probe the meshsize interact with the gel;47 we discuss adhesive interactions indetail in section 4. The nuclear pore is filled with intrinsically

disordered proteins called nucleoporins that form a mesh witha mesh size ∼2.5–5 nm in size that excludes proteins andprotein complexes larger than 30–100 kDa, unless they are cha-peroned by a nuclear transport receptor (NTR).48,49

Note that biological gels are often heterogeneous, with adistribution of mesh sizes. For example, some biofilmscontain channels that allow efficient passage of large particlesto cells deep in the biofilm.46,50 In mucus, nanoparticles oftenshow a broad diffusivity distribution in particle trackingexperiments, suggesting that some nanoparticles are trappedwhile others diffuse nearly freely.32,45,51 One important ques-tion is whether the regions that allow free diffusion are con-nected enough to allow efficient penetration through macro-scopic mucus layers, or whether these regions are simply largewater-filled pores surrounded by impassable polymeric bar-riers. Answers to this question are somewhat conflicting, butoverall it appears that mucoinert particles pass through macro-scopic mucus barriers.52–54

On a related note, collagen fibrils in ECM are often alignedwith each other; this directionality of the mesh drives an-isotropic diffusion behavior that may be important for transportin ECM.55,56 The importance of diffusion anisotropy is unclearin other gels, although filamentous bacteriophage can driveliquid crystallization of P. aeruginosa biofilms, suggesting thepotential for anisotropic transport in that context.57 Finally, incontrast to a chain-link fence, in gels thermal motion andstructural dynamics play a role in controlling transport.58 Gelpolymers and physical crosslinks constantly rearrange, withlocal deformations allowing large particles to escape steric bar-riers and caging effects.59–63

2.2 Modulation of gel structure by transporting particles

The transport of a particle through a gel may also be promotedby the solute directly interfering with the gel’s componentpolymers or crosslinks, thus changing the mesh size. Forexample, attaching the mucolytic protein papain to nano-particle surfaces somewhat enhanced penetration of the nano-particles through intestinal mucus (Fig. 3d).64,65 Treatment of

Fig. 2 Effects of steric hindrance and chemical interactions on gel penetration. (a) Particles above the mesh size are unable to penetrate the gel,even if they do not interact with the gel. (b) Small inert particles penetrate gels. (c) Under some conditions (see Section 3.4 for details), weak inter-actions with gel polymers can enhance partitioning into the gel and subsequent penetration. The schematic assumes that the bath is fixed at a con-stant solute concentration. (d) Binding to the gel causes enrichment of solute at the bath-gel interface but slowed gel penetration.

Feature article Nanoscale


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CF lung mucus with N-acetyl cysteine, a disulfide bondingreducer, likewise facilitated transport by increasing the meshsize,66 although surprisingly treatment with recombinantDNase (used in CF to degrade DNA in mucus) did not improvetransport.67 An elegant example of crosslink interference mayoccur in the nuclear pore (discussed in detail in Section 5), inwhich transporters reversibly disrupt physical crosslinks,allowing fast transport and rapid self-healing of the pore struc-ture (Fig. 3e).68 This mechanism of reversibly disrupting physi-cal crosslinks has not, to our knowledge, been found in anyother biological system, but it presents an intriguing mechan-ism for biology and engineering applications. Finally, mucin-binding particles may sequester mucin strands, reducing theconcentration of gel strands elsewhere in the gel. Thisreduction increases the effective mesh size and thus enhancestransport of other, non-mucin binding nanoparticles.69,70

Living cells also modulate gel structure as they move. Manytypes of human cells degrade and/or secrete various ECMelements in tightly regulated ways, with degradation some-times necessary for cell motility.71 Improper regulation of ECMstructure, including overexpression of ECM-degrading matrixmetalloproteinases or the collagen crosslinking factor LOX,has been associated with numerous types of cancer.37

Similarly, biofilm EPS is constantly remodeled by bacteria.42

Bacterial motion disrupts the gel structure,72 while lyticenzymes such as alginate lyase are both used by bacteria andhave potential as anti-biofilm treatments.73,74 The stomachpathogen Helicobacter pylori has also been shown to facilitateits motion by secreting high levels of ammonia into stomachmucus; the local increase in pH deprotonates carboxylic acidgroups in mucin, which reduces intermolecular hydrogenbonding and hydrophobic interactions while increasingelectrostatic repulsion between mucin strands. H. pylori is ableto easily swim through this locally weakened gel.75

2.3 Techniques to analyze particle transport

Experimental assays of particle transport generally require theparticles to be fluorescently labeled or radiolabeled. Fromthere, one common technique to measure transport is directvisualization of bulk particle transport into or through a gel,followed by qualitative analysis76 or quantitative fitting of con-centration timecourses or spatial profiles as the particles pene-trate the gel.77,78 Bulk transport visualization methods aresimple and easily visualized, but they are generally unable tomeasure heterogeneity or other structural details. Two relatedtechniques for analyzing particle transport in gels are fluo-rescence recovery after photobleaching (FRAP) and fluo-rescence correlation spectroscopy (FCS).55,79 In FRAP and FCS,

Fig. 3 Steric effects on diffusion in gels (a–c) and mechanisms to modulate steric hindrance (d, e). Thin lines represent thermal motion of the par-ticle. (a) Particles smaller than the mesh size diffuse freely in interstitial fluid. (b) Particles on the order of the mesh size have significant steric hin-drance but eventually penetrate gels. (c) Large particles are trapped. (d) Particles that cleave gel polymers may diffuse more quickly. Notched greencircles attached to particle are lytic enzymes; green segments of gel polymer are substrates for the enzymes. (e) Particles may reversibly disrupt gelcrosslinks (blue–blue contacts), allowing enhanced diffusion without irreversibly degrading the gel.



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the gel is pre-incubated with fluorescently labeled particles. InFRAP, a region of the gel is photobleached and the recovery offluorescence in the bleached spot is fit to models that calculatediffusion parameters.80 In FCS, the fluorescence autocorrela-tion function within a small gel region is measured and fit.79

With FRAP and FCS, observing and fitting parameters for sub-diffusion may be possible using appropriate fittingprocedures.81–84 Subdiffusion refers to diffusion behavior inwhich the mean squared displacement of individual particlesscales with tα for α < 1, rather than with t1 as in standarddiffusion; subdiffusion or transient subdiffusion is commonlyobserved in gel transport studies and can impact importantparameters such as passage time through a gel.85,86

An alternative to bulk transport analysis is single particletracking (SPT), in which the thermal motion of individual par-ticles is recorded and analyzed.87 SPT easily identifies bothsubdiffusion and heterogeneity in gel samples, which is usefulfor identifying subpopulations of particles that diffuse rela-tively unhindered.32 This technique’s main weakness is thatdiffusion out of a microscope’s focal plane renders it difficultto track individual particles over tens of microns, whichmeans that unlike in bulk diffusion or FRAP experiments,direct measurements of penetration through physiologicallysized gels is not feasible.54

Finally, nanoparticle dispersal has been imaged in vivo fora variety of mucus systems as well as brain ECM. These assaysdo not provide quantitative measurements of diffusion para-meters, but nonetheless serve as a valuable bridge betweenin vitro studies and translation to the clinic.47,52,88,89

3. Interactive filters3.1 Overview: effects of chemical interactions on gelpenetration and retention

Biological hydrogels selectively interact with foreign particlesbased on the solutes’ chemical properties. These interactionsoften lead to reversible binding, which may be desirable, un-desirable, or neutral depending on context. Since reversiblebinding improves retention within a gel, it may enable longerdrug action, as in mucoadhesive drug delivery systems.9,90

Similarly, binding of native immune factors to cervical mucusmay promote their retention at high concentration and preventmicrobial penetration through the cervical mucus plug duringpregnancy, which could lead to intrauterine infection andpreterm labor.39 On the other hand, reduced diffusivity mayslow the penetration of a gel (Fig. 2d), impacting the oral bio-availability of drugs or microbial killing by antibiotics.Similarly, binding can sequester a molecule and decrease itseffective concentration. Finally, diffusivity could simply be atunable regulatory parameter, neither intrinsically “good” nor“bad.” Whatever the effect of gel binding, unlocking the forcesunderlying it is crucial to understanding the function of nativesystems and optimizing drug function.

As with nanoparticles, bulk diffusion assays, FRAP, and FCSare commonly used to measure the diffusion coefficients of

small solutes such as drugs and proteins.46,91,92 SPT is moredifficult for smaller solutes because of the difficulty inherentin resolving individual molecules with fluorescencemicroscopy; this technique is rarely if ever used to studydiffusion of proteins or smaller molecules in gels.

To clarify what exactly is meant by slowing solute pene-tration and improving retention, we briefly present the stan-dard model for diffusion of a solute with first-order reversiblebinding to a gel. Under the assumptions that (1) binding andunbinding are fast, such that the solute reaches local equili-brium between bound and free states at each point, (2) boundsolute is immobile, (3) binding sites within the gel are farfrom saturated, and (4) binding to the gel does not disrupt gelstructure, we can define the effective diffusivity Deff as:

Deff ¼ DFð1þ NT=KDÞ�1 ð1Þ

where DF is the diffusion coefficient of the free solute in thegel (which may be lower than the diffusivity in water, due tosteric constrains or increased interstitial fluid viscosity), NT isthe total binding site density in the gel, and KD is the dis-sociation constant.

The timescale for a solute to penetrate a gel (or equiva-lently, the timescale of escape from a gel) τlag is related to theDeff by:

τlag � L2=Deff ð2Þ

where L is the length scale of the gel. These two equationsshow how increasing solute–gel binding strength or gelbinding site density increases τlag, thus slowing penetration orimproving retention.77 Fig. 4a shows this effect in action, asthe penetration of tobramycin (an aminoglycoside antibiotic)into a P. aeruginosa biofilm is hindered by binding of tobramy-cin to biofilm components.76

Given these results, it is no surprise that specific protein–ligand interactions are commonly exploited to regulate trans-port and retention within a gel, often for protective purposesor to control signaling.12,93 For example, sialic acid-terminatedO-linked oligosaccharide chains on secreted mucin molecules,particularly Muc5AC, act as decoy receptors for sialic acid tar-geting bacteria and viruses, including the influenza virus.94

This interaction slows penetration of sialic acid targeting bac-teria and viruses through the mucus layer to the vulnerableepithelium95,96 and competitively inhibits binding to cell-associated sialic acid.94 A mouse model with lung Muc5ACoverexpression has increased resistance to influenza, illustrat-ing this protective function of lung mucus.97 In the ECM,binding of insulin-like growth factor (IGF) to IGF binding pro-teins limits IGF transport, thus helping to regulate intercellu-lar signaling.13 Sequestration of transforming growth factor-βin the ECM is another well-studied case showing the impor-tance of binding to the gel matrix.98,99 In addition to non-covalent protein–ligand interactions, covalent linkage to bio-logical hydrogels is also possible; for example, certain muco-adhesive formulations use free thiol groups to form disulfidebonds to mucin.100



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While eqn (1) and (2) describe the simplest effects of gelbinding, interesting cases arise when the assumptions are vio-lated. For example, saturation of gel binding sites results inenhanced penetration because there are fewer binding sitesavailable per solute molecule to arrest diffusion.76 In anotherexample, Braga et al. showed that if bound solute is mobilewith diffusivity DB but the other assumptions hold, (we havechanged the notation slightly to stay consistent with thisreview):101

Deff ¼ ð1� f ÞDF þ fDB ð3Þwhere f is the fraction of time a solute is bound (or equi-valently, the equilibrium bound fraction of solute at equili-brium). It can be shown that f = NT/(NT + KD), so we recovereqn (1) in the limit of immobile bound solute (DB = 0). In theopposite limit of DB = DF, penetration is not slowed becauseDeff = DF. In the nuclear pore, the mobility of bound solutemay help explain why weak binding of NTRs to nuclear porecomponents does not arrest passage.102 On the applied side,Fig. 4b shows that penetration of the positively chargedprotein Avidin into articular cartilage ECM is facilited byelectrostatic interactions (Fig. 4b), potentially allowing Avidin

to act as a nanocarrier for osteoarthritis drugs.21,41 We empha-size the dramatic difference between Fig. 4a and b: whiletobramycin and Avidin both electrostatically interact with theirrespective gels, tobramycin penetration is inhibitedwith respect to a neutral counterpart, whereas Avidin pene-tration is facilitated. The difference arises because Avidin-ECMelectrostatic interactions do not hinder Avidin diffusion,and in fact these interactions do not constitute binding at allin some sense; Avidin transport is best described usingDonnan partitioning analysis.77,103 Mechanisms of mobilityfor interacting solutes such as nuclear pore transporters andAvidin are related to the nanoscale energy landscape encoun-tered by the solutes (Fig. 4c and d), and are described inSection 3.4.

Violation of criterion 4, that binding to the gel does notdisrupt gel structure, also has interesting effects. For example,high concentrations of tobramycin induce precipitation of algi-nate gels; this disruption of barrier integrity facilitates tobra-mycin transport at high concentrations.104 Also, reversiblebinding of particles to physical gel crosslinks could disruptexisting crosslinking (Fig. 3e), which could help overcome asteric barrier as discussed in Sections 2.2 and 5.

Fig. 4 Distinction between “binding” and “partitioning.” (a) Penetration of Cy5-labeled (fluorescent) tobramycin and ciprofloxacin into P. aeruginosabiofilms. Plots represent quantified timecourses of penetration, with representative images shown above plots. Cy5-tobramycin penetrates the gelless extensively than Cy5-ciprofloxacin. Adapted with permission from Tseng et al. (2013).76 Copyright (2013) Society for Applied Microbiology andJohn Wiley and Sons Ltd. (b) Penetration of Avidin and a neutral Avidin variant (NeutrAvidin), both fluorescently labeled with fluorescein isocyanate(shown in green), into bovine articular cartilage. Positively charged Avidin penetrates more than NeutrAvidin. Reprinted with permission fromBajpayee et al. (2014).21 Copyright (2014) Elsevier. (c, d) Schematic of difference between a and b in terms of nanoscale free energy landscape. (c)Cy5-tobramycin binds specific targets and must escape binding energy wells to continue diffusing, meaning that the energy landscape is not con-stant on the nanoscale. (d) Avidin diffuses in nearly uniformly charged cartilage; thus, binding energy wells are minimal and a Donnan treatment ofelectrostatics is appropriate.



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3.2 Biochemistry of reversible binding to the gel matrix

Reversible binding to biological hydrogels is important formolecules throughout the size spectrum;92 even proton trans-port can be slowed by gastric mucus, which protects epithelialtissue from stomach acid.105 Here, we discuss representativeexamples that illustrate the importance of various inter-molecular forces, as well as several cases showing how modu-lating gel binding could deliver novel approaches to fightinginfections.

The biochemistry underlying reversible binding to gels isdifficult to study for several reasons. First, crystal structures ofgel components do not usually exist since gel polymers aregenerally intrinsically disordered. It is also often not obviousto which component(s) of the gel a solute binds, since biologi-cal gels contain a complex mix of components.76 Despite thelack of structural information, existing literature on gelbinding and the composition of biological hydrogels yieldsuseful predictions and conclusions about transport in gels;these predictions usually center on how charge and hydro-phobicity influence diffusion. Other intermolecular forcessuch as van der Waals forces and hydrogen bonding are impor-tant, but their effect on binding is not easily predictable. Inone case, research has shown that hydrogen bonding is impor-tant for mucoadhesion of hydrophilic polymers such as poly(acrylic acid).90 In summary, analysis of a molecule’s overallchemical properties may predict that the molecule will bind tosomething within a gel.

An instructive instance in which charge interactions domi-nate is P. aeruginosa lung infections in CF patients, whichinvolve two biological gels: mucus and biofilm EPS.One primary class of antibiotics used to treat P. aeruginosainfections, aminoglycosides, are highly positively charged andthus bind to polyanions such as mucins, DNA, and actin,which are present at pathologically high levels in CF respirat-ory mucus. This binding impedes antibiotic penetration andinhibits activity.74,106 The P. aeruginosa EPS acts as a secondprotective barrier for biofilm-associated cells. The EPS alsocontains DNA and, in the case of mucoid strains, high levels ofthe polyanionic aminoglycoside-binding alginate, althoughalginate surprisingly seems to not significantly hinderpenetration.107–109

Although charge-mediated binding to polyanions is impor-tant for aminoglycosides and other cationic antibiotics such ascolistin,110 reversible gel binding does not seem to be a majorfactor in many other antibiotic treatments of biofilms;111 forexample, Fig. 4a shows that ciprofloxacin easily penetrates amodel P. aeruginosa biofilm. In addition to slowing antibioticpenetration, biofilms have other resistance mechanismsincluding slow metabolism and hypoxia in the biofilm interior(which are related to reaction-diffusion dynamics of nutrientsand oxygen, emphasizing the broad importance of diffusion ingels), and slow-dividing “persister cells.”17,112,113 The variousresistance mechanisms may be cooperative; slowed pene-tration could allow more time for bacterial adaptations to anti-biotic treatment.114

The importance of hydrophobic interactions in small mole-cule transport has mainly been studied in the context of oraldrug bioavailability, which is partially dependent on the abilityto diffuse through intestinal mucus to the epithelium. Manyhydrophobic drugs such as paclitaxel, testosterone, and cin-narizine bind to exposed hydrophobic sites on mucin, lipidspresent in mucus, or both; binding slows penetration andrenders intestinal mucus a barrier to absorption into thebloodstream.115

However, for many molecules it is not immediately clearwhether hydrophobic or electrostatic interactions drive inter-active properties. For example, cationic antimicrobial peptides(CAMPs), an important class of antibiotics and immune mole-cules, contain both cationic and hydrophobic residues; thecharge, or the hydrophobicity, or both, could lead to gelbinding. EPS–CAMP interactions are particularly important, asmany antimicrobial peptides are under investigation as poss-ible anti-biofilm treatments, but binding to EPS componentsoften inhibits their effectiveness.116–118 For example, the Debergroup designed a set of synthetic anti-P. aeruginosa CAMPsand investigated their interaction with alginate.119–122 Whilealginate is anionic, highly soluble, and contains no largehydrophobic domains, alginate blocked the penetration of syn-thetic anti-P. aeruginosa CAMPs when a hydrophobicitythreshold was exceeded.121 Furthermore, they showed that thisinteraction was mediated by a combination of electrostatic andhydrophobic interactions with alginate.119

Similarly, both synthetic and natural CAMPs have beenshown to bind mucins, DNA, and F-actin, and strongerbinding correlates with greater inhibition of CAMP activityagainst pathogenic bacteria in saliva and CF sputum.123–126

CAMPs are also investigated as anti-cancer treatments, andunsurprisingly can be inhibited by ECM: the GAG heparansulfate inhibits antitumor CAMPs.127

3.3 The effect of nanoscale heterogeneity on solute–gelinteractions

While knowing the composition of a gel gives an overall senseof the gel’s net charge and helps predict how solutes behave, itis also important to understand how nanoscale molecularheterogeneity, or spatially varying chemical properties, tunessolute–gel interactions. Heterogeneous surfaces are commonlyfound in gel-related molecules: protein surfaces and anti-microbial peptides contain charged, neutral hydrophilic, andhydrophobic residues, and larger objects such as viruses havecomplex surfaces (Fig. 5a).128 In addition, the gels themselvesoften have locally varying properties that render simplisticmodels of gel charge problematic. For example, despite the netnegative charge of ECM, both positively and negatively chargednanoparticles and liposomes have reduced diffusivity in re-constituted ECM gels compared to neutral hydrophilicparticles. The observation that anionic particles are slowedjust like cationic particles is probably due to alternate patchesof negative and positive charge in the gel that bind positiveand negative charges, respectively, on the particles.129,130



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As an example of the effect of nanoscale heterogeneity onthe solute side, when Li et al. examined the diffusion of twopeptides, with the same near-neutral net charge but a differentarrangement of these charges, these peptides diffused differ-ently into a reconstituted mucin gel. The block-charge peptideinteracted weakly with mucin, while the alternating-chargepeptide did not (Fig. 5b).131 This result is indicative of howvariations in molecular structure beyond simple net chargeaffect transport properties in gels, which could have appli-cations for drugs whose penetration is inhibited by gelbinding.

For example, the Smyth group covalently attached a 5 kDapolyethylene glycol (PEG) chain to tobramycin and found thatthe PEGylated variant was more effective in vitro againstP. aeruginosa biofilms than the unmodified antibiotic, likelydue to reduced EPS binding. While the PEGylated tobramycinremained highly positively charged as is required for its mech-anism of action, PEGylation sufficiently changed the context of

that charge to reduce gel binding. However, the improvementin activity of the PEGylated tobramycin against biofilms wasmodest, because the PEG chain compromised the biologicalactivity of the molecule.132 One possible strategy to avoidreduced activity is to make the PEGylation reversible via hydro-lysis, a strategy that has been investigated for gentamicin(another aminoglycoside) due to the potentially improvedpharmacokinetic properties offered by PEGylation, but notstudied in a biofilm context.133 Reversible PEGylation mayallow for gel penetration, as well as full activity of the hydro-lyzed form.

An alternate strategy would be to minimally change thestructure of tobramycin in order to reduce gel binding withoutcompromising activity. Recent experiments have suggestedthat modifying hydrophobicity could achieve this aim, becausehydrophobic interfaces can affect electrostatic binding.134 Wealso note that immobilized charged groups on hydrophobicsurfaces can tune hydrophobic interactions (Fig. 5c), whichcould be useful for changing the gel binding of partially hydro-phobic solutes such as CAMPs.135

For a more fully developed example of the utility of prevent-ing sequestration by gels, we consider the activity of lysozyme,a cationic antimicrobial protein, against P. aeruginosa in CF.Lysozyme is inhibited by F-actin,136 mucin, alginate, and DNA.As with tobramycin, some positive charge is required for lyso-zyme’s function; however, in a screen of charge-reduced vari-ants, at least one variant reduced its inhibition by all of thesepolyanions while retaining full antimicrobial activity.137,138

This mutant lysozyme also displayed superior antibioticactivity to wild type in a murine P. aeruginosa lung infectionmodel.139,140 There was substantial variation in P. aeruginosakilling by lysozyme both with and without the presence ofpolyanions throughout the screen, emphasizing that (as withthe charged peptide assay in Fig. 5b 131) net charge is notsufficient to determine the strength of electrostatic binding;context and charge placement are important as well.137,138

3.4 Mechanisms for mobility of interacting solute

We mentioned in Section 3.1 that gel interactions need notreduce effective diffusivity if the bound particles remainmobile (DB ∼ DF). We now present two mechanisms for fastdiffusivity of interacting particles: mobility of the boundcomplex and high density of interaction sites. In the firstmechanism, if a solute is bound to a site which itself isdiffusing, then the solute is taken along for the ride with DB =Dbound complex. Experimentally, Sprakel et al. (2007) observedtransient subdiffusion of a silica particle bound to a polymernetwork due to Rouse dynamics.60

Bound complex mobility would be most significant inweakly physically crosslinked gels comprised of flexible poly-mers because these properties allow fast polymer dynamics,and for larger solutes because small molecules diffuse morequickly than polymers (DF ≫ Dbound complex). Also, boundcomplex diffusion is faster on short timescales because cross-links and entanglements limit range of motion over long time-scales.60 Binding events with fast off rates will therefore take

Fig. 5 Nanoscale heterogeneity of solutes and effect on gel diffusion.(a) Surfaces of (i) human rhinovirus (PDB: 2rm2) and (ii) human albumin(PDB: 2bxi), with positive charges in blue and negative charges in red.Reprinted with permission from Cone (2009),128 (Copyright (2009)Elsevier). Rhinovirus and albumin are densely coated with opposingcharges. (b) Two fluorescently labeled peptides with the same netcharge but different spatial arrangement. The “block” peptide at leftinteracts weakly with mucin while the “alternating” peptide at right doesnot;131 schematic shows potential mechanism for this difference. (c)Effect of immobilized charges on nearby hydrophobic interactions,probed by measuring the adhesion of a hydrophobically functionalizedgold (Au) atomic force microscopy tip to surface monolayers. Amine(NH3

+) groups strengthen hydrophobic interactions between thesehydrophobic surfaces, while guanidinium (Gdm+) groups weaken oreliminate them. Adapted by permission from Macmillan Publisher Ltd:Nature (Ma et al., 2015).135 Copyright (2015).



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greater advantage of bound complex mobility. Transport ofNTRs through the nuclear pore neatly checks off each of theseconstraints: NTRs transiently bind weakly crosslinked andhighly mobile nucleoporins. Thus, bound complex mobilityhelps explain why NTR binding to nucleoporins does notresult in slow transport, as one would naively expect given eqn(1), and accurately predicts that strengthening NTR-nucleo-porin binding should inhibit transport.102,141,142

To understand the high interaction site density mechan-ism, we consider an energy landscape interpretation ofdiffusion, in which interactions are represented by electro-chemical potential wells (Fig. 4c and d). Escape of a solutefrom potential wells into interstitial fluid is slow, thus imped-ing diffusion (Fig. 4c), as with tobramycin penetration into bio-films. However, if the density of binding sites is high enough,then it is possible for a solute to transition from one freeenergy minimum to another without transitioning to bulkinterstitial fluid (Fig. 4d). The classic example of this is slidingdiffusion on DNA: certain DNA-binding proteins transitionfrom free diffusion in the nucleus or cytoplasm to one-dimensional sliding along DNA upon nonspecific DNAbinding.143–145

A similar effect likely takes place for the diffusion of Avidin(a positively charged 60 kDa protein) in articular cartilage ECM(Fig. 4b), in which proteoglycans allow full three-dimensionalmobility. High densities of proteoglycans and GAG chainscombined with weak nonspecific interactions enable cationssuch as Avidin to freely move along and between GAGs(Fig. 4d). In this case, since Avidin moves between interactionsites it does not make sense to refer to “binding” to any par-ticular site (Avidin actually also has true diffusion-arrestingbinding sites within ECM, for which the term “binding” doesmake sense).21 The partition coefficient between the gel and abath in this limit can be quantitatively predicted via Donnanequilibrium analysis, which assumes a constant potentialthroughout the gel (green dotted line in Fig. 4c and d).77 Notea subtlety of language found in the literature: partition coeffi-cient of a solute between two phases is usually defined as theconcentration ratio of the solute in each phase at equilibrium,but in studies of diffusion in biological hydrogels, the par-tition coefficient often refers only to the partitioning of freelydiffusing, unbound solute between the phases.77 We definepartitioning here in the latter sense.

Finally, differential partitioning into hydrogels can bedriven by forces other than Donnan equilibrium. Steric inter-actions are an obvious case, as they drive partitioning of largesolutes toward phases with larger mesh sizes. Aqueous two-phase systems (ATPSs) consisting of phase-separated dextranand PEG gels differentiate biomolecules based partly on theirhydrophobicity.146,147 Similarly, partitioning into membrane-less organelles such as Cajal bodies, P bodies, and P granules—which are likely formed via phase separation, sometimesincluding a sol–gel transition—is a key feature of cellularorganization.148,149 Finally, transport through the nuclear poredepends in part on a combination of hydrophobic and electro-static partitioning effects.102,150

4. Selective transport of objects onthe order of mesh size4.1 Adhesive interactions and polyvalent trapping

When large particles are not inert with respect to the gelmatrix, even low-affinity binding tends to dramatically slowparticle diffusion because the particles bind multivalently tothe gel (Fig. 6a).128 For example, while hydrophobic inter-actions moderately reduce the diffusivity of hydrophobic drugs

Fig. 6 Mechanisms of polyvalent trapping in gels. (a) Non-specificinteractions (hydrophobic, electrostatic, etc.) with gel can trap a particle,even if individual interactions are weak. (b) Binding to decoy receptors(such as sialic acid) that are present on gel polymers can trap a particle.(c) Gel-binding antibody bound to an otherwise inert particle mediatestrapping.



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in mucus, polystyrene beads are almost completely trappedbecause they form many hydrophobic bonds simultaneously.Similarly, aminated beads bind to polyanions in mucus, gener-ally reducing their diffusivity relative to carboxylated poly-styrene beads.43,44 Carboxylated beads in turn diffuse far moreslowly in mucus than do PEGylated beads, due either to hydro-phobic interactions resulting from incomplete coverage by car-boxyl groups or to interactions with patches of positive chargeon mucin molecules (similarly to the description of ECM, asdescribed in Section 3.3).51,129,151 PEGylated beads also diffusemore quickly than cationic and anionic nanoparticles in bio-films; the relative diffusivities of the charged nanoparticlesdepend on the species.44,46,152

Low molecular weight (2–5 kDa) PEG coatings are particu-larly effective at reducing adhesive trapping for two reasons.First, PEG is neutral yet hydrophilic, thus precluding strongelectrostatic or hydrophobic interactions.51 Second, high-density surface-grafted PEG forms a brush that physicallyresists the adsorption of gel components,153 while the low PEGmolecular weight precludes adhesion to gel components dueto chain entanglement.51 Neither of these reasons is gel-specific, which explains why PEGylation seems to be fairly uni-versally applicable to promoting diffusion in biological hydro-gels: PEGylated particles effectively penetrate many types ofmucus,51–53,89,154,155 biofilms,44 and ECM.47,156,157 In addition,recently Maisel et al. (2016) recently showed that PEG mole-cular weights as high as 40 kDa can be used for mucus pene-tration, with higher molecular weights requiring extremelyhigh PEGylation density to reduce entanglements.158

In addition to uniform surface coatings such as PEGylationor carboxylation, heterogeneous particle surfaces have beeninvestigated in the context of viral diffusion in mucus. Virusessuch as human papilloma virus (HPV), Norwalk virus, herpessimples virus 1 (HSV-1), and human immunodeficiency virus(HIV) quickly penetrate cervical or cervicovaginal mucus underpH-neutral conditions in the absence of antibodies,91,159,160

likely because these viruses are densely coated with both posi-tive and negative charges (Fig. 4a), yielding overall hydrophili-city without having any uniformly charged domains largeenough for electrostatic interactions.128 This explanation iscorroborated by the charged peptide diffusion assay, in whichthe alternating-charge peptide had weaker interactions withmucin than the block-charge peptide (Fig. 4b).131 Also, in con-trast to influenza, these viruses do not specifically bind sialicacid; as mentioned in Section 3.1, sialic acid binding hindersinfluenza transport and this may be compounded by multi-valency (Fig. 6b). At any rate, some non-sialic acid dependentviruses are unable to penetrate the mucus barrier: adeno-viruses and adeno-associated viruses (AAVs) used as genetherapy vectors diffuse thousands of times more slowly in CFsputum than in water,161 potentially explaining why the manygene therapy trials for CF employing adenoviruses or AAVshave all failed. Interestingly, mutating an AAV to reduceheparin binding enhanced penetration through CF sputum,highlighting the potential for rational design of gene deliveryvectors to enhance transport.162

The mucosal immune system exploits polyvalency byemploying antibody-based trapping of viruses. For example,anti-HSV-1 immunoglobulin G (IgG) located in cervical mucuscan prevent HSV virions from penetrating the mucus andinfecting cells. HSV trapping occurs because the Fc region ofIgG binds mucin with low affinity; while this binding does notsignificantly hinder free IgG transport, when many IgG mole-cules coat one HSV virion, the weak polyvalent binding tomucins adds up to an interaction that is strong enough tonearly completely trap the virion (Fig. 6c).160 Recent resultssuggest antibody trapping may even be more important thansialic acid-mediated trapping for preventing influenza pene-tration.163 It should be noted that strong antibody binding tomucus and/or membrane-bound mucins may or may notoccur;164–166 however, theoretical treatments have shown thatweak antibody-mucin binding is sufficient for trapping.167,168

Finally, exogenously added anti-PEG IgG and IgM appear tobe able to trap PEGylated nanoparticles in mouse cervicovagi-nal mucus via the same polyvalent trapping mechanism as forviruses.169 As mucosal anti-PEG antibodies may be common inthe human population but are probably not present in animalmucus models, antibody-mediated trapping (and immuno-genicity of PEG in general170) is a potential hurdle for clinicalapplications of PEGylated nanoparticles.169

4.2 Implications of gel penetration studies for nanoparticledesign

Nanoparticles are used for a myriad of functions in medi-cine171 including gene therapy,53 antimicrobial172 and anti-cancer treatments,173 insulin delivery for diabetes,174 and sus-tained drug release,175 and many of these contexts requirediffusion through a gel layer discussed in this review. Propersurface functionalization is therefore key to developingeffective therapeutics. Two major lessons emerge from thestudies of PEGylation, uniformly charged nanoparticles, andviruses described above. First, even weak adhesive interactionscan trap a particle if there is a high density of binding sites. Ofparticular note is the ability of the immune system to generateantibodies against essentially all foreign substances, whichmeans that antibody trapping should be considered whereveran immune response could be present.

Second, a good strategy across gel types to avoid both hydro-phobic and electrostatic binding is to have a dense hydrophilicbut overall neutral coating, which has been implemented intwo different ways: neutral hydrophilicity and dense oppositecharges. In addition to PEGylation, N-(2-hydroxypropyl) meth-acrylamide polymer (pHPMA)174,176,177 and poly(vinyl alcohol)(PVA)178 were recently tested as alternative mucoinert coatings,with some success. A dense coating of opposite charges hasshown effectiveness in multiple different contexts, includingviruses (as discussed above), self-assembled nanoparticlescomprised of distinct ratios of polycationic chitosan and poly-anionic poly(acrylic acid) (also in mucus),26,179 zwitterionicdilauroylphosphatidylcholine monolayer-coated nanoparticles(in intestinal mucus),180 and liposomes with zwitterionicdipalmitoyl phosphatidylcholine membranes (in biofilms and



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respiratory mucus as an inhaled treatment carrying amikacin,an aminoglycoside; currently in clinical trials).172 Not all ofthese coatings provide ideal protection against gel interactions,and in particular polymer brushes form a useful physicaladsorption barrier in addition to a biochemical one. Yu et al.found that while a zwitterionic liposome diffused faster thanpolystyrene nanoparticles in cervical mucus, PEGylating theliposome still significantly increased its diffusivity.181

The utility of promoting gel transport is application depen-dent, and gel adhesion may be unavoidable or even desirable.9

Mucoadhesive drug delivery systems are useful for promotinglong contact times with mucosal surfaces, thus allowing forsustained drug release, particularly on surfaces with slow turn-over.9 Cationic liposomes may have increased contact timewith biofilms and promote sustained antibiotic release, andtheir affinity with bacterial membranes gives them increasedantibacterial efficacy.175 Furthermore, even slowly diffusingnanoparticles will eventually penetrate a gel depending on thetimescale of treatment, and even if the particles themselves donot penetrate, released free drug likely will.

Several groups have designed nanoparticles that changeproperties over time in order to exploit both fast initialdiffusion and the favorable properties of cationic particles (geladhesion and/or cell penetration). Two approaches have beenused: (1) a dissociable mucoinert coating with a cationiccore,174 and (2) alkaline phosphatase-mediated phosphate clea-vage that increases overall surface charge over time, thus switch-ing from mucopenetration to charge-mediated trapping.182,183

5. The nuclear pore: a case study intransport at the molecular level

Having illustrated the main factors that control transport behav-ior in biological polymeric matrices, we now discuss the nuclearpore in detail as a case study. Nuclear pore translocation encom-passes every concept discussed above and has been studied inmolecular detail, which is not possible for complex hetero-geneous macroscopic gels such as mucus, biofilms, and ECM.

The nuclear pore complex has a diameter of approximately100 nm, and the central channel is filled with intrinsically dis-ordered proteins called nucleoporins. These nucleoporinscontain many instances of short hydrophobic repeats calledFG repeats, which likely act as crosslinkers. The nuclear poreinhibits nuclear translocation of proteins larger than roughly30–100 kDa and even some smaller proteins such as his-tones.48,184 However, NTRs efficiently carry large cargo throughthe nuclear pore.49 This transport ability of NTRs derives inlarge part from exposed hydrophobic patches on NTR surfaces,which have hydrophobic and/or π–π interactions with the FGrepeats on nucleoporins.68 The mechanism by which theseinteractions mediate selective transport is still under debate,and multiple models exist.

In the selective phase model, the nuclear pore is rep-resented as an FG repeat-crosslinked hydrogel whose pore sizedetermines the size cutoff above which inert particles cannot

penetrate the hydrogel.102 NTRs locally dissolve the gel bybinding FG repeats and disrupting the crosslinks, allowing forpenetration of large objects (Fig. 3e).68 While individual FGcrosslinks are weak and highly dynamic,185,186 even transientcrosslinks may contribute to barrier function, and these transi-ent crosslinks may be dissolved by hydrophobic NTR regions.Brownian dynamics simulations suggest that while FG cross-links are dynamic, at each point in time the crosslinks perco-late through the center of the pore, forming a hydrogel insome technical senses.187 Purified FG-rich nucleoporindomains self-assemble into hydrogels with transport selectivitysimilar to that of the nuclear pore.68

The other main family of models, the virtual gate and/orentropic barrier models, notes that there is a free energybarrier to entering the nuclear pore because nuclear pores areextremely small relative to the size of the nucleus or cyto-plasm.188 Thus, entering the pore imposes a loss of transla-tional entropy and corresponding increase in free energy;entropic exclusion by nucleoporins causes an even greaterincrease in free energy.185,186 In these models, NTR binding toFG repeats functions like Donnan partitioning: it locallyincreases the concentrations of NTR near and inside thenuclear pore, without immobilizing the NTRs due to fastunbinding from FG repeats.141,142 This local enrichment ofNTRs overcomes the barrier to nuclear pore entry and thusenables effective transport of NTR-cargo complexes.189 Notethat the virtual gate and selective phase models are notmutually exclusive; Donnan-like transport facilitation effects,entropic exclusion, and dissolution of FG repeat crosslinksmay all be important for nuclear pore transport in vivo.

Finally, the fact that hydrophobic interactions with FGrepeats are required for transport through the nuclear poredoes not mean that charge is not important as well. Our grouphas found that nucleoporins and NTRs tend to have comp-lementary net charges (positive and negative respectively),thus implying that charge assists in the recruitment and trans-location of NTRs;150 this hypothesis is supported by theore-tical189 and structural141 analysis.

6. Outlook

We have taken a whirlwind tour through the forces and con-cepts that guide diffusion through ECM, mucus, biofilm EPS,and the nuclear pore. In all of these systems, size filtration pre-vents the penetration of overly large particles via a stericbarrier; in turn, size filtration can be modulated by gel clea-vage or reversible crosslink disruption. Interaction filteringenables more precise tuning of gel selectivity. For particlesbelow the mesh size, the nuclear pore displays little selectivity;diffusion in the ECM is mainly determined by electrostatics;both hydrophobic and electrostatic interactions are importantin mucus; and electrostatic interactions are certainly impor-tant in biofilms, while hydrophobic interactions may or maynot be important depending on the species and environment.Electrostatic interactions can have multiple effects that facili-



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tate or repress particle penetration depending on whether theinteractions constitute binding or Donnan partitioning; theseinteractions are useful for optimizing drug delivery. Similardiffusion rules apply to biological hydrogels that have notbeen discussed in great detail here. For example, in thevitreous humor, cationic nanoparticles diffuse more slowlythan anionic nanoparticles and PEGylation enhancestransport.190–192

Chemical selectivity acts on the nanoscale, where smallvariations in the spatial arrangement of charge tune gelbinding affinity.131 Furthermore, the relationship betweencharge–charge and hydrophobic interactions is not fullyunderstood, and the chemical heterogeneity of biological gelslends the problem an additional layer of practical uncertainty.These difficulties notwithstanding, the cases of PEGylatedtobramycin132 and charge-reduced lysozyme137–140 suggest thatengineering drugs with hydrogel interactions in mind canimprove efficacy. Engineering drug interactions with biologicalgels may also lead to new functional biocompatible hydrogels,sidestepping the need for complex synthetic formulations. Forexample, complexing LL-37 (a CAMP) with a calcium alginatehydrogel reduced the toxicity of LL-37 toward mammalian cellsand allowed for sustained LL-37 release kinetics.193 Covalentlycrosslinked methacrylated bovine submaxillary mucin hydro-gels also showed sustained release kinetics of both polymyxinB (a cationic antibiotic) and paclitaxel (a hydrophobic cancerdrug).194

For large particles, polyvalent weak interactions with the gelmatrix can lead to adhesion and trapping. Synthetic nano-particles, and probably some viruses as well, use net-neutralhydrophilic coatings to avoid trapping and promote mucuspenetration.128 On the other hand, weak interactions mayfacilitate gel penetration through transient crosslink dis-solution. This mechanism may occur in the nuclear pore andreconstituted gels formed from nucleoporin.68 Studies of thenuclear pore’s unique transport properties may lead to newinnovations; for example, nuclear pore-mimicking nanoporeshave been developed with novel potential biotechnologyapplications.195

Given the importance of gel permeability in living systems,it may serve as a biomarker for disease states, particularly formucosal diseases in which mucus is easily accessible. Forexample, we showed that cervical mucus stratifies reproductivestates, distinguishing between pregnant women at high and lowrisk for preterm birth via both rheological and permeability-based assays (ref. 196 and submitted; Fig. 1a depicts the visualdifference between mucus from high- and lowrisk patients).Overall, understanding particle transport in biological gels willlead to powerful applications and innovations in diagnostics,biomimetic gels, pharmacology, and nanomedicine.

Acknowledgements

This work was supported by the Department of Defense,Defense Threat Reduction Agency, under award HDTRA-13-1-

0038, NIH R01-EB017755, NSF Career PHY-1454673, theMRSEC Program of the National Science Foundation underaward DMR – 0819762, and the Burroughs Wellcome Fundunder award 1012566. J. W. was supported in part by theNational Science Foundation Graduate Research Fellowshipunder Grant No. 1122374. The content of the information doesnot necessarily reflect the position or the policy of the federalgovernment, and no official endorsement should be inferred.We wish to thank Prof. Alan Grodzinsky at MIT for his valuablefeedback and helpful discussions, and Erica Shapiro, WesChen, Tahoura Samad, Caroline Wagner, Kathryn Smith-Dupont, and Lisa Walker for careful reading of the manuscript.

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