Advanced in developmental organic and inorganicnanomaterial: a review
Khalisanni Khalid, Xuefei Tan, Hayyiratul Fatimah Mohd Zaid, Yang Tao,Chien Lye Chew, Dinh-Toi Chu, Man Kee Lam, Yeek-Chia Ho, Jun Wei Lim &Lai Chin Wei
To cite this article: Khalisanni Khalid, Xuefei Tan, Hayyiratul Fatimah Mohd Zaid, Yang Tao,Chien Lye Chew, Dinh-Toi Chu, Man Kee Lam, Yeek-Chia Ho, Jun Wei Lim & Lai Chin Wei (2020)Advanced in developmental organic and inorganic nanomaterial: a review, Bioengineered, 11:1,328-355, DOI: 10.1080/21655979.2020.1736240
To link to this article: https://doi.org/10.1080/21655979.2020.1736240
Advanced in developmental organic and inorganic nanomaterial: a reviewKhalisanni Khalida,b, Xuefei Tanc,d,e, Hayyiratul Fatimah Mohd Zaidf, Yang Taog, Chien Lye Chewh, Dinh-Toi Chui,j,Man Kee Lamk, Yeek-Chia Ho l,m, Jun Wei Lim n,o, and Lai Chin Wei p
aMalaysian Agricultural Research and Development Institute (MARDI), Serdang, Malaysia; bDepartment of Chemistry, Faculty of Science,University of Malaya, Kuala Lumpur, Malaysia; cCollege of Materials and Chemical Engineering, Heilongjiang Institute of Technology, Harbin,PR China; dState Key Laboratory of Urban Water Resource and Environment, School of Environment, Harbin Institute of Technology, Harbin,PR China; eDalian SEM Bio-Engineering Technology Co., Ltd, Dalian, PR China; fFundamental and Applied Sciences Department, Centre ofInnovative Nanostructures & Nanodevices (COINN), Institute of Autonomous System, Universiti Teknologi PETRONAS, Bandar Seri Iskandar,Malaysia; gCollege of Food Science and Technology, Nanjing Agricultural University, Nanjing, China; hSime Darby Plantation Research(Formerly Known as Sime Darby Research), R&D Centre – Carey Island, Pulau Carey, Malaysia; iFaculty of Biology, Hanoi National Universityof Education, Hanoi, Vietnam; jCentre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo and OsloUniversity Hospital, Norway; kDepartment of Chemical Engineering, Universiti Teknologi PETRONAS, Seri Iskandar, Malaysia; lCivil andEnvironmental Engineering Department, Univesiti Teknologi PETRONAS, Seri Iskandar, Malaysia; mCenter for Urban Resource Sustainably,Institute of Self-Sustainable Building, Universiti Teknologi PETRONAS, Seri Iskandar, Malaysia; nDepartment of Fundamental and AppliedSciences, Universiti Teknologi PETRONAS, Seri Iskandar, Malaysia; oCentre for Biofuel and Biochemical Research, Institute of Self-SustainableBuilding, Universiti Teknologi PETRONAS, Seri Iskandar, Malaysia Lim; pNanotechnology & Catalysis Research Centre (NANOCAT), Universityof Malaya (UM), Kuala Lumpur, Malaysia
ABSTRACTWith the unique properties such as high surface area to volume ratio, stability, inertness, ease offunctionalization, as well as novel optical, electrical, and magnetic behaviors, nanomaterials havea wide range of applications in various fields with the common types including nanotubes,dendrimers, quantum dots, and fullerenes. With the aim of providing useful insights to helpfuture development of efficient and commercially viable technology for large-scale production,this review focused on the science and applications of inorganic and organic nanomaterials,emphasizing on their synthesis, processing, characterization, and applications on different fields.The applications of nanomaterials on imaging, cell and gene delivery, biosensor, cancer treatment,therapy, and others were discussed in depth. Last but not least, the future prospects andchallenges in nanoscience and nanotechnology were also explored.
ARTICLE HISTORYReceived 12 January 2020Revised 16 February 2020Accepted 17 February 2020
Nanomaterials is defined as a set of substances where atleast one of its dimensions is between 1 and 100 nm,nanomaterials follow the central principle ofnanoscience and nanotechnology, the application ofwhich covers a wide interdisciplinary of research areaand development activity that grows explosivelyworld-wide [1]. Nanomaterials have the ability to transforminto functionalized substitute that can be further re-accessed. Common types of nanomaterials includenanotubes, dendrimers, quantum dots (QDs), and full-erenes. The rising awareness of nanomaterials is due toits unique optical properties which are significantlyimpactful for various fields such as electronics, mecha-tronics, medicine, pharmaceutical, ionic liquids, poly-mer, and many more. Commercial applications of
nanomaterials include nanoscale titanium dioxide incosmetics, sunscreen, and self-cleaning windows;nanocoatings and nanocomposites in windows, sportsequipment, bicycles, and automobiles; nanoscale silicabeing used as filler in cosmetics and dental fillings; andothers such as stain-resistant and wrinkle-free textiles,electronics, paints, and varnishes [2].
Redesigning materials at the molecular level state,also known as engineered nanomaterials, where mod-ification is made in their small size and novel proper-ties, are generally not visualized in their conventionaland bulk counterparts. A distinct propery of thesenanomaterials is their relatively large surface areawhich triggers the novel theory of quantum effects.Nanomaterials provide a much greater surface area tovolume ratio compared to their conventional forms,
CONTACT Khalisanni Khalid [email protected]; [email protected] Malaysian Agricultural Research and Development Institute (MARDI),Headquarters, Persiaran MARDI-UPM, Serdang, Selangor 43400, Malaysia
which is beneficial as this can provide greater chemi-cal reactivity affected by their specialty [3].Considering the reaction at the nanoscale level, thematerial properties and characteristics which lead tonovel optical, electrical, and magnetic behaviors canbe more vital due to the quantum effects [4].Nanostructured materials are classified as zero-dimensional (0-D), one-dimensional (1-D), two-dimensional (2-D), and three-dimensional (3-D)nanostructures [5]. These dimensionalities of nano-materials are characterized using an ultrafine grainsize less than 50 nm or limited to 50 nm. Variousmodulation dimensionalities can be formed such as0-D (e.g. atomic clusters, filaments, and cluster assem-blies), 1-D (e.g. multilayers), 2-D (e.g. ultrafine-grained overlayers or buried layers), and 3-D (e.g.nanophase materials composed of equiaxed nan-ometer-sized grains). Figure 1 reveals the changes inproperties when a bulk material is broken down intonanoparticles (NPs).
The aim of this review is to bring togetherscience and applications on inorganic and organicnanomaterials specifically emphasizing on synth-esis and preparation, processing, characterization,and applications of organic and inorganic nano-materials that provide a novel approach in the fieldof nanomaterial sciences.
2. Evaluation studies of inorganic andorganic nanomaterials
Most nanomaterials that are commonly com-posed of silicon and whose chemical (molecular)
properties range from sub-nanometer to few nan-ometer levels are well-defined crystallographicallymaterials used in supermolecular chemistry[6,7,8,9]. The raw materials used in makingglass, quartz, and desiccants consist of particulateproperties that define additives and bulk proper-ties. Hardness of materials is associated withproperties of ceramics or metals and is rarelyallied with polymer. For instance, materials canbe value-added into a quality product by addingadditive of small and hard particles in conveyinghardness [10]. A similar approach applied forUV-radiation absorption, magnetization, color,and reflectivity can be supplementary toa sensitive polymer-based NP. Nanoparticulateadditives are already well established, wherenanoparticulate carbon was developed early inthe twentieth century known as carbon blacktypically made up of more than 10% of a typicalcar tire. Obviously, the term ‘nano’ was onlycoined much later. Likewise, several most estab-lished chemical products would be called ‘nano’which are introduced today. All of these productshave been around for decades and are generallysafely recognized product. Chemists design mole-cular products using thousands of well-established building blocks or subunits.Therefore, this will easily identify the suitablemodifications to the dye’s functional core part(or the conjugated system), to alter the moleculesolubility and material affinity. Last, the idea ofchemical modification by incorporating solidmagnetization properties on a NP in a productcan serve as a coating application on the particlesurface.
2.1. Organic and inorganic nanoparticles
Organic NPs have been widely investigated, withliposomes, polymersomes, polymer constructs, andmicelles being employed for imaging or drug andgene delivery techniques [11]. Meanwhile, inor-ganic NPs have also attracted researchers’ atten-tion in recent years attributed to their uniquematerial- and size-dependent physicochemicalproperties, which are incomparable with tradi-tional lipid- or polymer-based NPs. What makesinorganic NPs attractive? It is their physical prop-erties (e.g. optical and magnetic), in addition to
Figure 1. The changes in properties when a bulk material isbroken down into nanoparticles (NPs).
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their chemical properties such as inertness, stabi-lity, and ease of functionalization [12].
2.2. Hybrid organic–inorganic nanomaterials
With the possibility of combining interesting prop-erties out of different components, hybrid organic–inorganic nanomaterials have attracted researchersin developing new and smart nanocomposite mate-rials as they demonstrate many novel properties ofhigh interest, such as high catalytic activity, stimulat-ing optical properties, and various physical proper-ties [13]. However, it is still necessary to integratea polymer matrix or an organic component, whichfurther improves the mechanical, thermal, and che-mical properties. This strategy maximizes the inter-face between the components, shapes the percolationthreshold of the NP filler, and optimizes the proces-sability of the integrated polymer-based component[14]. Despite this, the challenge associated in synthe-sizing these nanomaterials is the incompatible prop-erties that affect the integration of these components.
2.3. Metal-containing organic dendrimers
The presence of these organic dendron ligands inmetal-containing organic dendrimers has contribu-ted to various applications in drug delivery agents,chemical sensors, and nanoscale catalysts [15].These organic dendron ligands act as a good stabi-lizer for metal and semiconductor NPs [16]. Asidefrom that, these dendron ligands provide a stericcrowing effect where closely packed thin ligandstoward the shell are formed by long flexible alkylchain ligands. Steric crowding effect is an idealapproach as this fills up the spherical ligand layercaused by the dendron ligands which are naturallyin a cone shape on the surface of the NP.Furthermore, these dendron ligands are also com-posed of flexible branches, also known as inter- andintramolecular entanglement where slow diffusionprocess of these small molecules and ions from thebulk solution transfers to the interface between thenanocrystal and the ligand [17].
Till date, AuNPs are one of themost well-exploredNP materials because of the simplicity in their synth-esis, surface plasmon properties, and amenability tosurface functionalization. As for quantum dot(QD) semiconductor NPs, they are beneficial for
applications related to biomarkers and to sensingelements due to their interesting quantum size effectsand narrow emission bands. This also includes tuningwithin the whole visible range in electronic transition(e.g. cadmium chalcogenides), and synthesis-relatedapproach can be carried out easily due to theirremarkable size control and high crystallinity.
Previous research has reported that the synthesisand characterization of conjugated polythiophenedendrons and dendrimers exhibited broad absor-bance and bandgap tunability by varying the gen-eration and connectivity of the macromolecule [18].These materials exhibited interesting nanostructureson mica and graphite surfaces due to p–p transitionstate and van der Waals interactions. This included2-D crystallization and nanowire formation.
2.4. Cell biology nanomaterials
Nanoscale range molecules in cell biology, such asproteins, carotenoids, DNA, and others, havealready been studied in depth for a long time. It isknown that nanomaterials of size <100 nm can easilyinfuse into the cells, those with the size of <50 nmcan enter most cells, while those of <20 nm cantransport and permeate along the blood vessels adja-cent to the tissue, and leap through the blood–brainbarrier. We have reached the stage where NPs can becreated and strategized for the use in biomedicineapplication which is known as ‘theranostic’approach for diagnostics (imaging) and therapeutics(drug or gene delivery). Their specialty is mainly dueto two beneficial factors such as: (1) high surfacearea to volume ratio, which allows for the attach-ment of multifunctional components (e.g. fluores-cent moiety and targeting molecules) and (2) thepossibility of ubiquitous tissue accessibility [19].
3. Inorganic nanomaterial
3.1. Magnetic nanoparticles (mNPs)
One of the most profound inorganic nanomaterialsis magnetic nanoparticles (mNPs) which are illu-strated in Figure 2. They usually consist ofa magnetic core (e.g. magnetite (Fe3O4) or maghe-mite (g-Fe2O3)) [20]. Other metals such as cobaltand nickel are also used, but have limited applica-tions due to their toxicity and vulnerability to
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oxidation [21]. Iron is primarily stored in the pro-tein ferritin in the human body. Iron oxide mNPshave the ability to process excess iron to supply inthe human body. The presence of these cationicmNPs remains localized for a long period of timein the endosomes [22]. Then, elemental compoundssuch as iron and oxygen join the body storagewhich are being metabolized or consumed byhydrolytic enzymes during the postcellular uptakein the endosome and lysosomes. The iron home-ostasis in the human body is well controlled byadsorption, excretion, and storage where the ironlevel is maintained and postulated. The role of ironoxide NPs helps in processing any excess iron in thebody [23]. Iron plays an important role in virtuallyall living tissues; however, it has limited bioavail-ability. In some cases, it can be toxic to cells in theform of free iron or as opposed to being associatedwith hemoglobin.
As for advanced biomedical materials, super-paramagnetic iron oxide (SPIO) NPs owna unique superparamagnetic property [25]. WhenSPIO NPs are being exposed to an external mag-netic field, a large magnetic moment will be trig-gered and subsequently disappeared when themagnetic field is removed. This large momentresults in a higher signal change per unit of parti-cles; thus, only smaller quantities are needed toproduce good signal feedback [26].
The implementation of biocompatible surfacecoating to the inorganic iron core providesa stable behavior under physiological conditions(i.e. inhibits aggregation). A variety of substancesinclude synthetic and natural polymer (e.g. pro-teins or dextran) and amphiphilic molecules suchas fatty acids or phospholipids [27] which can beused as coating materials. The controlled surface
can be further functionalized by coupling withfragments of DNA strands, proteins, peptides, orantibodies. Findings related to mNPs have beenincreasingly exploited as efficient delivery vectors,leading to opportunities in magnetic resonanceimaging (MRI) contrast enhancement, mediatorsof hyperthermia cancer treatment, and targetedtherapies [28].
3.1.1. Magnetic resonance imaging (MRI)Due to its noninvasive nature as well as capabil-ities of providing high 3-D resolution and tomo-graphic, MRI is a powerful imaging tool and offersthe advantage of high spatial resolution of contrastdifferences between tissues. Compared to the con-ventional gadolinium chelates, the mNP-basedcontrast agents offer excellent image enhancementdue to their large magnetic moment as well asimproved cellular internalization and slower clear-ance from the target site [29]. U.S. Food and DrugAdministration has approved the use of iron oxidemNPs for use as MRI signal enhancers. For MRIpurposes, iron oxide cores are commonly used asso-called T2 contrast agents, due to their ability toshorten T2 relaxation times. These agents can bedivided into either SPIOs, with diameters of morethan 50 nm, or ultrasmall SPIOs (USPIOs), withdiameters of less than 50 nm, which then tend tohave longer plasma half-lives of 14–30 h [30,31].Currently, there are several formulations for clin-ical applications such as bowel (Lumiren andGastromark) and liver or spleen imaging (endo-derm and feridex). As with all NPs, the tissuedistribution is heavily influenced by size; therefore,larger SPIOs tend to rely on passive targeting, suchas uptake by the cells of the reticuloendothelialsystems (RES), rather than direct labeling, andthe USPIOs benefit from slower opsonization andRES clearance.
The next generation of active targeting contrastagents is current ongoing research, which providesexciting new opportunities for imaging, diagnosis,and treatment. The first targeting agents weremonoclonal antibodies that exploit molecularrecognition to deliver mNPs [28]. However, oneof the drawbacks of using monoclonal antibodiesis their large size, which can cause poor diffusionthrough typical biological barriers. MRI agentsusing mNPs have been evaluated intensively to
Figure 2. Multifunction magnetic nanoparticles [24].
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improve the diagnosis of solid tumors. This is anactive area of research, since clinicians requirea contrast agent that specifically targeted malignanttumors to allow a more accurate and precise diag-nosis of stages of the disease. Direct tumor targetingusing antibodies has been successful for rectal car-cinoma and breast cancer, as despite the size issue,antibodies remain beneficial in their high specifi-city. A recent application utilizing antibody-directed targeting is more efficient as the presenceof shorter and smaller single-chain fragments(≤20%) provides a high binding affinity and speci-ficity. Antibody-directed targeting consists of anti-body heavy- and light-chain variable domainsconnected by a flexible peptide linker [32].Conventional iron oxides, however, still providea poor signal enhancement compared to that withother imaging modalities (e.g. fluorescence andpositron emission tomography). Further researchis required for developing high and tunable nano-magnetism iron oxide NP [33,34].
3.1.2. HyperthermiaThe growth of tumor cells is more in temperature-sensitive cells compared to other normal healthycells. Intracellular hyperthermia is a method devel-oped using mNPs (in particular SPIOs) that havebeen potentially utilized to treat cancers [35]. Thistreatment consists of a delivery agent which acts asa nanoscale heater function to heat up the cell andinstigate necrosis [36,37]. When the nanoscaleheater was subjected to an alternating magneticfield, it converts the electromagnetic energy intoheat energy, which can be dissipated to the sur-rounding medium. Magnetic materials with tem-perature range within 42°C to 60°C provide aneffective treatment as these materials can poten-tially substitute as an in-vivo temperature controlswitch to prevent overheating of the neighboringhealthy tissue [38].
Tumors can be targeted passively via generalbiodistribution, as tumor tissue tends to have‘leaky’ vasculature, which allows NPs to accumu-late. Alternatively, solid tumors can be directlyinjected with mNPs and further exposure to analternating magnetic field for inducingtumor regression. It has been reported that themNPs can be heated effectively (10–30 nm ironoxide particles) in human cancer models (e.g.
breast cancer) [39]. However, the optimizationin controlling the heat distribution is needed tobe explored to deliver the best performance of it.For example, a hyperthermia research usestumor-targeted mNPs to evaluate their enhance-ment of drug payload [40]. The likelihood oftargeting chimeric L6 monoclonal antibody hastarget-specific breast cancer using dextran-coated mNPs. On the other hand, hyperthermiaresearch has also involved targeting gene expres-sion in tumor [41]. It is essentially important tocontrol and evaluate these gene expressions intumor [42-47] .
3.1.3. Magnetic transfectionThe term ‘magnetic transfection’ is usedwhennucleicacid delivery is being influenced by a magnetic fieldacting on nucleic acid vectors that are conjugated tomNPs. Magnetic transfection technique can also beapplicable for both ‘large’ nucleic acids and smallconstructs [48]. These mNPs aim to bind these nega-tively charged DNAs to the mNPs through electro-static interactions and subsequent release of themafter cell internalization [49]. Magnetic transfectionhas significant advantages over conventional trans-fection methods such as reduced process time andthe increased efficiency in the transfection rate withlower vector doses. Due to the limited half-life in-vivo, these therapies still lack specificity instigated bythe poor diffusion process across the cell membrane,resulting in poor magnetic transfection efficacies.Therefore, it is recommended that these mNPsshould be used as carriers to overcome some ofthese problems.
RNA interference is a natural cellular processused to silence gene expression, which can beexploited artificially [50]. The use of wide-scaletherapeutic RNA silencing requires the develop-ment of a suitable transfection method that canadminister siRNA molecules into human cellsin vivo. NPs are attractive delivery vehicles forsiRNA due to their multifunctionality, allowingthem to overcome many problems associatedwith systemic siRNA application for human celltransfection. A study has shown that NPs can pre-vent rapid excretion of siRNA by the kidney [51].This is a fate that succumbs naked siRNA mole-cules. Magnetic force pulls mNPs toward targetcell attachment to NPs and results in minimal
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renal filtration, extending the unmodified siRNAhalf-life within the circulation [52]. In 2007,a study demonstrated cancer cell transfection byNP-delivered siRNA, reporting the uptake ofmNPs into tumors [53]. In this case, membranetranslocation was facilitated by an attached myris-toyl-coupled polyarginine peptide. In addition todelivering siRNA treatment, these NPs werefurther functionalized with a near-infrared dye toallow noninvasive imaging of their localizationwithin the body. The imaging was performed byboth MRI and near-infrared in vivo optical ima-ging. Imaging results from this study showedNPe accumulation in tumors, which is most likelypassive due to the enhanced permeability andretention effect associated with the leaky vascula-ture of tumors. The study concluded thatNPs further functionalized with tumor-targetingmoieties would increase tumor localization inorder to deliver siRNA treatment.
3.2. Gold and silver nanoparticles
Colloidal gold or gold nanoparticles (AuNPs) canbe easily synthesized for the use of various applica-tions due to its versatility as indicator and detectionprobe. While AuNPs share most of their attractivequalities with regard to bioapplications with otherNPs (e.g. size, inertness, ease of synthesis, and bio-compatibility), AuNPs are very attractivecandidates for biological imaging techniques asthey can be visualized based on the interactionbetween the NPs and light, whereby the particlesstrongly absorb and scatter visible light [54]. Uponlight absorption, the light energy excites the freeelectrons in the gold particles to a collective oscilla-tion, the so-called surface plasmon. This absorptionlies in the visible region for gold, silver, and copper,with the surface plasmon resonance (SPR) ofAuNPs being visible down to 3 nm. AuNPs giverise to both absorption and scattering, the size ofwhich depends on AuNP size [55]. Particles smallerthan 20 nm essentially show absorption, but largersizes of 80 nm increase the ratio of scattering toabsorption. On the other hand, silver nanoparticles(AgNPs) have excellent antimicrobial activityagainst viruses, bacteria, and other eukaryoticmicroorganisms. Therefore, AgNPs have wideapplications in dental materials, textile fabrics,
coating stainless steel materials, water treatment,medicine for burn treatment, and others.
3.2.1. Biological imagingAuNPs have been employed as a contrast agent inelectron microscopy for several decades. Owing tothe high atomic number of gold, the colloidal goldparticles are electron dense which render themamong the best for electron microscopy. However,AuNPs were involved in several other imaging tech-niques that rely on the plasmon band [56]. AuNPsgreater than 20 nm can be used in optical micro-scopy under phase contrast or differential interfer-ence contrast mode. Other techniques includefluorescence microscopy, photothermal coherencetomography (similar to ultrasound with good depthpenetration), multiphoton SPR microscopy, andX-ray scattering [57].
Perhaps, the most referenced application of AuNPsin bioimaging is in immunostaining. Essentially, anti-body-conjugated AuNPs are designed to bind againstantigens on fixed and permeabilized cells, being sub-sequently visualized via TEM or light microscopy[56]. Typically, an excess of gold is used so thatvirtually all entities are labeled to improve contrast.As cells are fixed and permeabilized, targets outside aswell as inside the cells can be labeled. As an ultra-structural marker for detection of proteins, peptides,or amino acids, gold can be used for immunostainingthick or thin sections prior to embedding, or forimmunostaining ultrathin sections after embedding[58]. By virtue of its particulate nature, gold as animmunolabel facilitates a semiquantitative analysis ofantigen densities on ultrathin sections. Various com-binations of different-size gold particles, or dualimmunolabelling with enzymatic immunolabels,together with colloidal gold or silver-intensified gold,serve well for ultrastructural immunocytochemicallocalization of two antigens in the same tissue section[59]. Compared to fluorescence microscopy, AuNPsare more stable as they do not suffer from photo-bleaching, and in most cases of TEM imaging,AuNPs offer excellent lateral resolution with highcontrast.
3.2.2. Cell delivery vehiclesMost delivery strategies, such as using cancer-targeting moieties conjugated to NPs for deliveryinto cancer tissue, are very similar to those used
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for magnetic and other types of NP. Indeed, goldhas been used for many years to deliver moleculesinto cells. For delivery applications, AuNPs areemployed for their small size, colloidal stability,ease of synthesis and conjugation, and their inert,biocompatible nature. Introduction into cells caneither be forced, for example, with gene guns, orbe achieved via cellular uptake. With regard to thegene gun technique, DNA is adsorbed onto thesurface of AuNPs, which are then essentially shotinto cells [60]. The force required typically pro-vided via gas pressure or electric discharge. Whilegene guns were more commonly employed inplant cell biology to breach the plant cell wall.Gene guns have also been used for DNA deliveryinto animal cells. Alternatively, specific or nonspe-cific endocytosis cellular uptake can be relied uponfor AuNP delivery. Specific uptake depends onreceptor–ligand binding, for example, using trans-ferrin adsorbed onto the AuNPs as a means ofinstigating cellular uptake, and is far more effectivethan passive nonspecific uptake. Following uptake,Figure 3 illustrates the NPs which are stored inendosomal/lysosomal vesicles inside the cells.
There have been efforts to avoid endosomaluptake and subsequent vesicle storage. The nucleartranslocation of AuNPs (10–35 nm) coated withnucleoplasmin (a Xenopus oocyte protein that
contains a well characterized nuclear localizationsequence) was performed using microinjection onchemically modified cells that bypassing theplasma membrane entry [62]. Further workshowed nuclear targeting was only achieved whenexposing cells in culture to AuNPs (20 nm) deri-vatized with bovine serum albumin and functio-nalized with a variety of short peptide sequencesthat exhibit nuclear localization sequences, whenthe whole peptide was present, thus including thesequence for plasma membrane translocation(otherwise the particles were trapped in the endo-some). Following the advent of cell-penetratingpeptide research, whereby direct plasma mem-brane translocation was achieved, conjugatingt-t peptide to AuNPs also achieved excellent cel-lular uptake levels [63].
3.2.3. BiosensorWhile imaging and delivery shown above tend touse passive methods, plasmon-related sensing usesAuNPs in a more active role. Basically, the NPs arerequired to specifically register the presence ofanalyte molecules and provide a concentrationreadout. This is typically achieved by changes inthe optical properties of the AuNPs. The plasmonresonance frequency is a very reliable intrinsicfeature of AuNPs that can be used for sensing
[64]. The binding of molecules to the particle sur-face can change the plasmon resonance frequencydirectly, which is visible by scattered light. In addi-tion, the influence of the inter-AuNP distance onthe plasmon resonance, when this distance isreduced to less than the particle diameter, is thecrucial factor in the sensor application, and linkingthe NPs with a biological analyte results in a colorchange that makes the basis of sensing [65]. Thefirst colorimetric sensing of nucleic acids wasreported and is now the most recognized exampleof a gold-based biosensor [66]. The DNA linkedthe AuNPs together with an interparticle distanceof 0.34 nm, which caused a red-to-purple colorchange depending on whether the gold particlesare free in colloidal suspension (red) or attached tothe DNA target (blue-violet) [56]. The colorchange is temperature reversible. Therefore, whenthe sample is heated, even a single sequence mis-match will result in a different melting tempera-ture, thereby causing a difference in color change.AuNPs possess excellent fluorescent propertiesand display antiphotobleaching behavior understrong light illumination. AuNPs exhibit strongnative fluorescence under relatively high excitationpower. For example, if cells stained with AuNPsare illuminated with strong light, AuNP fluores-cence can be recorded for cell imaging. A furtherphenomenon is that the fluorescence of manyfluorophores is quenched when they are in closeproximity to gold, and this effect can be used forseveral sensing strategies [67].
3.3. Quantum dots
QDs are colloidal nanometer-sized crystals, com-prising atoms of elements from groups II to VI(e.g. Cd, Zn, Se, Te) or III to V (e.g. In, P, As) inthe periodic table [68]. In terms of bioapplications,the most typical QDs employed were composed ofCdSe and encased in a ZnS shell in order to protectfrom the highly toxic cadmium. By confining theelectrons in variable sizes, the energy bandgap of theabsorption spectra allows their corresponding emis-sion wavelengths to be tuned from the ultraviolet tonear-infrared (NIR) region [69]. Smaller QDsdemonstrate blue fluorescence emission (within380–440 nm), while larger particles demonstratered fluorescence emission (within 605–630 nm).
QDs have further advantages over organic dyemolecules, aside from their tuneable fluorescence.They are robust and stable light emitters due to theirinorganic makeup and are less susceptible to photo-bleaching than organic dye molecules. This photo-stability makes them extremely useful in observingcells over longer periods of time.
3.3.1. Biological imagingDue to their high photostability and limited cyto-toxicity, QDs appeared as a very promising probefor longer-term experiments in living cells, pro-vided several issues are addressed. Initially, watersolubility is an important requirement forin vitro and in vivo imaging. Thiol groups (SH)are generally anchored to the ZnS shell withterminal carboxyl (COOH) in order to increasethe hydrophilicity of QDs [70] followed by cellinternalization. As with both magnetic and goldNPs, the cell uptake is typically enhanced viaparticle surface functionalization. Several meth-ods have been proposed to deliver QDs to thecell cytoplasm. Aside from the physical methods,such as microinjection and electroporation, manyattempts have been made using lipid- or poly-mer-mediated endocytosis and peptide-mediatedendocytosis [71]. The main issue here is that theQDs remain trapped in the endosomes for sev-eral days. An alternative pathway is to exploitpinocytosis. This is activated by an increase inosmotic pressure in the cell culture medium. Tocounter-balance this, the cell uptake medium inthe pinosomes is placed together with QDs in themedium. Pinosomes can subsequently be dis-rupted by a second osmotic shock, therebyreleasing the QDs into the cell cytoplasm [72].
3.3.2. Single-cell imagingTracking of single cells was initially developed tostudy membrane receptor dynamics. The first stu-dies included diffusion of transmembrane proteinsusing micrometer-sized beads of AuNPs [73].Recently, similar experiments have been per-formed using QDs to target membrane proteinsand study the mobility and kinetics of receptors,transmembrane proteins, and synapses [74]. QDscan be detected and tracked with the sameapproach as for traditional organic dyes. In thecellular context, tracking QDs is technically
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difficult. First, the background noise due to cellautofluorescence and to the surrounding QDsdiminishes precision. To reduce this noise, QDsemitting in the red field are used, where the cellautofluorescence disappears [75]. Furthermore,using small levels of QDs helps to improve thesignal-to-noise ratio. The second difficulty arisesfrom the need to track in a 3-D space. In recentyears, various techniques have been developed toacquire image stacks by reconstructing the3-D structure of the cell. For example, the totalinternal reflection fluorescence microscopy, whereonly particles closest to the glass coverslip areexcited typically 100–200 nm of a cell [76]. Themost popular reconstruction of a z-series, how-ever, is the scanning confocal microscope, whichlimits excitation to a particular volume.
3.3.3. In vivo imagingQD–peptide conjugates were the first used in vivoto target tumor vasculature in mice shown inFigure 4. Histology indicated that the tissue-specific peptide coating on (CdSe) Frontiers ofNanoscience ZnS QDs increased NP accumulationat vascular sites following intravascular injection[77]. Although this does not describe QD imagingin a live animal, this study demonstrated thepotential of using QDs for molecular-level detec-tion. This indicated that untargeted or passivetargeting of QD probes demonstrated weak or nosignal, but antibody-conjugated QDs resulted inintense fluorescent signals. Despite using targeting
moieties with QDs, such as antibodies or peptides,targeted to tumors in live animals for cancer ima-ging, light penetration and autofluorescence ofdeep tissue remain a major hurdle. As with single-cell imaging above, the use of red field emittingQDs has minimized light absorption by blood andwater and improved tissue depth [78].
3.3.4. Targeted therapiesSimilar to mNPs employed in transfection thera-pies in vivo, QDs are also used as delivery andreporter systems. A big advantage of NP transfec-tion, compared to other types of delivery vehicles,is that they can be functionalized with many dif-ferent oligonucleotides and cell-binding ligands atonce, potentially allowing multiple gene knock-downs and higher affinity for the target cell simul-taneously. Studies reported that one siRNA perparticle in conjunction with >15 peptides, or twosiRNA per particle in conjunction with <10 pep-tides, gave optimal knockdown and targeting [80].
Further study showed that by using QDs designedwith tumor targeting, facilitated by the attachment ofa tumor-homing peptide (F3), which binds tonucleolin expressed on the surface of cancer cells[81]. The addition of F3 increased the QD tumorcell uptake by two orders of magnitude compared tonontargeting QDs. Diagnostic imaging was facili-tated via the QD core with emission in the NIR,and treatment delivery was facilitated by the attach-ment of siRNA. This siRNA silenced the enhancedgreen fluorescent protein gene, but the authors notedthat their design could also be applied to silenceoncogenes for cancer treatment. Results showedthat siRNA attached to the QD by a disulfide bondincreased gene knockdown compared to siRNAattached to the QD by a covalent bond. This researchdemonstrated the importance of such acute attentionto molecular design in the development of an effi-cient NP-based RNAi platform.
3.4. Carbon nanotubes
Hollow and porous NPs, such as nanotubes,nanoshells, and hollow spheres, can be loadedwith a large amount of cargo, thus enhancingsignal and sensitivity. Carbon nanotubes arecylindrical graphene sheets. Although mostapplications of carbon nanotubes have focusedFigure 4. The QDs emitting different wavelengths [79].
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on microelectronic devices, due to their uniqueelectronic and physical properties, carbon nano-tubes have shown some attractive properties forbiomedical use, including easy translocationacross the cell membrane and relatively low toxi-city [82]. Both single-walled carbon nanotubes(SWCNTs; 1–3 nm in diameter and 5–30 nm inlength) and multiwalled CNTs (MWCNTs;10–150 nm in diameter and 200 nm to severalmicrons in length) have been investigated forvarious bioapplications. As with QDs, it hasbeen indicated that SWCNTs have strong opticalabsorbance in the NIR region where biologicalsystems are known to be highly transparent [83].
3.4.1. Neuronal tissue engineeringCNTs have been rapidly developing as a technologyplatform for designing novel neuroimplantabledevices. They combine incredible strength withextreme flexibility, in addition to exhibiting physicaland chemical properties that allow them to effi-ciently conduit electrical current in electrochemicalinterfaces. Thus, CNTs can be employed in tissueengineering scaffolds, organized as fibers or tubes,with diameters similar to those in neuronal pro-cesses such as axons and dendrites [84]. The firstapplication of nanotubes to neuroscience researchemployed MWCNTs for growth of rat brain neu-rons [85]. It was noted that on unmodified tubesneurons extended only one or two neurites, whichexhibited very few branches. In contrast, neuronsgrown on tubes coated with a bioactive molecule(4-hydroxynonenal) demonstrated multiple neuriteswith extensive branching.
3.4.2. Imaging and cancer treatmentSimilar to mNPs and AuNPs, SWCNTs have beenutilized in thermal necrosis of cancer cells.Intratumoural injection of tubes, alongside NIRirradiation, resulted in thermal death of humanepidermoid mouth carcinoma KB tumor cells inxenografted mice with minimal side effects up to6 months after treatment, with excretion via urinein 3 months. Similarly, thermal cancer therapy wasapplied to mice bearing kidney tumors usingMWCNTs, and complete tumor regression wasobserved with no recurrence within 3 months.Tumor targeting has also been employed, using
antibodies and folate receptors (which are knownto be highly expressed in a range of tumor cells).
When considering targeting, CNTs have alsobeen studied for their use in delivering therapeuticdrugs to tumors. SWCNTs exhibiting paclitaxelconjugated to PEG chains were injected into breasttumor xenografts in mice. Particles were well dis-persed, with inhibition of tumor growth by almost60%. Similar studies using other drugs, such asdoxorubicin-loaded SWCNTs, RGD peptides to tar-get-specific integrins, and anticancer agent cisplatin,all had varying effects on tumor growth and someretention in the liver, kidney, and spleen [86]. Thereare other types of inorganic NPs, such as silica,nanoshells, nanorods, and hybrid particles, but theparticle systems described above are the four mostcommonly used when considering bioapplications.Such systems, due to their multifunctionalapproach, hold a great promise in diagnostics,drug and gene delivery, sensing and biosensing,and both in vitro and in vivo imaging.
While each of the particles described exhibitssome features that are original to them, the bioap-plications do overlap with many sharing function-alities and targeting groups. Each particle type isdesigned with the view to boosting cellular uptakeefficiency, for image/signal enhancement or cargodelivery and to target-specific tissues/cells [87]. Itis envisaged that diseases may be managed bymultifunctional NPs that encompass both imagingand therapeutic capabilities, thus allowing simul-taneous disease monitoring and treatment.
4. Organic nanoparticles
4.1. Use of nanoparticles in nucleic acid delivery
Safe and effective gene delivery systems are tre-mendously important in gene therapy for tacklingvarious genetic diseases, viral infections, and car-diovascular disorders. Gene therapy is a process bywhich genetic material in the form of oligonucleo-tides or plasmids is introduced into specific targetcells to recover or induce the expression ofa normal protein to treat human disorders. Genetherapy can also deliver antisense oligonucleotidesor small interfering RNA to interrupt the functionof target genes and trigger silencing [88]. In recentdecades, many methods for gene delivery have
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been developed for a wide range of cells and tis-sues. In addition to these therapeutic applications,gene delivery is widely used in vitro as a researchtool to investigate gene function/regulation withina cellular and physiological context. Initial systemsfor gene delivery have been developed, with thepotential to significantly impact biotechnology,diagnostic applications, and basic research.
Gene delivery systems should enable the forma-tion of stable complexes with nucleic acids, pro-viding a low cytotoxicity, and disassembleintracellularly to release the nucleic acid [89].Environmental interactions are manipulatedthrough the incorporation of functional groupsthat may stabilize the vector in the extracellularmilieu, target a specific tissue or cell type, ormaintain the vector within the delivery location.Intracellular trafficking addresses the need to deli-ver the plasmid to the nucleus for expression; thus,bioactive groups can be incorporated to facilitateendosomal escape or nuclear localization.
Gene delivery systems include viral and nonviralvectors. Viral vectors are themost effective, but thereis an overlapping concern about their application,which is limited by their DNA loading capactiy, andin therapeutic application by their oncogenicity andimmunogenicity [90]. Nonviral vectors are morereproducible, do not present DNA size limits, andare safer and less costly than viral vectors. Nonviraltransfection systems are usually composed of catio-nic peptides, cationic polymers, or cationic lipids,although the combination of some of them is alsopossible. These so-called modular nonviral vectorsconsist of a vector backbone modified with func-tional groups that mediate environmental interac-tions and intracellular trafficking to overcome themultiple barriers to gene transfer. For both in vitroand in vivo applications, nonviral vectors must bedesigned by taking into consideration their interac-tions with serum components, the extracellularmilieu within the tissue or cell culture media, andbinding to the cell surface. Many of the aforemen-tioned vectors interact with serum proteins that caninactivate the complex or promote clearance fromthe desired tissue, which limits the opportunity forcellular internalization. Cellular internalization canbe improved by the addition of receptor ligands tothe NP, thus increasing binding to cell-surface recep-tors and allowing the targeting of specific cell types.
Once the DNA is introduced into the cell, itmust pass a series of barriers that could damageit, before reaching the nucleus. To avoid this,extensive research is underway to define the idealcarrier to facilitate cell entry and transport to thenucleus of the DNA in a protected manner. Thecarrier should be sufficiently small to allow cellularinternalization and pass through nuclear pores,must have flexible tropism, and should be able toescape the endosome–lysosome process that fol-lows endocytosis [91]. It must also not be cytotoxicnor elicit an immune response. All these condi-tions can be accomplished by nonviral nanovec-tors including cationic molecules such as cationiclipids and synthetic or natural cationic polymers,which have been widely developed to condenseDNA and to efficiently deliver therapeutic geneswithin mammalian cells.
While gene delivery has been tested in many celltypes using a number of different conditions andreadout systems to test for transfection efficiencyand toxicity, the lack of a reference standardmakes it difficult to directly compare differentapproaches. Thus, this section presents selectiveapproaches with illustrative examples, rather thanan exhaustive comparison of the different methodsthat have been developed over the years.
4.2. Engineered biomaterials for vectorbackbones
Numerous cationic lipids and polymers have beendeveloped to package DNA for cellular internali-zation and protect it from degradation, leading tothe identification of some structure–functiondesign relationships. Cationic lipids are composedof three basic constituents: a polar headgroup,a linker, and a hydrophobic moiety. The cationicheadgroup promotes the interaction with DNA,whereas the hydrophobic moiety provides self-association to form either micelles or liposomesin the presence of a helper lipid, such as dioleyl-phosphatidylethanolamine. Lipoplexes forma multilayered structure consisting of the plasmidsandwiched between the cationic lipids [92].Despite the fact that cationic lipids have lowimmunogenicity, they show in vitro and in vivocytotoxicity and relatively low gene transfer effi-ciency [93].
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4.2.1. Cationic polymersCationic polymers can be classified into two groups,namely natural polymers (e.g. proteins and pep-tides) and synthetic polymers (e.g. polyethyleni-mine, dendrimers, and polyphosphoesters). Theyspontaneously associate with plasmids by electro-static interactions due to protonatable amine resi-dues to form condensed complexes calledpolyplexes. With these electrostatic forces betweenthe polymer and the nucleic acid, the complexshould maintain a stable and condensed nanosizestructure, promoting cellular endocytosis, and pos-sibly enhance the transfection efficiency of thera-peutic genes. Furthermore, DNA plasmids can becondensed inside the NPs for protection againstnuclease degradation [94]. High-molecular-weightpolymers tend to form relatively stable, small com-plexes compared to low-molecular-weight poly-mers. Low-molecular-weight polymers, however,can enhance transfection efficiencies, likely due toa decreased cytotoxicity and the increased ability ofthe plasmid to dissociate from the cationic polymer.Block copolymers can potentially regulate theassembly and structure of the complex, while pro-viding for multiple functions due to each compo-nent. Poly(2-diethylaminoethyl methacrylate) orpoly(2-dimethylaminoethyl methacrylate), whichhave primary and secondary amines to facilitatecomplexation and intracellular trafficking, can becombined with poly(ethylene oxide) (PEO) or poly(propylene oxide) to prevent aggregation andreduce toxicity [95]. Although the condensing vec-tors seem to be an excellent substitute for viralvectors, some drawbacks inherent in the conden-sing system limit their application for systemicdelivery. These include toxicity of the cationic poly-mer or lipid, rapid clearance by the reticuloen-dothelial system, inability of the complex to escapefrom the endosome/lysosome compartments in thecells, and lack of intracellular unpacking of thenucleic acid construct from the electrostaticcomplex.
Noncondensing lipids and polymers possesseither a neutral or net negative charge. They canbe used to engineer nanoscale vectors for tissue-and cell-specific delivery and allow for enhancedtransfection efficiency with significantly less toxicityconcerns. Nucleic acids are encapsulated withinsuch vectors either by physical entrapment within
the matrix or through hydrogen bonds betweenpolymer and nucleic acid bases [96]. Physicalencapsulation offers protection from the enzymesand other plasma proteins during its transit fromblood to the site of action. Cellular uptake is facili-tated since masking the negative charge of DNAprevents electrostatic repulsion with the negativelycharged cell surface. Moreover, in contrast to con-densing lipids and polymers, the absence of positivecharges on noncondensing systems limits theirrecognition by the mononuclear phagocyte systemand hence limits their early clearance and opsoniza-tion by IgM and the innate immune response [97].
4.2.2. Solid lipid nanoparticlesSolid lipid nanoparticles (SLNs) shown in Figure 5have also been tested. They are generated byexchanging the liquid lipid of emulsions fora solid lipid, making them solid at room and bodytemperature, thus inducing a reduction in particlesize. The capacity of SLN:DNA vectors to inducethe expression of a foreign protein after intravenousadministration has been demonstrated. By neutra-lizing the acidic pH inside the endosome, DNAmolecules, protected against acid attack of protons,remain intact inside the cell. The most importantdifference with liposomes is that the core matrixused for release and delivery of bioactive substancesbecomes lipophilic instead of aqueous for liposomes[98]. Some advantages of SLNs are their low toxi-city, good storage stability, and the possibility ofsteam sterilization and lyophilization.
4.2.3. Cationic cholesterol disulfide lipidsThe use of cholesterol and some of its derivativesfor the synthesis of gene delivery carriers has been
Figure 5. Schematic diagram of solid lipid nanoparticles [99].
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recently tested. Cationic cholesterol disulfide lipids(CHOSS) were synthesized by binding cholesterolto cationic head groups via a disulfide linkage.Headgroups which were used included histidine,pyrimidine, or methyl imidazole, which demon-strated low cytotoxicity and high transfection effi-ciency. CHOSS can bind to DNA with high affinityforming stable lipoplex NPs. Transfection experi-ments with COS-7 cells confirmed the low cytotoxi-city and particularly high uptake capability of theseparticles [100]. Interestingly, the particles were spe-cifically localized to the periphery of cell nucleifollowing uptake. Once inside the cell, the releaseof the DNA may occur via cleavage of the disulfidebond in the reducing intracellular environment.
4.2.4. Biomimetic engineeringAnother approach has been created which isknown as biomimetic nanocapsules. They consistof an oily core made up of a mixture of trigly-cerides and polyglyceryl-6 dioleate surroundedby a shell of free polyethylene glycol (PEG) andHS-PEG. They have been used for DNA encap-sulation to improve the resistance to rapid clear-ance. Through PEG coating, the positive chargesof such complexes are masked and thus preventopsonization by immune proteins. Furthermore,because PEG leads to destabilization of the lipidendosomal membrane, DNA nanocapsules arebetter than lipoplexes when the limiting step inthe transfection of endosomes has to be avoided[101]. Another important consideration is thebiodegradability of the nanospheres once theyhave traversed the required specific sites. Earlybiopolymer NP design focused primarily on theuse of nonbiodegradable synthetic polymers suchas polyacrylamide and poly(methylacrylate). Therisks of chronic toxicity due to the intracellularand/or tissue overloading of nondegradablepolymers were soon considered as a major lim-itation for the systemic administration of poly-acrylamide and poly(methylacrylate) NPs inhumans. As a consequence, the focus has shiftedtoward NPs designed using synthetic biodegrad-able polymers including polyalkylcyanoacrylate,poly(lactic-co-glycolic acid), and polyanhydride[102]. The therapeutic potential of these biode-gradable colloidal systems was investigated forvarious applications.
There is another limitation for the bionanopar-ticle-based administration of hydrophilic mole-cules including nucleic acids (oligonucleotidesand genes) [103]. This limitation is mainly becausethe polymers forming these NPs are mostly hydro-phobic, while biomolecules including nucleic acidsare hydrophilic. This leads to difficulties of effi-cient encapsulation and protection against enzy-matic degradation. Therefore, the preparation ofNPs using more hydrophilic and naturally occur-ring materials has been explored.
4.2.5. Dendrimer-based DNA engineeringDendrimers have attracted interest for drug andgene delivery systems because they possessa number of unique and interesting characteristicssuch as defined structures, inner cavities able toencapsulate guest molecules, and controllable mul-tivalent functionalities in their inner or outer parts[104]. Figure 6 shows the repetitively branchedmolecules of dendrimers composed of poly(amido amine) monomers. These propertiesmake dendrimers an important option for thedevelopment of nanoscale nonviral vectors fornucleic acid delivery. Dendrimers can interactwith various forms of nucleic acids (i.e. DNA,RNA, and oligonucleotides). These interactions,primarily electrostatic, lead to complexes, whichprotect the nucleic acid from degradation. The
Figure 6. PAMAM dendrimers [106].
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properties of these complexes depend on manyfactors, such as stoichiometry, the concentrationof dendrimer amines and nucleic acid phosphates,solvent properties like pH, and salt concentration[105]. However, the inherent difficulty of synthe-sizing new dendrimers that are suitable carriers fordrug delivery has led researchers to focus primar-ily on the modification of existing dendrimers,instead of the development of novel dendrimersfor gene delivery systems.
Poly(amido amine) (PAMAM) dendrimers havebeen tested as genetic material carriers and havebeen also modified with PEG, amino acids, orligands in order to enhance their gene deliverypotency. Numerous reports have been publisheddescribing the use of amino-terminated PAMAMor PPI (poly(propileneimine)) dendrimers as non-viral gene transfer agents, enhancing the transfec-tion of DNA by endocytosis and, ultimately, intothe cell nucleus [107]. The observed high transfec-tion efficiency of dendrimers may not only be dueto their well-defined structure but may also becaused by the low pKa of the amines, which per-mits the dendrimer to buffer the pH change in theendosomal compartment. Dendrimers holda promising future for various biomedical applica-tions for the coming years, as they possess uniqueproperties, such as a high degree of branching andmultivalency. Also, as research progresses, newerapplications of dendrimers will emerge, and thefuture should witness an increasing number ofcommercialized dendrimer-based DNA deliverysystems.
4.2.6. Protein-based engineered colloidalThe first naturally occurring material used for thepreparation of NPs consisted of two proteins,albumin and gelatin. Protein-based colloidal sys-tems are very promising because they are biode-gradable, nontoxic, and less immunogenic. Theyhave greater stability in vivo and during storageare relatively easy to prepare and to monitor sizedistribution [108]. In addition, because of thedefined primary structure of proteins, protein-based NPs offer various possibilities for surfacemodification and covalent drug attachment. Forall these reasons, a number of proteins have beenused to develop protein-based NPs for drug deliv-ery. These include albumin, collagen, gelatin,
fibroin, sericin, and keratin. As an example, gela-tin is one of the most versatile natural biopoly-mers derived from collagen, and it has beenwidely used in food products and medicines.Many researchers have used gelatin NPs asa gene delivery vehicle. With solvent displace-ment, type B gelatin, derived from alkaline hydro-lysis of collagen, which has an isoelectric point ataround, can physically encapsulate nucleic acidconstructs at neutral pH. Furthermore, the physi-cal encapsulation in gelatin NPs preserves thesupercoiled structure of the plasmid DNA andimproves the transfection efficiency upon intra-cellular delivery [109].
An approach which has recently been used forgene delivery includes genetically engineered pro-tein-based polymers, which incorporate peptidemotifs such as elastin, silk, and collagen. Thisapproach has the advantage that the properties ofthe resulting NPs can be tailored to avoid cyto-toxicity and rapid clearance, while ensuring deliv-ery of the DNA package to the intended target[110]. Another promising approach, which hasprimarily been tested in vitro, combines the prop-erties of lipids and peptides to achieve high effi-ciency of transfection with minimal toxicity evenin the presence of serum. This approach consistsof the use of oligoarginine–lipid conjugates. Thelipidic part of these complexes consists of 3,5-bis-(dodecyloxy)benzamide (BDB) and a PEG spacerwhich is introduced between the amide group ofBDB and the C-terminal of oligo-Arg.
Polysaccharide-derived NP surfaces help toimprove the biocompatibility of cell toxic material,together with new immobilization approaches,which are currently in development for novel bio-nanoparticle-derived pharmaceutical formulations[111]. NPs from naturally occurring polysacchar-ides were designed for the administration of pep-tides and proteins, as well as nucleic acids. Animportant example of this approach is the use ofchitosan, a natural biodegradable cationic polysac-charide derived from chitin consisting ofD-glucosamine and N-acetyl-D-glucosamine. It isproduced by deacetylation of chitin extracted fromthe shells of crabs, shrimp, and krill. This linearpolymer has been shown to be biocompatible andnonimmunogenic and to possess mucoadhesiveproperties, making it an excellent biopolymer for
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the preparation of NPs as vectors for DNA deliv-ery [112]. Indeed, chitosan can spontaneously bindto DNA via ionic interactions to form NPs. Thebiochemical properties of chitosan make ita suitable vehicle for gene transfection. Theamino groups confer to the molecule a high chargedensity and are readily available for chemical mod-ification and salt formation with acids. In vivo, it isdegraded by lysozyme and it has been also shownto partially protect DNA from nuclease degrada-tion [113]. Cellular transfection with chitosan–DNA complexes has demonstrated that onceinside the cell, complexes of 100–250 nm can befound accumulated in the nucleus. Finally, chito-san–DNA nanospheres have been shown to benontoxic in both experimental animals andhumans.
4.3. Environmental interactions
Interactions with serum components can be mini-mized by modifying the surface of polymericNPs with hydrophilic polymer chains ina process called passive targeting as it allows themodified NPs to avoid the body’s clearancemechanisms, increasing the chances that they willreach their intended target. There are several waysto achieve passive targeting such as surface mod-ification of polymeric NPs with hydrophilic poly-mer chains or incorporation of environmentallyinsensitive polymers into the nanoparticles.A dense, hydrophilic shell of long chains is formedthat protect the core from interacting with solutes,including nonspecific hydrophobic interactionswith the reticuloendothelial system. This poly-meric protection is referred to as ‘steric stabiliza-tion'. Modifications of the cationic polymers, suchas PEGylation, have been incorporated to facilitatedelivery and enhance transgene expression. At thesame time, the terminal hydroxyl groups ofPEGcan be derivatized, leading to monofunctional,homo-, or hetero-bifunctional and even multi-armPEG, allowing further conjugation of selectedligands.
The type B gelatin-based NPs has been used asa noncondensing gene delivery system for tumortargeting [114]. They prepared unmodified and PEG-modified gelatin NPs by ethanol precipitation, leadingto particles in the range of 200–500 nm.
Tetramethylrhodamine-dextran, a hydrophilic fluor-escently labeled molecule, was first used as modeldrug for in vitro cell uptake studies. The control andPEG-modified type B gelatin NPs were taken up bycells through nonspecific endocytosis [115]. Within12 h of PEG-modified gelatin NP internalization byNIH-3T3murine fibroblasts, the payload was releasedand accumulated around the perinuclear region.Further experiments using encapsulated plasmidDNA, encoding for enhanced green fluorescence pro-tein (EGFP-N1), confirmed the long-lasting transgeneexpression potential of PEG-modified type B gelatinNPs compared to other methods. In addition, neithergelatin nor PEG-modified gelatin NPs demonstratedany toxicity. To complement the in vitro evaluations,the biodistribution profiles of unmodified and PEG-modified 125Iodine (125I)-labeled gelatinNPs following intravenous administration throughthe tail vein in Lewis lung carcinoma-bearingC57BL/6 J mice were further examined [116]. PEG-modifiedNPs remained in circulation for an extendedperiod and preferentially accumulated in the tumorfor up to 24 h postadministration as well as the liver.Conversely, unmodified NPs were rapidly clearedfrom the circulation and remained mostly in theliver and spleen. These results show that PEG-modified gelatin NPs can be passively targeted to thetumor mass following systemic administration andhave the potential to be an effective vector for antic-ancer gene therapy. In another study, the potential ofPEG-modified gelatin NPs for passively targetinggene delivery to tumors were further demonstrated.Interestingly, intravenous administration of an encap-sulated reporter plasmid DNA into tumor-bearingmice eventually led to a higher level of transfectionof the tumor than following intratumoral administra-tion [117]. A more active tumor-targeting strategytakes advantage of the fact that tumors express ele-vated levels of glutathione (GSH) compared to normalcells [118]. GSH is a tripeptide, generally expressed inthe cell cytoplasm and functions as an antioxidant toprevent damage related to the reactive oxygen species.The intracellular GSH concentration (5–10 mM) isgenerally higher than the extracellular concentrations(1–10 mM). During active proliferation of tumorcells, GSH and peroxide levels are further elevated inthe cytoplasm. Introduction of thiol groups isa common modification that can allow for intracellu-lar delivery through the reduction of disulfide cross-
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links [119]. This approach has been tested in vitro andleads to increased transfection efficiency compared toother NP-based systems.
Chitosan–DNA nanospheres have also beentested for clinical applications. Several deliveryroutes of chitosan–DNA NPs have been investi-gated in animal models including intranasal andoral delivery, resulting in transduced gene expres-sion in the stomach and intestinal epithelium ofa peanut allergen gene providing protectionagainst allergen-induced anaphylaxis [120]. Inaddition, such NPs have also been designed fortissue engineering and local gene delivery in peri-odontal tissue regeneration.
4.4. Intracellular trafficking
Following extracellular transport to the targetcell, the vector is internalized primarily byendocytosis and then must escape the endoso-mal compartment to avoid lysosomal degrada-tion before transport to the nucleus andcrossing the nuclear membrane for subsequenttranscription [121]. Nuclear localization occurseither following cell division or through trans-port through the nuclear pore complex. Therate-limiting step for this process is dependentupon the vector. Lipoplexes exhibit poor cellularuptake, whereas polyplexes exhibit limitednuclear import. Small-molecule peptides andproteins such as transferrin, antibodies, orgalactose (for hepatocyte targeting) have beenattached to either the plasmid or to the cationiclipid or polymer to promote endosomal escapeand nuclear localization [122].
Fusogenic peptides have been incorporated todisrupt membranes, which may facilitate eithercell entry without endocytosis or endosomalescape following endocytosis. Nuclear targetingcan be enhanced by the attachment of nuclearlocalization signals (NLS), which are oligopep-tides that bind to importins, cytoplasmic recep-tors responsible for binding and transportthrough the nuclear pore complex. For example,NLS addition to the polysaccharide chitosanimproved transfection efficiency. However, suc-cess with NLS has been varied: while somepapers have reported that inclusion of NLS-containing proteins or peptides increases gene
transfer and expression, such as for chitosan,others have found no such enhancement [123].
Numerous opportunities remain to increase genetransfer by virus mimicking: that is the design ofvectors to interface with specific cellular processes,with functional groups derived from the under-standing viruses or cellular processes. Nonviral vec-tors engineered with functional groups that allowfor directed motion along the cytoskeleton couldincrease accumulation at the nucleus and decreasethe amount of DNA required [124]. Peptide nucleicacids (PNAs) have been developed that are able tobind tightly to specific DNA sequences.Incorporation of PNAs could potentially target thetransgene to a specific chromosomal location assome viruses do. Alternatively, engineered zinc-finger proteins with nuclease activity were devel-oped to recognize a unique chromosomal site andinduce a double-strand break [125]. At this breaksite, the chromosome can recombine with an extra-chromosomal sequence of interest. Finally, thenucleic acid itself could be engineered to avoidsilencing by methylation, thereby extending theduration of transgene expression.
4.5. Immunoassays
Immunoassays play a vital role in laboratoryresearch, clinical diagnostics, and food and envir-onmental monitoring. They combine immunologyand chemistry to create scientific tests such asenzyme immunoassays for the specific and sensi-tive detection of the analytes of interest. Theseassays are based on the principle of the specificityof the interaction between the antibody and itscognate antigen. Immunoassays are relatively easyto perform contributing to their widespread use.Radioimmuneassays (RIAs) and enzyme immu-noassays such as ELISA (enzyme-linked immuno-sorbent assay), luminescent immunoassays, andfluorescent immunoassays (FIAs) are all currentlyused. The antibodies can be labeled in several waysincluding radioisotopes, fluorescent dyes, orenzymes that catalyze fluorogenic or luminogenicreactions, thus allowing visualization of the anti-body–antigen interaction [126].
In the past, RIAs were widely used but have beenslowly replaced by assays using fluorescent mole-cules and enzymes as labels to avoid the obvious
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disadvantages of radioisotopes. Nevertheless, the useof enzymes and fluorescent labels is often lessstraightforward and do not yet achieve the sensitivityand limits of detection of radioisotope-based assays.When first developed in the 1970 s, ELISAs andWestern blot assays provided a significant increasein sensitivity over existing detection methods [127].Today, a wide variety of enzyme-linked antibodieswith choices of chemiluminescent, bioluminescent,chemifluorescent, fluorescent, and more traditionalcolorimetric detection systems are commerciallyavailable from various suppliers. Despite recentadvances, there is an urgent need to improve immu-noassay sensitivity for multiple applications such asthe deciphering of normal and pathophysiologicalbiological processes, early disease detection, or thedetection of environmental hazards, all instanceswhere marker levels may be very low.
Antibody–enzyme (Enz-Ab) conjugates aremost often prepared by crosslinking enzymes tothe antibody via their functional groups such asthe primary amines and sulfhydryls, or by cross-linking through sugar moieties attached to one ofthe proteins. Chemical activation of the targetresidues on both the enzyme and the antibodytends to be random and difficult to control.Thus, the use of homobifunctional reagents suchas glutaraldehyde often results in very low yields,and the resulting Enz-Ab complexes can be het-erogeneous and highly polymerized [128]. SuchEnz-Ab complexes can compromise the sensitivityof the subsequent immunological assay due tosteric hindrance, which affects antigen-bindingcapacity. In addition, large-molecular-weight oli-gomers formed by uncontrolled cross-linking mayproduce insoluble aggregates that are impossible tomanipulate in immunoassays. In the case of gly-coproteins, some of these difficulties can be cir-cumvented by periodate oxidation. This methodhas been used to cross-link horseradish peroxidase(HRP), a glycoprotein, to functional groups on anantibody in a more controlled way [129].However, this approach is limited to enzymesthat contain carbohydrate moieties and thus isnot applicable to all enzymes including the com-monly used reporter enzyme alkaline phosphatase.
In FIAs, signal amplification is typicallyachieved by coupling fluorophores, such as organicdyes, to the antibody probes. The sensitivity of
FIAs is mainly determined by the number oflight quanta emitted/analyte molecule. Increasingthe fluorescent dye to Ab ratio results in improvedsignal amplification and therefore sensitivity [130].As for Enz-Ab conjugates, labeling antibodies withlarge numbers of fluorophores usually leads toreduced specificity and binding affinity as well asa reduced quantum yield due to dye self-quenching effects. For these reasons, the F/Pratio is normally kept around 4–8, thus limitingthe sensitivity of the assays.
4.6. Functionalization of nanoparticles bybiological entities
The creation of NPs with the desired physiobio-chemical properties remains a challenge. First, it isimportant that the antibody molecules are stablyattached to the particle surfaces while maintainingtheir ability to interact with the target analyte. Inaddition, NP aggregation and their nonspecificbinding with biological molecules remaina serious issue. For example, proteins can be eitherhydrophilic or hydrophobic, with either negativeor positive charges, making it very difficult toavoid nonspecific interactions. To address thesetechnical challenges, numerous surface modifica-tions and immobilization procedures have beenexplored and developed [131].
A common surface modification strategy is post-coating and modification of the NP surface withdifferent functional groups, including carboxylate,amine, PEG, or combinations of different function-alities. Alternatively, the direct synthesis on the sur-face of the NPs of a mixed monolayer of PEG anda functional group has been tested. While the ethy-lene glycol chains, which are water soluble andneutral in charge, function as a shielding compo-nent to minimize nonspecific binding, the func-tional group can be used as a capture agent forantibody conjugation acts. Immobilization of bio-molecules on surfaces can be achieved in a numberof ways via adsorption, by chemical linkage, or byaffinity-based interactions.
In adsorption, molecules are adsorbed at theinterface via physical forces such as van derWaals, electrostatic, or hydrophobic interactionsdepending on the chemical nature of the surfacesand molecules. Consequently, the conditions used
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for immobilization are highly sensitive to ionicstrength, pH, and temperature, leading under cer-tain conditions to subsequent dissociation of bio-molecules [132]. Moreover, this method ofimmobilization may interfere with proper foldingand can lead to multilayer adsorption and loss oforientation or enzymatic activity. However,because adsorption of biomolecular targets is rela-tively simple to perform, it is widely used. Thedisadvantages of adsorption can be overcome tosome extent by covalently linking biomolecules tothe particle surface via amide, ester, ether, or sul-fide bonds. Because of the large variety of reactivegroups found on organic NPs made of proteins,polysaccharides, lipids, or polymers, the well-defined methods of chemical modification areoften not position specific and therefore lackorientation [133]. In some cases, the reactivegroups are located close to the active center, affect-ing activity and function.
Streptavidin–biotin interactions are widely usedfor biotechnology applications, in particular immu-noassays. Although biotin–streptavidin binding isnot covalent, its high-affinity constant producesa highly specific and nearly irreversible immobiliza-tion. The streptavidin–biotin interaction is thestrongest noncovalent receptor–ligand interaction(Ka = 1015 M−1) currently known, with a strongeraffinity than in any known antigen–antibody inter-action [134]. Bond formation between biotin andstreptavidin is very rapid, and once formed, it isunaffected by most extremes of pH, temperature,organic solvents, and other denaturing agents.Streptavidin contains four subunits, each witha single biotin-binding site, allowing signalamplification.
A variety of biomolecules can be biotinylatedand subsequently used with streptavidin labeledprobes [135]. Biotinylated antibodies, which serveas recognition agents, are used in a variety ofdifferent assays, and therefore, many conjugatesare now commercially available. As such, strepta-vidin linkage is a very convenient and efficientconjugation method to attach recognition agentsonto NPs. An easy way of immobilizing streptavi-din onto a surface is based on electrostatic inter-action. The positively charged streptavidinnaturally adsorbs to a negatively charged surfaceand can then be stabilized by cross-linking using
a bifunctional reagent such as glutaraldehyde[136]. Resulting streptavidin-coated particles canthen be functionalized by conjugation to biotiny-lated recognition molecules.
An interesting alternative to the streptavidin–biotin system called ‘protein-assisted nanoassem-bler’ has been recently described. This approachallows the robust self-assembly of multifunctionalsuperstructures consisting of different single-function particles such as labels, carriers, recogni-tion, and targeting agents, including antibodies.This bioengineering method employs two uniqueproteins from Bacillus amyloliquefaciens, barnaseand barstar, to rapidly bring together the struc-tural components directly in solution [137]. Theproperties of the superstructures can be designedon demand by linking different agents of varioussizes and chemical nature, as a function of thespecific purpose. It has been demonstrated thatusing barnase and barstar, it is possible to assem-ble colloidally stable trifunctional structures bybinding together magnetic particles, QDs, andantibodies. Indeed, the bonds between these pro-teins are strong enough to hold macroscopic(5 nm–3 mm) particles together. Specific interac-tion of such superstructures with cancer cellsresulted in fluorescent labeling of the cells andtheir responsiveness to a magnetic field. Thisvery recent and versatile method can be used formultiple nanotechnology applications includingimmunoassays.
4.7. Bioanalytical applications
Numerous efforts have been made to optimize anti-body labeling in order to further improve the per-formance of immunoassays. In a careful study, theoptimization of biotin labeling of a mouse IgG wasdone by varying the classical parameters of the label-ing protocol [138]. The immobilization of biotin-tagged mouse IgGs on avidin-coated plates wasthen investigated by incubating the bound antibodieswith goat anti-mouse IgGs linked to fluorescentbeads. The optimum conditions were successfullyapplied in sandwich immunoassays for two differentanalytes, resulting in the detection of as little as 2 and5 ng of troponin I and N-terminal probrain natriure-tic peptide (BNP), respectively. In an interesting butstill little used approach, Simons et al. described the
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covalent in vacuo cross-linking of HRP to anti-rabbitimmunoglobulin G (IgG). The advantageous featureof this co-lyophilization-based procedure is that thecross-linking to form Enz-Ab conjugates is accom-plished without the use of chemical modifying oractivating reagents, reducing the potential activityloss due to chemical modification. The resultingsoluble multienzyme–IgG immunoconjugate exhib-ited a 100-fold increased sensitivity for antigen detec-tion compared to a commercial conjugate preparedby conventional chemical cross-linking methods.
The uses of NPs provide an interesting andpowerful avenue to achieve significant signalamplification by allowing the linkage of a singleantibody molecule with up to thousands of repor-ter molecules such as fluorophores or enzymes[139]. In an interesting example of this strategy,a polypeptide containing 20 lysine residues eachconjugated to an HRP molecule was attached to0.44 mm streptavidin polystyrene sphericalNPs introducing hundreds of HRP molecules andmaking a signal amplifying detector conjugate.Such highly labeled NPs were further functiona-lized with IgG molecules to achieve a molar ratioof 1 IgG to 105 HRP complexes. These immuno-conjugates efficiently bound to plasma anti-HIV-1antibodies that had been captured by HIV antigenson 5 mm carboxyl magnetic microparticles andproduced a detection signal with five to eighttimes more sensitivity as compared to conven-tional HRP conjugated goat anti-human IgG [140].
Similarly, sulfate functionalized polystyreneNPs were used to electrostatically attract positivelycharged antibodies. Protein immobilization main-tained the Y-shaped orientation of the molecules aswell as their immunological activity, optimizing thesensitivity of the immunosensor. Such complexestested in sandwich immunoassays conducted todetect cardiac troponin I showed a fivefold higheractivity over the control [141].
The sensitivity of these assays is generally lim-ited by the ratio of label (fluorescent or enzyme)molecules per biomolecule (L/P ratio). The L/Pratio is typically 4–8 for a conventional, cova-lently coupled fluorescent immunolabel, forexample, an IgG labeled with fluorescein isothio-cyanate (IgG-FITC) conjugate. A higher L/P maylead to a decrease of the specific binding affinityof the biomolecule, and additionally cause self-
quenching effects [142]. Increasing the effectivedye/biomolecule ratio while minimizing dye self-quenching and maintaining the biomolecule’sbinding properties is thus an important goal inassay development. Several ways to increase theF/P ratio have been investigated, in particularthose which are based on coprecipitation or self-assembly without the formation of covalentbonds. One approach has been the substitutionof labeling molecules by micro- or nanocrystal-line dyes. For example, perylene, a fluorescentpolycyclic aromatic hydrocarbon consisting oftwo molecules of naphthalene that have beenfused together, has been used as label. A higherF/P ratio can be obtained by precipitating fluor-escent perylene microparticles in the presence ofthe antibodies. After the immunoreaction, a largenumber of fluorescent molecules contained inthese particles can then be dissolved ina suitable solvent for detection. An analogousroute was to link antibodies to polyelectrolyteencapsulated microcrystalline fluorescent mate-rial [143]. The surface of these particulate struc-tures is typically engineered by the layer-wiseassembly of oppositely charged polyelectrolytes,the outer layer consisting of biorecognition mole-cules, for example, immunoglobulins. Because ofthe exceptionally high F/P ratio of the detectionantibodies, a dramatically amplified immunoas-say was achieved. Despite the advantages of thesetwo methods, a key limitation lies in therestricted number of materials that can be pre-cipitated or crystallized for encapsulation. Giventhe limitations of existing fluorescence-based bio-chemical assays, the development of new strate-gies and biolabeling systems will be necessary.
A novel signal amplification technology based ona new class of biofunctional fluorescent nanocrys-tals has been developed, consisting of a two-stepapproach to encapsulate the fluorogenic precursorfluorescein diacetate (FDA) nanocrystals followedby conjugation of the antibody [144]. Figure 7shows the overall process of amplification technol-ogy. Distearoylphosphatidylethanolamine modifiedwith PEG (2000) amine is coated on the surface ofthe FDA nanocrystals to provide an interface forantibody coupling. Anti-mouse antibodies are sub-sequently attached to the nanocrystalline FDA bio-labels by adsorption.
346 K. KHALID ET AL.
A high molar ratio of fluorescent molecules tobiomolecules is achieved in this nanocrystal biola-bel system. The analytical performance of thenanocrystal-based label system has been evaluatedin a model sandwich immunoassay for the detec-tion of mouse IgG. The high sensitivity of thisassay may be explained by the boosting effect ofthe high ratio of dye/antibody, but washing out thedye molecules after the affinity reaction may alsocontribute to improve the signal, the mean dye-to-dye distances being large enough to diminish thequenching effects.
Other high-load systems have been successfullydesigned and used as immunolabels such as fluores-cent conjugated dendrimers, fluorophore-loaded latexbeads, and liposome-encapsulated fluorophores. Forexample, liposomes used in immunodetection anddrug delivery systems can be tagged with antibodiesto form immunoliposomes. Numerous proceduresfor the conjugation of antibodies to liposomes havebeen developed, falling into four general categoriesdefined by the particular functionality of the antibodybeingmodified, namely aminemodification, carbohy-drate modification, disulfide modification, and non-covalent conjugation. Interestingly, studies showedinserted a monosialoganglioside (GM1), which exhi-bits a specific affinity toward cholera toxin (CT), intothe phospholipid bilayer during the liposome synth-esis [146]. These GM1-sensitized, sulforhodamineB dye-entrapping liposomes were then used for thedetermination of CT. Subsequently, this group
reported the successful preparation of biotin-tagged,carboxyfluorescein-encapsulated liposomes by usingthe reversed-phase evaporation method from a lipidmixture containing biotin-X-DHPE. Such liposomeshave been successfully used to improve biosensingsystems. Carboxy-encapsulated fluorescein biotin-tagged liposomes were used as a novel alternativeanalytical method for the detection of low concentra-tion of biotin.
4.8. Nanoparticles and the fundamental study ofcell adhesion mechanisms
Adhesion is a highly important and fundamentalphenomenon in biology. Living cells are endowedwith different receptors expressed at the plasmamembrane that allow the continuous perceptionof the extracellular environment. These ubiqui-tously present receptors are quite diverse in func-tion and include, in particular, receptors thatanchor cells to the extracellular matrix (integrins)or those involved in cell–cell interactions (such asselectins or cadherins).
The study of cell adhesion has to be tackledusing multiple approaches, from molecular, devel-opmental, or cell biology to biophysics [147]. Thefield of the study of cell adhesion is thus, bynature, multidisciplinary, involving a large numberof research groups producing an ever-increasingnumber of publications as our understanding ofthis complex process grows.
Figure 7. Overall process of amplification technology [145].
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Intercellular contacts, created by morphologi-cally distinct structures, are made of the clusteringof cell-surface transmembrane adhesive receptorsinto multiprotein assemblies, or junctions, con-nected to the cytoskeleton and intracellular signal-ing pathways [148]. Numerous molecules areinvolved which regulate various mechanisms suchas differentiation, migration, or apoptosis.
Adherens junctions consist of complex intercel-lular structures formed by localized clusters of transdimers between classical cadherins from apposedcells. Cadherins are single-pass transmembrane gly-coproteins and signal transducing moleculesinvolved in Ca2þ-dependent homophilic cell–celladhesion. During development, cadherins contri-bute to the regulation of a large number of pro-cesses, including tissue morphogenesis such asmesenchymal–epithelial transition, cell sorting andtissue rearrangement through convergence exten-sion, neurite elongation, and synaptogenesis. Inaddition, deregulation of cadherin-mediated adhe-sion has been associated with alterations of tissuehomeostasis. Thus, cadherins are key morphoregu-latory molecules in developmental processes, as wellas essential contributors to cell–cell cohesion withinadult tissues and organs [149]. A comprehensiveview of cadherin recruitment and dynamics atcell–cell contacts and its regulation is of majorimportance for the understanding of the controlof cell fate in normal as well as pathological situa-tions, such as carcinogenesis.
Classical cadherins (type I and type II) consistof an extracellular segment typically containingfive tandem repeats of an approximately 110-amino acid module, numbered EC1 to EC5 fromthe outermost domain, a transmembrane region,and a highly conserved cytoplasmic domain.Cadherin engagement triggers a series of stillonly partially understood intracellular signalingevents that lead to the reorganization of the actincytoskeleton via cytoplasmic proteins such as cate-nins, plakoglobin, and p-120 that, in turn, triggerchanges in cell morphology and motility [150].The formation of adherens junctions is likely torepresent the first step in this signaling cascade.
Despite detailed studies of cadherin-mediatedadhesion in multicellular organisms, the molecularunderstanding of the adhesive states of cadherin isless clear. Cadherin expression is cell type or tissue
specific, and a cell type may express more than onetype of cadherin. Cells expressing cadherins sortout and aggregate only with cells expressing iden-tical cadherins. This is the basis of tissue pattern-ing and architecture in both cell-to-cell contactand cell migration. How these molecules interactwith each other and the mechanisms by whichthey transfer specific intracellular signals remainpoorly understood.
Structural studies have shown that cadherin–cadherin contacts are mediated by the cadherinextracellular domain. A detailed structural descrip-tion of adherens junctions is emerging from theelucidation of the structure of individual molecu-lar partners. However, despite numerous studiesover the past 25 years, the details of trans dimer-ization are still under debate. The crystal struc-tures of the entire (EC15) ectodomain fromclassical type I C-cadherin and more recently E-and N-cadherins reveal a ‘strand swap’ trans inter-face in which the N-terminal b-strand from theEC1 domain of each paired cadherin exchangeswith that of the partner molecule. A second func-tionally important trans interface, involving thelinker region between the EC1 and EC2 domains,has also been identified and constitutes a kineticintermediate on the path to the formation ofstrand swapped dimers. However, a widelyaccepted model as summarized in recent reviewarticles is that the functional unit of cadherinadhesion is a cis dimer formed by binding of theextracellular domains of two cadherins on thesame cell surface [151]. The interplay betweentrans binding and lateral cis interactions amongproteins on the same membrane theoretically playsa crucial role in the clustering of cadherins intojunctions, but evidence for these trans- and cis-cadherin-binding states remains controversial.
4.9. Polymeric beads
4.9.1. Bead–bead and bead–cell assaysCell aggregation assays were often used in earlystudies to evaluate the role of cadherins on cell–celladhesion. Such studies are challenging in live cells,and this approach provides mostly qualitative data asit is difficult to take into account the number ofcadherin molecules involved. Moreover, cadherinsare multimodular and involved in complex
348 K. KHALID ET AL.
multimolecular structures, making it difficult to elu-cidate their biological properties. For this reason,glass and polymeric NPs have been often used instandard assays to assess the biological activity ofcadherin recombinant fragments. Fluorescent pro-tein A-coated 0.9 mM polystyrene beads functiona-lized with chimeric C-cadherin fragments andstreptavidin-coated 2.8 mm polystyrene beads deco-rated with E-cadherin fragments were used toaddress the involvement of the different ectodomainmodules in bead aggregation assays. Figure 8 revealsthe bead-based assay principles with regard to thecell aggregation.
Bead–cell-binding assays have also been used todissect the molecular mechanisms of signal trans-duction [153]. N (neural)-cadherin chimera-loadedlatex beads of 6-mm diameter self-aggregate andspecifically bind, in an aCa2þ-dependent manner toN-cadherin-expressing cells. N-cadherin full-lengthchimera-coated beads fully mimic cadherin-mediated cell–cell interactions, inducing the accu-mulation of N-cadherin, catenins, and F-actin as wellas membrane remodeling at the bead–cell contact.Streptavidin spherical polystyrene beads (2.8 mm indiameter) decorated with E/EC12 have been shownto efficiently interact with HC11 cells and activatemembrane dynamics events. Indeed, subsequent tothese contacts, the beads were rapidly engulfed by
the E-cadherin-expressing cells, and this internaliza-tion appears to be highly specific and sensitive topoint mutations [154].
4.9.2. Single-molecule assaysMost approaches used to study cadherin–cadherininteractions provide information from the beha-vior of multimolecular systems with an oftenincompletely defined geometrical organization.Many contradictory results and unanswered ques-tions suggest that it would be a hopeless task toderive clear molecular properties from these data.An understanding of the intrinsic kinetic proper-ties of cadherin interactions requires the measure-ment of parameters at the single-molecule level.During the past 15 years, new biophysicalapproaches have been developed that allow thestudy of ligand–receptor interactions with unpre-cedented accuracy, down to the single bond level[155]. Reported results include information onbond mechanical properties, association behaviorsof surface-attached molecules, and the dissectionof energy landscapes and reaction pathways.Indeed, monitoring single bond formation anddissociation has made it possible to bypass difficultproblems such as force sharing between multiplebonds or assessing the effect of partial geometricalmatches on the kinetics of bond formation [156].
Figure 8. Bead-based assay [152].
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Several of these methodologies have been used tostudy cadherin interactions at the molecular level.A surface force apparatus has been used to inves-tigate the mechanisms of cadherin binding bymeasuring the force/distance between cadherinectodomains. Single-molecule atomic force micro-scopy (AFM) was used to study the mechanicalresistance of cadherin interactions. However,interpretation of the data was not straightforward:the relationship between the unbinding force, asmeasured by AFM, and the dissociation rate iscomplex and dependent on the cantilever stiffnessand rate of sample displacement [157]. At thepresent time, these experiments have yet to giveus a comprehensive view of the mechanismsunderlying cadherin interactions.
Two approaches, the flow chamber and theBiomembrane Force Probe (BFP), offer a way tobring into contact two surfaces covered with mole-cules and a means of measuring the duration of theinteraction as well as the rupture force under stress.The molecules to be tested are linked in the case ofthe BFP approach onto two micron-sized glassbeads, whereas in the flow chamber approach,they are linked onto a polystyrene bead and a flatsurface [158]. These methodologies allow the ana-lysis of the association of surface-attached ratherthan soluble molecules reproducing a molecularorientation relevant to physiological conditions,highly sensitive detection of molecular interactions,and the kinetic study of bond formation and dis-sociation, under stress. These two approaches pro-vided the first quantitative data describing thedissociation kinetics of individual E-cadherin andC-cadherin interactions.
5. Conclusion and perspective
Nanomaterials, notable for their extremely smallfeature size, have the potential for wide-rangingindustrial, biomedical, and electronic applications.They are excellent adsorbents, catalysts, and sensorsdue to their large specific surface area and highreactivity. NPs play an important role in many bio-technology applications and promise to take centerstage for many new and emerging applications inthe coming years. Interesting future developmentsinclude not only biomedical applications such asimproved delivery of drugs to tumor cells and the
use of dendrimers for regenerative medicine but alsofields such as water purification and disinfection,food production, and packaging. It will be attractiveto find a simple, efficient, and controllable way toproduce nanomaterials in mass production as wellas to bridge their applications in optoelectronics.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Yeek-Chia Ho http://orcid.org/0000-0002-2820-9696Jun Wei Lim http://orcid.org/0000-0003-0158-8822Lai Chin Wei http://orcid.org/0000-0002-7549-5015
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