Presentation on theme: "of a protein immobilized on a Qdot surface"— Presentation transcript:
1 There is certainly a lack of experimental tools with which to discern the orientation of a protein immobilized on a Qdot surfaceFor specific targeting, it is highly desirable that the delivery proteins (e.g. antibody) areproperly oriented and fully functionalTherefore, Qdot bioconjugation step is extremely important in obtaining success in bioimaingGold NanoparticlesFor over 30 years, nanometer-sized gold particles have been used to stain cells and tissuesamples for electron microscopyThe basic principle of interactions between gold particles and biomolecules, like proteins, hasbeen well studied for immunocytochemical staining applicationsAlthough nanosize metals like gold and silver do not fluorescence they can effectively scatterlight due to the collective oscillation of the conduction electrons induced by the incidentelectric field (light)This is known as “surface plasmon resonance”Thus, colloidal gold particles exhibit a range of intense colors in the visible and NIR spectralregionsSurface plasmons, also known as surface plasmon polaritons, are surface electromagnetic wavesthat propagate in a direction parallel to the metal/dielectric (or metal/vacuum) interfaceSince the wave is on the boundary of the metal and the external medium (air or water for example),these oscillations are very sensitive to any change of this boundary, such as the adsorption ofmolecules to the metal surface
2 The excitation of surface plasmons by light is denoted as a surface plasmon resonance (SPR) for planar surfaces or localized surface plasmon resonance (LSPR) for nanometer-sizedmetallic structuresThis phenomenon is the basis of many standard tools for measuring adsorption of material ontoplanar metal (typically gold and silver) surfaces or onto the surface of metal nanoparticles.It is behind many color based biosensor applications and different lab-on-a-chip sensors.In the Otto setup, the light is shone on the wall of a glass block, typicallya prism, and totally reflected. A thin metal (for example gold) film ispositioned close enough, that the evanescent waves can interact with theplasma waves on the surface and excite the plasmonsIn the Kretschmann configuration, the metal film is evaporated onto theglass block. The light is again illuminating from the glass, and anevanescent wave penetrates through the metal film. The plasmons areexcited at the outer side of the film. This configuration is used in mostpractical applications.Surface plasmons have been used to enhance the surface sensitivity of several spectroscopicmeasurements including fluorescence, Raman scattering, and second harmonic generation.However, in their simplest form, SPR reflectivity measurements can be used to detect DNA orproteins by the changes in the local index of refraction upon adsorption of the target molecule tothe metal surface.
3 Gold nanoparticle have high absorption and scattering cross section For example, the absorption cross section of a 5 nm diameter gold particle is about 3 nm at awavelength of 514 nm, which is about two orders of magnitude higher than that of organicfluorescent molecules at room temperatureScattering cross section of gold nanoparticle is much larger than polymeric spherical particlesof similar size, especially in the red region of the spectrum, i.e. red to NIR range, havingpotential in deep tissue imagingFor example, using composite core-shell gold (dielectric silica core and gold shell) particles,it is possible to tune the scattering from 600 to 1200 nmDue to excellent biocompatibility, gold nanoparticles have been widely used inimmunohistochemistry (gold-based staining) and in ultra-sensitive DNA detection assaysHowever, a few literature reports are available on gold nanoparticle-based cancer imagingGold nanoparticles, because of their strong SPR properties, have attracted considerable attentionin bioimaging in recent yearsThe SPR signal originates from the collective oscillation of conduction electrons uponinteraction with absorption photonsSPR frequency depends on various factors, particle size, shape, dielectric properties, aggregatemorphology, surface functionalization, the refractive index of the surrounding medium
4 (a) Gold Nanoparticles Synthesis Various methods have been reported for the synthesis of gold nanoparticlesThere are two general approaches (so-called “top-down” and “bottom-up”) that primarilycategorize most reported synthesis strategiesSynthesis of gold nanoparticles by employing laser ablation technique is an example of a“top-down” approach, where the embryonic (nascent) partcles are formed from the ionizedgold atoms via nucleation and growth processesThe challenge still remains how to stabilize particles in the solution phaseUsing a surfactant-based capping agent (sodium dodecylsulfate, an ionic surfactant), Kondowet al. have successfully stabilized ultrafine particlesThe capping agents, in general, control the particle size and size distribution,prevent aggregation, and stabilize particle solution (such as in aqueous-based medium)
5 ablation of a metal surface immersed in liquid could produce nanoparticles of the metal in the liquidgas-phase metal clusters are most conviently preparedby laser ablation (In liquid phase, metal atoms are justejected from metal surface)Metal atoms are ablated from a metal rod by laserirradiation and are aggregated into metal clusterswith a sufficiently larger sizesSelf-aggregation of the nanoparticles suspended in theliquid should be prevented by hindering directcontact of the nanoparticlesFor example, Takami and co-workers have preparedsmall metal clusters by laser ablation of a metal platein liquid helium, where the clusters are encapsulatedin helium bubblePreparation of stable nanoparticles in a solution containing a surfactant by use of laser ablationon a metal plate immersed in the solutionThe surfactant which surrounds each nanoparticles prevents direct contact of the nanoparticles- Sodium dodecyl sulfateSDS
6 II. Particle Growth and Aging: +Ascorbic Acid+Au3+Ag++Ag+Ag+Au3+ReductionAg+MicellesCTAB++AgingParticle growth+ NaOHSeeding+Color Green+210C, 12 h350C, 5 min210C, 24 h+Color Red
7 Preparation of Spherical Nanopaerticle: In the “bottom up” approach, gold nanoparticles and gold nanocomposites (composite of goldand silica) have been chemically synthesized by reducing gold precusors. Various reductionmethods have been reported. The major synthesis is as followsi) Reduction of gold precusors (e.g. hydrochloroauric acid HAuCl4) using appropriate reducingagents, such as citrate, sodium borohydride, ascorbic acid.The citrate reduction of gold (III) ions has been widely usedWhile sodium citrate reduces (AuCl4)- ions in hot aqueous solution, it forms a colloidThe reported average particle size is about 20 nmPreparation of Spherical Nanopaerticle:EFTEM image
8 Both the citrate ions and the oxidation (e. g Both the citrate ions and the oxidation (e.g. acetone dicarboxylate) act as capping agents.- reduced gold is not stable- sulfur or oxygen capping such as citrate anion stabilize the reduced gold by providingelectrons to the reduced goldIn conjunction with citrate ions, amphiphilic surfactants have also been used that allowed particle size tunning upon varying the gold/stabilizer ratio+MicellesCTABReductionAgingParticle growthAg+Ascorbic AcidSeedingColor GreenNaOH
9 ii)Two-phase synthesis of gold nanoparticles has been reported in which a phase-transfer agent is used to transfer [AuCl4]- ions from an aqueous phase to an organic phase (toluene)containing alkanethiol stabilizerThe Au(III) in organic phase is reduced by the addition of aqueous sodium borohydride.Resulting Au clusters are then capped immediately by alkanethiolsThis method (Brust-Schiffrin) produces monodisperse particles (approx 1.4 nm) in thediameter range nm
10 (b) Surface Functionalization and Bioconjugation gold nanoparticles have been widely used as immunostaining agents for labeling cell, tissuesection, blots, etc.In general, protein conjugated gold nanoparticles are mostly used as labeling probesAlthough the actual mechanism of macromoleculs (e.g. protein) binding to gold particles ispoorly understand, some of accepted mechanism areProtein binding via electrostatic (ionic) interaction. Negatively charged gold nanoparticles canbind to positively charged protein domains via electrostatic interactionProtein binding via hydrophobic interaction. Hydrophobic domains present in the proteinstructure can interact with the metal surface of the particleProtein binding via chemical interaction. Protein molecules containing sulfohydryl (-S-H-)can chemically interact with the gold atoms. This is also called dative bindingOther biomolecules, such as protein A, antibodies, lectins, avidins (or streptavidins), etc. havealso been conjugated to gold nanoparticles to be used as sensitive probes.(c) Protein-A Gold ConjugateProtein A-gold conjugates are generally prepared by adsorbing protein-A onto the gold surfaceFollowing a similar method, many other immunoglobulin binding proteins can also be attachedto gold nanoparticles (Protein A’s ability to bind immunoglobulins)Protein A is a kDa MSCRAMM surface protein originally found in the cell wall ofthe bacteria Staphylococcus aureusIt has found use in biochemical research because of its ability to bind immunoglobulinsIt binds proteins from many of mammalian species, most notably IgG’s
11 - It binds with the Fc region of immunoglobulins through interaction with the heavy chain These probes have been used as “universal” probes for labeling cells, tissue sections andvarious blotsIn a typical tissue labeling experiment, primary antibodies are specifically targeted to thetissue antigens- In the following step, protein A-gold conjugates bind to the antibodiesThe advantage of this labeling technique is that the same protein A-gold conjugate can beused for various immunochemical procedures(d) Antibody-Gold ConjugateAntibody-gold conjugated probes are prepared by coating antibodies directly onto thegold nanoparticle surface.These probes have been successfully used for the detection, localization and quantificaitonof antigens on the target specimensThis is powerful technique for detection of pathogens, intracellular foreign substances,monitoring cellular metabolic processes, etc.
12 lectin lectin lectin lectin (e) Lectin-Gold ConjugateLectin-coated gold nanoparticle probes have been used for the detection of sugar-bindingreceptors that are expressed on the cell membranesLectins are sugar-binding proteins which are highly specific for their sugar moietiesThey typically play a role in biological recognition phenomena involving cells and proteinsLectin molecules have specific carbohydrate binding sites.In this assay, a specific carbohydrate molecule is sandwiched between the lectin molecule andthe cellular receptorThe objective of this assay is to localize glycoproteins (proteins that contain oligosaccharidechains), glycolipids (carbohydrate-attached lipids), etc. on cell surfaces(f)Avidin (or streptavidin)-Gold ConjugateAvidin-gold conjugated probes have been used to localize, detect and quantify biotin moleculeslectinGoldlectinGoldlectinGoldlectinGoldSugarSugar bindingreceptor
13 Surface Plasmon Resonance Scattering and Absorption of anti-EGFR Antibody Conjugated Gold Nanoparticles in Cancer Diagnostics: Applications in Oral Cancer Ivan H. El-Sayed, Xiaohua Huang, Mostafa A. El-Sayed Nano Lett., 5 (5), , 2005.
14 AbstractSPR scattering imaging or absorption spectroscopy generated from antibody conjugated gold nanoparticles can be useful in molecular biosensor techniques for the diagnosis and investigation of oral epithelial living cancer cells in vivo and in vitro.Comparisons of cells without gold nanoparticles,with colloidal gold nanoparticles,With gold nanoparticles conjugated to monoclonal anti-epidermal growth factor receptor (anti-EGFR) antibodiesColloidal gold nanoparticles are found in dispersed and aggregated forms and provide anatomic labeling information, but their uptake is nonspecific for malignant cells.The anti-EGFR antibody conjugated nanoparticles specifically and homogeneously bind to the surface of the cancer type cells with 600% greater affinity than to the noncancerous cells.
15 IntroductionQuantum dots are widely used and studied due to their unique size-dependent fluorescence properties. (potential human toxicity and cytotoxicity of the semiconductor material)Alternative consideration: Colloidal gold nanoparticlesThe ability of gold nanoparticles : resonantly scatter visible and near-infrared light upon the excitation of their surface plasmon oscillation. The scattering light intensity is extremely sensitive to the size and aggregation state of the particles.They scatter light intensely, much brighter than chemical fluorophores.They do not photobleach and they can be easily detected in as low as10-16 M concentration.
16 Introduction The advantage of gold nanoparticles ease of preparation ready bioconjugationpotential noncytotoxicityScattering images and the absorption spectra recorded from anti-EGFR antibody conjugated gold nanoparticles incubated with cancerous and noncancerous cells are very different and offer potential techniques for cancer diagnostics.
17 MethodThe preparation of Gold NPs : the citrate reduction of chloroauric acid.The preparations of the anti-EGFR/gold conjugates :Dilution of the gold NPs in 20 mM HEPES buffer (pH 7.4) to a final concentration with optical density of 0.8 at 529 nm. .Adding of 40 uL anti-EGFR monoclonal antibodies (host mouse) to 960 uL of the same HEPES buffer to form 1 mL dilute solution.Mixing of 10 mL of the gold solution with the dilute antibody solution for 20 min.Adding of 0.5 mL of 1% poly(ethylene glycol) to the mixture to prevent aggregationCentrifugationRedispersion of the anti-EGFR/gold pellet in PBS buffer (pH = 7.4)
18 Method * Incubation of gold nanoparticles The cover slips were coated with collagen type I in advance for optimum cell growth.One nonmalignant epithelial cell line HaCaT (human keratinocytes)two malignant epithelial cell lines HOC 313 clone 8 and HSC 3 (human oral squamous cell carcinoma) were cultured on glass cover slips in DMEM plus 5% FBS at 37 C under 5% CO2.For the incubation of colloidal gold, nanoparticles (~ 0.3 nM) were added into the medium and the cells were grown for 48 h.The cells on the cover slips were rinsed with PBS buffer and fixed with 1.6% paraformaldehyde and sealed.For the incubation of conjugated nanoparticles, the cells were grown on the cover slips for 48 h and then the cell monolayer was immersed into the conjugated nanoparticle solution for 40 min, rinsed with PBS buffer, fixed with paraformaldehyde, and sealed.
19 MethodThe light scattering images were taken using an inverted Olympus IX70 microscope.When the light beam direction is optimized, the center illumination light beam does not enter the light collection cone of the microscope objective, and only the scattered light of the side beam by the sample is collected.Gold nanoparticles are introduced into cells by the endocytosis process during cell differentiation and proliferation processes.Smaller nanoparticles cross the cytoplasmic membrane more easily, but their scattering light cross-section is smaller than larger nanoparticles. They also give more greenish scattered color which cannot be easily resolved from the scattered green light from the cellular organelles.Larger nanoparticles have higher scattering cross-section but have smaller labeling efficiency, possibly due to steric hindrance.After experimental determination of the particle uptake efficiency,the cellular labeling efficiency, and the light scattering intensities of the nanoparticles, gold nanoparticles with the average sizes of 35 nm was selected.
20 Results For the size : HOC cancer cells are almost four times Diamond shapeDiamond shapeFor the size :HOC cancer cellsare almost four timeslarger than HaCaT or HSC.For the shape : HaCaT and HSC cells show almost homogeneous diamond shapeswhile HOC cells have other shapes for some cells.Figure 1. Light scattering images of HaCaT noncancerous cells, HOC cancerouscells , and HSC cancerous cells without gold nanoparticles.Dim greenish light : This green light is due to autofluorescence and scattered lightfrom the cell organelles in cell cytoplasm and membrane.The weak greenish scattered light from the cells shows large difference in the sizesand shapes of the three different types of cells.
21 Figure 2 Light scattering images of one noncancerous two cell cancerous cells after incubation with unconjugated colloidal gold nanoparticles.The incorporated gold nanoparticles scatter strong yellowish light and makeindividual cells easily identifiable.The images show that the particles are inside the cells in the cytoplasm regionbut do not seem to adsorb strongly on the nuclei of the cells.In most HaCaT noncancerous cells the gold nanoparticles demonstrate aspotted pattern inside the cytoplasm, while the nanoparticles are homogeneouslydistributed in the cytoplasm of HOC and HSC cancerous cells.The HSC specimens give the strongest scattering light due to the large amount ofaccumulated gold nanoparticles.
22 Absorption maximum around 548 nm Figure 2. The absorption spectra of one noncancerous two cell cancerous cells after incubation with unconjugated colloidal gold nanoparticles.Nanoparticles have an SPR absorption maximum around 548 nm, independent of the cell type.The NPs inside all cells have a major peak around 545 nm, characteristic of the surface plasmon absorption of the individual nanoparticles inside the cytoplasm of the cells that are red shifted by 16 nm compared to the colloid nanoparticle suspension at 529 nm.This suggests that the nanoparticle surface has a different dielectric environment when present inside the cells.The broad long wavelength tails: the presence of aggregates, which is likely induced by the saltsSPR absorption : red : larger particle: aggregationblue: smaller particle
23 * Absorption maximum around 548 nm Figure 2. The absorption spectra : nanoparticles have an SPR absorption maximum around 548 nm, independent of the cell type.The capping material could also be dissolved inside cells and thus leads to aggregation of the resulting metallic nanostructures.In HSC cells, the aggregates have the absorption maximum around 715 nm. In HaCaT cells, the size of these large aggregates is smaller as concluded from the shorter wavelength surface plasmon absorption maximum.The absorption of the aggregates inside HOC is not as resolved due to the shorter wavelength (679 nm), which is close to the absorption maximum of the surface plasmon absorption of the individual nanoparticles.The different sizes of the aggregates inside different kind of cells may reflect the difference in the cell cytoplasm medium or differences in the intracellular processing of the nanoparticles by the cells.The ability to resolve aggregates within cells by SPRA spectroscopy suggests that different capping agents could be utilized to monitor intracellular processes as aggregates are formed.
24 Figure 3. Light scattering images of cells after incubation with anti-EGFR antibody conjugated gold nanoparticles. The conjugated nanoparticles bind specifically with high concentrations to the surface of the cancer cells.The light scattering pattern of gold nanoparticles is significantly different when anti-EGFR antibodies were conjugated to gold nanoparticles before incubation with the cells.The HaCaT noncancerous cells are poorly labeled by the nanoparticles and the cells could not be identified individually. The nanoparticles are also found on the HaCaT noncancerous cells due to part of the specific binding, but mostly due to the nonspecific interactions between the antibodies and the proteins on the cell surface, and thus the nanoparticles are randomly distributed.When the conjugates are incubated with HOC and HSC cancerous cells for the same amount of time, the nanoparticles are found on the surface of the cells, especially on the cytoplasm membranes for HSCcancer cells.- This contrast difference is due to the specific binding of overexpressed EGFR on the cancer cellswith the anti-EGFR antibodies on the gold surface.The nonspecific interaction between the anti-EGFR antibodies and the collagen matrix also exists,which is shown as the reddish scattering light of the gold nanoparticles on the collagen background.
25 Figure 3. Absorption spectra of cells with anti-EGFR antibody conjugated gold nanoparticles. Conjugated nanoparticles did not show aggregation tendency (no long wavelength broad tail is observed).0.08545 nm543 nm552nm0.06545 nm0.01The nanoparticles bound to HOC and HSC cancer cells have similar absorption maxima at around 545 nm, which is 9 nm red shifted compared to the isolated anti-EGFR/Au solutions at 536 nm.Red shift: the specific binding of the anti-EGFR antibodies on the gold surface to EGFR on the cell surface : the interparticle interaction resulting from the arrangement of the conjugates on the cell surface in two dimensions.For HaCaT noncanerous cells, the particles with maximum at 545 nm are found to have a maximum absorption of 0.01.- The rest of the nanoparticles have their maximum at 552 nm. This red shift indicates that these nanoparticles are nonspecifically bound.The maximum absorbance of the conjugated particles to cancer cells is 0.06 for HOC cells and 0.07 for HSC cells.Binding ability of the anti-EGFR antibody conjugated nanoparticles to HOC and HSC cancerous cells is 600% and 700%, respectively, over the HaCaT noncancerous cells.
26 ConclusionsThere is a distinct difference in the distribution of anti-epidermal growth factor receptor antibody conjugated nanoparticles when incubated with cancerous and noncancerous cells.Conjugated nanoparticles bind homogeneously and specifically to the surface of the cancer cells with an absorption maximum at 545 nm.Both SPR scattering imaging and SPR absorption spectroscopy from anti-EGFR antibodies conjugated gold nanoparticles are found to distinguish between cancerous and noncancerous cells. This makes either technique potentially useful in cancer diagnostics.
28 188.8.131.52 Dye-doped Silica Nanoparticles Amorphous silica nanopartcles that are produced via stober’s sol-gel or microemulsion techniquefound in application in bioimagingUnlike Qdots or gold nanoparticles, silica does not have inherent strong fluorescence that can beexploited for sensitive imaging applicationsHowever, silica nanoparticles can be made fluorescence by incorporating fluorescent dyemolecules inside the silica matrix (dye-doping)Another approach could be attaching fluorescent dye molecules (via covalent binding) on thesilica surfaceFor bioimaing applications, it is preferable that dye molecules remain encapsulated bythe silica matrix for the following reasonsSilica-based nanoparticles exhibit several attractive features, e.g. silica is water dispersible andis resistant to microbial attackThe size of silica particles remains unchanged by changing solvent polarity (i.e., resistant toswelling) and therefore, silica porosity remains unaltered in a wide selection of solvents,including aqueous-based neutral and acidic solutions.A silica matrix is optically transparent, allowing excitation and emission light to pass throughefficientlyFluorescent dyes can be effectively entrapped inside the silica particles.spectral characteristics of dye molecules remain almost intactsilica encapsulation provides a protective layer around dye molecules, reducing oxygen moleculepenetration (which causes photodegradation of dye molecules) both in air and in aqueous medium.As a result, photostability of dye molecules increases substantially compared with bare dyes insolution
29 using conventional silane based chemistry surface of a silica nanoparticle can be easily modified to attach biomolecules such as protein…using conventional silane based chemistryFor exapmple, carboxylated silica nanoparticle can be covalently attached to the amine groupsof proteins, antibodies etc. through the formation of stable amide bondOligoglutaraldehydeNH2(CH2)3SiONH2(CH2)3SiOC oAPTESOligoglutaraldehydeNH(CH2)3SiOAmide bondSilicon oxide surfaceAnti-E.coli O157:H7 antibody( -NH2 )general synthesis strategy of fluorescent silica nanoparticles is the incorporation of organicof organic or metalloorganic dye molecules inside the silica matrixMetalloorganic dye, tri(2,2’-bipyridyl)dichlororuthenium (II) (Rubpy), has been entrappedinside silica nanoparticles using a reverse microemulsion-based synthesis approach wherepositively charged Rubpy moleculecs were electrostatically bound to the negatively chargedsilica matrix
30 SynthesisSol-gel, reverse microemulsionStober’s methodIn a typical stober’s method, Alkoxysilane compounds, tetraethylorthosilicate (TEOS) ,tetramethylorthosilicate (TMOS),or various TEOS or TMOS derivatives, etc undergo base-catalyzed hydrolysis andcondensation in an ammonia-ethanol-water mixtureFollowing Stober’s protocol with a slight modification, fairly monodisperse organic dyedoped fluorescent silica nanoparticles have been synthesizedSince organic dyes are normally hydrophobic, doping them inside the hydrophilic silicamatix is not straightforward.typically, a reactive derivative of organic dye(e.g. amine-reactive fluorescein isothiocyannateFITC, carboxyl group) is first reacted with an amine-containing silane compound (APTS)- Then FITC conjugated APTS and TEOS are allowed ot hydrolyze and condense to formFITC conjugated silica nanoparticle- Note that particles so formed will have some amount of bare dye molecules on the particlesurface that is covalently attached- These bare dyes, due to their hydrophobic nature, will somewhat compromise the overallparticle aqueous dispersibility and also they will be prone to photobleaching- therefore, an additional coating with pure silica is usually applied around the dye-conjugatedsilica nanoparticle
31 Surface treatment of silicon chips with APTES-glutaraldehyde a . APTES reacts with silica leaving a primaryamine group on the surfaceOligonucleotide immobilization on plasma-cleaned silicon nitride surfaceSurface treatment of silicon chips with APTES-glutaraldehydeb. Glutaraldehyde treatment yields analdehyde that can form an imine linkagewith the primary amines on the proteinc. Reaction between the amine group on theprotein and the aldehyde group on thesurface attached are
32 Surface treatment of silicon chips with GOPS-CDI a) Treatment with GOPS forms an epoxy layerb) Reaction between hydroxyl groups of epoxy and 1, 1’carbonyldiimidazole (CDI) results in formation ofreactive imidazole carbamate groupsc) Imidazole carbamate groups react easily with aminecontaining ligand (such as avidin or biotin) forming astable carbamate linkage.Residual imidazole carbamate groups can behydrolyzed to hydroxyl groups with water or watersolutions at higher pH value.
33 (b) Surface Functionalization and Bioconjugation This surface modification involves a few steps.Firstly, the particle surface should be modified to obtain appropriate functional groups such asamines, carboxyls, thiols, etc.- secondly, using suitable coupling reagents, nanoparticles are attached to the bio-recognitionmolecules (such as antibodies, folate,..)Lastly, bioconjugated particles are targeted to cancersNote that all these steps are usually carried out in aqueous-based solutionsA few bioconjugation methods are briefly mentioned belowBioconjugation with carboxylated particles.- The surface of the nanoparticle is modified to obtain carboxyl groups (-COOH) by using acarboxylated silane reagent- Biomolecules such as proteins, antibodies, etc. containing free amine functional groups arethen covalently attached to the carboxyl functionalized nanoparticle,using carbodiimide-coupling chemistyii) Bioconjugation with aminated particles: Many cancer cells overexpress folate receptors.- Cancer targeting with folate-conjugated nanoparticle has been recently reported- Folates are chemically attached to aminated silica nanoparticles using carbodiimide chemistry- APTES treatment: aminated silica nanoparticle can be achieved
34 Attached with folic acid NH2(CH2)3SiOAPTESFolic acidSilica nanoparticleAttached with folic acidiii) Bioconjugation with avidin-biotin bindingAvidin-coated nanoparticles are typically attached to biotinylated molecules such as antibodies,proteins, etciv) Bioconjugation through disulfide bonding:Sulfohydryl-modified nanoparticles are conjugated to disulfide-linked oligonucleotidein this method, oligonucleotides are attached to nanoparticles through di-sulfide bond formationV) Bioconjugation using cyanogen bromide chemistrynanoparticles with hydroxyl groups (such as silica) can be activated with cyanogen bromideto form a reactive –OCN derivative of the nanoparticles.The OCN derivative then readily reacts with proteins (via amine groups), forming a“zero-length” bioconjugate as there is no spacer between the particle surface and the proteinmolecule
35 2.5 Optical imaging of Cancer with Nanoparticles These ultra-sensitive and specific probes (nanoparticles) provide a viable alternative to rapidlyand non-invasively image the uptake, distribution and binding of nanoparticles to tumorsTo establish the widespread use, it is important to understand the delivery, interaction andrecognition mechanism of these contrast agents with cancer cellsVarious delivery vehicles with varying specificity have been used to target cancer tissues,mainly for drug delivery applications, some of which are folates, antibodies, lectins, growthfactors, cytokines, hormones and low-density lipoproteinsmost of these carriers can be similarly used for molecular imaging applicationsThese can be broadly classified as active and passive targeting2.5.1 Active Targetingthis refers to the conjugation of targeting ligands to nanoparticles to provide preferentialaccumulation into the tumor antigents and blood vessels with high affinity and specificityThis relies on specific interactive forces between lectins-carbohydrate, ligand-receptorsand antibody-antigensLectins can recognize and bind to glycopreteins that occur on the surface of cells.- These proteins can bind to certain carbohydrates in a specific manner.- Direct and reverse lectin targeting have made used of this specific interactions to receptorsor antigens expressed by the plasma membrane
36 Folate receptor-based interactions are an excellent example of ligand-receptor based active targeting.Folate receptors are overexpressed on the surface of various cancers like those of the brain,ovary, kidney, breast and lungsConfocal microscopic studies have demonstrated the selective intake and receptor-medicatedendocytosis of folate-conjugated nanoparticles by tumor cells
37 Antibody-mediated tumor targeting has been performed for detecting the presence of antigenic moieties on the surface of cancer cellsTumor targeting ligands like monoclonal antibodies are attached to nanoparticles to targetthe specific receptors.These moieties are minimally present on the surface of normal tissuesOnly certain antigens are actually tumor-specific and are referred to as tumor-specific antigens