International Journal of
Molecular Sciences Review
Gold Nanoparticles: A Powerful Tool to Visualize Proteins on Ordered Mesoporous Silica and for the Realization of Theranostic Nanobioconjugates Marco Piludu 1, *, Luca Medda 2 , Maura Monduzzi 3 1 2 3
*
ID
and Andrea Salis 3, *
ID
Department of Biomedical Science, University of Cagliari, Monserrato, CA 09042, Italy Department of Chemistry, CSGI, University of Florence, Sesto Fiorentino, FI 50019, Italy;
[email protected] Department of Chemical and Geological Sciences, CSGI, University of Cagliari, Monserrato, CA 90042, Italy;
[email protected] Correspondence:
[email protected] (M.P.);
[email protected] (A.S.); Tel.: +39-070-675-4060 (M.P.); +39-070-675-4352 (A.S.)
Received: 21 June 2018; Accepted: 5 July 2018; Published: 8 July 2018
Abstract: Ordered mesoporous silica (OMS) is a very interesting nanostructured material for the design and engineering of new target and controlled drug-delivery systems. Particularly relevant is the interaction between OMS and proteins. Large pores (6–9 nm) micrometric particles can be used for the realization of a drug depot system where therapeutic proteins are adsorbed either inside the mesopores or on the external surface. Small pores (1–2 nm) mesoporous silica nanoparticles (MSNs), can be injected in the blood stream. In the latter case, therapeutic proteins are mainly adsorbed on the MSNs’ external surface. Whenever a protein-OMS conjugate is prepared, a diagnostic method to locate the protein either on the internal or the external silica surface is of utmost importance. To visualize the fine localization of proteins adsorbed in mesoporous silica micro- and nanoparticles, we have employed specific transmission electron microscopy (TEM) analytical strategies based on the use of gold nanoparticles (GNPs) conjugates. GNPs are gaining in popularity, representing a fundamental tool to design future applications of MSNs in nanomedicine by realizing theranostic nanobioconjugates. It may be pointed out that we are at the very beginning of a new age of the nanomaterial science: the “mesoporous golden age”. Keywords: ordered mesoporous materials; immunogold staining; gold nanoparticles; protein localization; nanomedicine; theranostics; nanobioconjugate
1. Introduction: Ordered Mesoporous Silica (OMS) Materials in Nanomedicine 1.1. The Beginning As long ago as 1990, when they were discovered [1], ordered mesoporous silica (OMS) materials drew the attention of most material scientists and engineers. Due to their singular structural properties [2], OMSs have been intensely studied in recent years for the design of new biomaterials. They are mainly characterized by ordered channels and cavities with uniform pores (2–50 nm) [3] (Figure 1). The high surface area makes these materials suitable for new and innovative applications. OMSs have largely been investigated for catalysis [4] and biocatalysis purposes [5,6] and have shown potential features to be employed in nanomedicine [3,7–9]. As a result of the development of strategic synthesis procedures OMS structural features can be modified and fitted to specific aims, extending enormously their range of applications.
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Figure 1. Transmission electron microscopy (TEM) images of Ordered mesoporous silica (OMS) Figure 1. Transmission electron microscopy (TEM) images of Ordered mesoporous silica (OMS) Figure 1. Transmission (TEM) images based of Ordered mesoporous silica (OMS) nanoparticles. (a) Originalelectron SBA-15microscopy materials and (b) MCM-41 mesoporous silica nanoparticles nanoparticles. (a) Original SBA-15 materials and (b) MCM-41 based mesoporous silica nanoparticles nanoparticles. (a) Original SBA-15 materials and (b) MCM-41 mesoporous silica nanoparticles (MSNs). Reprinted with permission from [10]. Copyright 2018,based Elsevier. (MSNs). Reprinted with permission from [10]. Copyright 2018, Elsevier. (MSNs). Reprinted with permission from [10]. Copyright 2018, Elsevier.
Their capacity to house into the pore channels specific therapeutic molecules have paved the Their capacity to house into the pore channels specific therapeutic molecules have paved the capacity to house into the pore channels specific therapeuticsilica molecules have paved systems the way way Their for the developing of innovative applications of mesoporous as drug delivery way for the developing of innovative applications of mesoporous silica as drug delivery systems for the developing of innovative applications mesoporous as drug delivery systemsmolecules [3,11–15]. [3,11–15]. OMS can be chemically modifiedof to load and silica release specific therapeutic [3,11–15]. OMS can be chemically modified to load and release specific therapeutic molecules OMS can be modified to load and release specific therapeutic according to according to chemically controlled conditions [16,17]. Biomedical application of OMS molecules is largely associated to according to controlled conditions [16,17]. Biomedical application of OMS is largely associated to controlled [16,17]. of OMStois their largely associated to theirSurface fate in their fate conditions in biological mediaBiomedical which, inapplication turn, is related surface properties. their fate in biological media which, in turn, is related to their surface properties. Surface biological media which, to their surface properties. Surface cytotoxicity, functionalization functionalization (Figure in2)turn, playsis arelated key role in determining biodegradation, and functionalization (Figure 2) plays a key role in determining biodegradation, cytotoxicity, and (Figure 2) plays through a key role in determining biodegradation, cytotoxicity, and biodistribution through biodistribution interactions which may be mediated by the macromolecules occurring in biodistribution through interactions which may be mediated by the macromolecules occurring in interactions which may be mediated by the macromolecules occurring in biological media [18–20]. biological media [18–20]. biological media [18–20].
Figure 2. Schematic representation of the sequential steps of functionalization process of MSN surface Figure 2. 2. Schematic Schematic representation representation of of the the sequential sequential steps steps of of functionalization functionalization process process of of MSN MSN surface surface Figure with specific biomolecules. MSN particles before (a) and after (b) functionalization process. with specific with specific biomolecules. biomolecules. MSN MSN particles particles before before (a) (a) and and after after (b) (b) functionalization functionalization process. process.
Important steps toward the development of targeted drug depot systems are the understanding Important Important steps steps toward toward the the development development of of targeted targeted drug drug depot depot systems systems are are the the understanding understanding of the mechanisms that regulate cell interaction of functionalized mesoporous silica nanoparticles of the mechanisms mechanisms that that regulate regulate cell cell interaction interaction of of functionalized functionalized mesoporous mesoporous silica silica nanoparticles nanoparticles of the (MSNs). Previous works have investigated by means of light microscopy the cellular uptake of MSNs (MSNs). Previous works have investigated by means of light microscopy the cellular uptake (MSNs). Previous works have investigated by means of light microscopy the cellular uptake of of MSNs MSNs in vitro. The strategy was to attach fluorescent dye molecules to the MSNs surface to visualize and in vitro. The strategy was to attach fluorescent dye molecules to the MSNs surface to visualize in vitro. The strategy was to attach fluorescent dye molecules to the MSNs surface to visualize and and locate the particles inside the cellular compartment [18,21–25]. Additional analyses through electron locate locate the the particles particles inside inside the the cellular cellular compartment compartment [18,21–25]. [18,21–25]. Additional Additional analyses analyses through through electron electron microscopy have highlighted, at ultrastructural level, the main morphological events that take place microscopy microscopy have have highlighted, highlighted, at at ultrastructural ultrastructural level, level, the the main main morphological morphological events events that that take take place place during cellular MSN internalization [18,25]. It was shown that cellular uptake of MSNs is strictly during during cellular cellular MSN MSN internalization internalization [18,25]. [18,25]. ItIt was was shown shown that that cellular cellular uptake uptake of of MSNs MSNs is is strictly strictly linked to the surface charge [25,26]. In this context, proteins have been indicated to be the right linked the surface surface charge charge [25,26]. [25,26]. In linked to to the In this this context, context, proteins proteins have have been been indicated indicated to to be be the the right right candidates to tailor the OMS behavior in a given biological system. For instance, surface protein candidates to tailor the OMS behavior in a given biological system. For instance, surface protein candidates to tailor the OMS behavior in a given biological system. For instance, surface protein functionalization of MSNs improves their biocompatibility when injected in biological media [27,28]. functionalization functionalization of of MSNs MSNs improves improves their their biocompatibility biocompatibility when when injected injected in in biological biological media media[27,28]. [27,28]. Moreover, the interaction between MSN and cellular surface can easily be modulated and controlled Moreover, the interaction between MSN and cellular surface can easily be modulated and controlled Moreover, the interaction between MSN and cellular surface can easily be modulated and controlled through specific protein functionalization [29] (Figure 3). Serum proteins, antibodies represent some through specific protein proteinfunctionalization functionalization[29] [29](Figure (Figure3).3).Serum Serumproteins, proteins, antibodies represent some through specific antibodies represent some of of the wide variety of proteins that can be used to tailor chemical properties of MSNs in order to of wide variety of proteins can be used to chemical tailor chemical properties ofin MSNs to thethe wide variety of proteins that that can be used to tailor properties of MSNs orderin to order increase increase their biocompatibility or to set controlled release toward targeted tissues [30,31]. Particularly increase their biocompatibility to set controlled toward targeted tissues [30,31]. Particularly their biocompatibility or to setorcontrolled releaserelease toward targeted tissues [30,31]. Particularly the the adsorption of specific peptides and proteins characterized by important biological functions, the adsorption of specific peptides and proteins characterized by important functions, adsorption of specific peptides and proteins characterized by important biologicalbiological functions, represents represents key factors in the design and engineering of new nanosized drug-delivery systems. represents key design and engineering new nanosized drug-delivery key factors in thefactors design in andthe engineering of new nanosizedofdrug-delivery systems. Moreover, systems. it is well Moreover, it is well known that antibodies are biomacromolecules produced by immune system that Moreover, it is well known that antibodies are biomacromolecules produced by immune system that known that antibodies are biomacromolecules produced by immune system that are able to recognize are able to recognize a large variety of pathogens such as virus and bacteria [32] or to interact with are able to recognize a large variety of pathogens such as virus and bacteria [32] or to interact with cancer cells [33,34] through specific binding. In recent years, several therapeutic applications of the cancer cells [33,34] through specific binding. In recent years, several therapeutic applications of the
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a large variety of pathogens such as virus and bacteria [32] or to interact with cancer cells [33,34]
antibodies havespecific been developed for the cure of autoimmune disordersofor the treatment through binding. In recent years, several therapeutic applications thefor antibodies have beenof cancer disease [32]. developed for the cure of autoimmune disorders or for the treatment of cancer disease [32].
FigureFigure 3. TEM micrograph interaction Hela cell surfaces. 3. TEM micrographofofMSN MSN interaction onon Hela cell surfaces. 1.2. The Age Golden Age 1.2. The Golden physico-chemical techniques,such such as as N isotherms and thermogravmetric Several Several physico-chemical techniques, N22-adsorption -adsorption isotherms and thermogravmetric analysis (TGA), provide important information on protein loading on OMS, but cannot directly analysis (TGA), provide important information on protein loading on OMS, but cannot directly visualize proteins adsorbed on the OMS’ surfaces. Different analytical methods are needed to deeply visualizeinvestigate proteins the adsorbed onofthe OMS’with surfaces. Different analytical methods aremechanisms needed to deeply interaction proteins OMS and to elucidate the cellular interaction investigate the interaction of proteins with OMS and to elucidate the cellular interaction mechanisms of protein-MSN conjugates. The strategic use of gold nanoparticles (GNPs) with dimension smaller than 100 nm has opened of protein-MSN conjugates. the way to the development of new and intriguing procedures. The unique properties of opened The strategic use of gold nanoparticles (GNPs) analytical with dimension smaller than 100 nm has GNPs make them suitable for biodiagnostic and bioanalitycal assays so that in the last years have the way to the development of new and intriguing analytical procedures. The unique properties of received great attention as biosensors [35–37]. As a matter of fact, GNPs can be identified through GNPs make them suitable for biodiagnostic and bioanalitycal assays so that the last years have different methodologies using their light absorption and scattering properties [38]. in Because of their receivedelectron great attention as biosensors Assuitable a matter of fact, and GNPs can be density properties GNPs are[35–37]. extremely for labelling can easily beidentified revealed by through microscopy using techniques. due to their and peculiar surface chemistry they can beBecause bound to of their differentelectron methodologies theirMoreover, light absorption scattering properties [38]. a large variety of molecules, proteins included. All these features, added together, provide a formidable electron density properties GNPs are extremely suitable for labelling and can easily be revealed by investigative tool to permit qualitative and quantitative analyses of protein location, and distribution electron microscopy techniques. Moreover, due to their peculiar surface chemistry they can be bound at the ultrastructural level. Additionally, due to their optical features, they have been suggested for to a large variety of molecules, proteins included. All theseIndeed, features, added together, provide a specific purposes that go beyond the imaging methodologies. GNPs are good candidates formidable investigative to permitnanobioconjugates qualitative andtoquantitative analyses of protein and for the developmenttool of theranostic be used in medicine therapy [39]. It islocation, well known nanoparticleslevel. can be Additionally, heated when exposed to light Photothermal therapy distribution at that the gold ultrastructural due to theirsource. optical features, they have been based on the use of the GNPs has already been tested in specifically targeted GNPs that can be used suggested for specific purposes that go beyond the imaging methodologies. Indeed, GNPstoare good destroy selectively malignant cancer cells, following laser exposure [40,41]. candidates for the development of theranostic nanobioconjugates to be used in medicine therapy [39]. In this review, we aim to describe the principal innovative transmission electron microscopy It is well(TEM) known that gold nanoparticles can when exposed to light source. Photothermal analytical strategies, based on the usebe ofheated nanogold conjugates, to monitor proteins adsorbed therapy based onThis thetopic use of the GNPs already been tested in specifically targeted GNPs that can on OMS. is related to thehas exploitation of GNPs’ potentiality in different nanomedicine applications, spanning from controlled drug delivery depots to diagnostic and theranostic purposes be used to destroy selectively malignant cancer cells, following laser exposure [40,41]. (Scheme 1). This just the of the iceberg. In this review, weis aim to tip describe the principal innovative transmission electron microscopy (TEM) analytical strategies, based on the use of nanogold conjugates, to monitor proteins adsorbed on OMS. This topic is related to the exploitation of GNPs’ potentiality in different nanomedicine applications, spanning from controlled drug delivery depots to diagnostic and theranostic purposes (Scheme 1). This is just the tip of the iceberg.
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Scheme1.1.Diagram Diagram illustrating main gold nanoparticle applications. Scheme illustrating thethe main gold nanoparticle applications.
2. “Seeing “Seeing Is Labelling Methods to Monitor Proteins 2. Is Believing”: Believing”:Immunogold Immunogold Labelling Methods to Monitor Proteins 2.1. Protein Protein Localization Tissues: The Case of Histatin 2.1. LocalizationininAnimal AnimalCells Cellsand and Tissues: The Case of Histatin Since their staining (IGS) techniques became the elective procedures Since their development developmentimmunogold immunogold staining (IGS) techniques became the elective procedures for macromolecules’ macromolecules’ detection and tissues. IGSIGS techniques represent a fundamental for detectionininanimal animalcells cells and tissues. techniques represent a fundamental tool in in ultrastructural ultrastructural biological aimed to to describe protein finefine localization in animal tissues. tool biologicalassays assays aimed describe protein localization in animal tissues. IGS procedures were first developed by Faulk and Taylor in 1971 [42] to meet suitable protein IGS procedures were first developed by Faulk and Taylor in 1971 [42] to meet suitable protein analytical analytical requirements during ultrastructural morphological investigations in animal They able, requirements during ultrastructural morphological investigations in animal tissues.tissues. They were were able, for the first time, to improve the standard routinely TEM studies adding details at the for the first time, to improve the standard routinely TEM studies adding details at the molecular molecular level about protein location and distribution. Before that, TEM methodologies were level about protein location and distribution. Before that, TEM methodologies were generally based generally based on pure morphological analysis of biological samples. Shape, size, and number of on pure morphological analysis of biological samples. Shape, size, and number of subcellular subcellular organelles were the main data accessible following TEM examination. Since then, IGS organelles were the main data accessible following TEM examination. Since then, IGS techniques have techniques have extensively been used to localize proteins in animal tissues and revolutionized the extensively been used to localize proteins in animal tissues and revolutionized the waycombined to investigate way to investigate at ultrastructure level where morphological details were unequivocally at ultrastructure level where morphological details were unequivocally combined to to a significant amount of chemical data. The advantage of this methodology is the aptitude aofsignificant goldamount of chemical The advantage this methodology is theproviding aptitudeevidences of gold-conjugated conjugated antibodiesdata. to recognize and bindof specifically proteins, thus about antibodies to recognize and bind specifically proteins, thus providing evidences about protein content protein content and distribution. Colloidal gold, as electron dense marker, represents the best and distribution. as electron dense represents the best probe to candidate probe toColloidal visualize gold, and locate, by means of marker, TEM, proteins exploiting the candidate highly specific visualize andantibody-GNP locate, by means of TEM, proteins exploiting highly specific of antibody-GNP binding of conjugates. GNPs provide an the unequivocal signalbinding that can easily be conjugates. GNPs provide an unequivocal that can easily be distinguished in both biological distinguished in both biological and inorganicsignal samples. IGS was used by Piludu and coworkers to locate the presence and distribution of specific and inorganic samples. proteins in several animal [43–46]. The purpose of these and investigations to provide IGS was used by Piludutissues and coworkers to locate the presence distributionwas of specific proteins additional data concerning the secretion process of antimicrobialwas peptides (AMP)additional in human data in several animal tissues [43–46]. The purpose of different these investigations to provide salivary glands. To date, process the AMPofhave been antimicrobial well characterized during the last 20 years, however concerning the secretion different peptides (AMP) in human salivary glands. only a few data were reported about their localization and secretion in human tissues. The IGS To date, the AMP have been well characterized during the last 20 years, however only a few data procedures permitted, for the first time,and to localize and study tissues. the distribution several AMP in were reported about their localization secretion in to human The IGS of procedures permitted, human tissues and to definitely establish the involvement of the different human salivary glands in for the first time, to localize and to study the distribution of several AMP in human tissues and to their production and secretion. In particular, the electron microscopic immunogold labelling definitely establish the involvement of the different human salivary glands in their production and extended enormously the previous biochemical investigations highlighting the fine visualization of secretion. In particular, the electron microscopic immunogold labelling extended enormously the AMP at the subcellular level (Figure 4). previous biochemical investigations highlighting the fine visualization of AMP at the subcellular level (Figure 4).
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Figure 4. Electron micrograph of immunogold staining of ultrathin sections of human deep posterior Figure 4. Electron micrograph of immunogold staining of ultrathin sections of human deep posterior lingual gland showing histatin localization. Gold nanoparticles are mainly visualized in the labelled lingual gland showing histatin localization. Gold nanoparticles are mainly visualized in the labelled secretory granules (LSG). Note that adjacent cells contain poorly or unlabelled granules (UG). L = secretory granules (LSG). Note that adjacent cells contain poorly or unlabelled granules (UG). lumen. N =N nucleus. Reprinted withwith permission fromfrom [43].[43]. Copyright 2006,2006, Elsevier. L = lumen. = nucleus. Reprinted permission Copyright Elsevier.
2.2. Proteins Adsorbed on Ordered Mesoporous Silica Materials: The Case of Lysozyme 2.2. Proteins Adsorbed on Ordered Mesoporous Silica Materials: The Case of Lysozyme Although immunogold procedures were initially developed for biological assays, IGS was then Although immunogold procedures were initially developed for biological assays, IGS was then employed for the functionalization of OMS materials to develop smart nanodevices for biomedicine employed for the functionalization of OMS materials to develop smart nanodevices for biomedicine applications. As previously stated, OMS materials have peculiar structural features that make them applications. As previously stated, OMS materials have peculiar structural features that make them suitable hosts to immobilize into their pores a large variety of macromolecules with important suitable hosts to immobilize into their pores a large variety of macromolecules with important biological biological functions [16]. Among the OMS materials, SBA-15 has largely been studied as a potential functions [16]. Among the OMS materials, SBA-15 has largely been studied as a potential carrier for carrier for different purposes. Previous investigations studied the interaction and adsorption different purposes. Previous investigations studied the interaction and adsorption mechanisms of mechanisms of several therapeutic molecules onto SBA-15 [3,47,48]. These studies pointed out the several therapeutic molecules onto SBA-15 [3,47,48]. These studies pointed out the potentiality for potentiality for obtaining smart delivery systems. Interestingly, specific chemical surface obtaining smart delivery systems. Interestingly, specific chemical surface functionalization of SBA-15 functionalization of SBA-15 was shown to enable peculiar biocompatibility conditions [10,18,49]. was shown to enable peculiar biocompatibility conditions [10,18,49]. In order to gain direct visualization of proteins adsorbed onto SBA-15 materials, we used for the In order to gain direct visualization of proteins adsorbed onto SBA-15 materials, we used for first time the IGS techniques in our investigations [50]. We studied the location and distribution of the first time the IGS techniques in our investigations [50]. We studied the location and distribution the human lysozyme adsorbed onto mesoporous silica SBA-15 particles using a post embedding of the human lysozyme adsorbed onto mesoporous silica SBA-15 particles using a post embedding immunogold staining procedure. The advantage of this method is due to the high resolution of the immunogold staining procedure. The advantage of this method is due to the high resolution of the gold labelling, and its unambiguous electron dense mark that can simply be evidenced by gold labelling, and its unambiguous electron dense mark that can simply be evidenced by transmission transmission electron microscopy. Human lysozyme loaded and unloaded SBA-15 microparticles electron microscopy. Human lysozyme loaded and unloaded SBA-15 microparticles (around 300 nm) (around 300 nm) were embedded in LR gold resin blocks. After resin polymerization, the embedded were embedded in LR gold resin blocks. After resin polymerization, the embedded samples were samples were cut into ultrathin sections (90 nm thick), and then processed for immunogold staining cut into ultrathin sections (90 nm thick), and then processed for immunogold staining through two through two consecutive incubation steps of the sections with specific antibodies. The first step of consecutive incubation steps of the sections with specific antibodies. The first step of this procedure this procedure was characterized by the interaction of a primary antibody, specific to human was characterized by the interaction of a primary antibody, specific to human lysozyme, whereas in lysozyme, whereas in the following step a gold-conjugated secondary antibody, specific to primary the following step a gold-conjugated secondary antibody, specific to primary antibody, was added. antibody, was added. TEM analysis highlighted the presence of specific lysozyme reactivity that was TEM analysis highlighted the presence of specific lysozyme reactivity that was visualized as definite visualized as definite black spots into cylindrical channels or located on the surface of SBA-15 black spots into cylindrical channels or located on the surface of SBA-15 particles (Figure 5). particles (Figure 5).
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Figure 5. TEM immunogold labelling of ultrathin sections (~60–80 nm thick) of SBA-15 with antiFigure 5. antibody. TEM immunogold labelling of ultrathin sections (~60–80 nm SBA-15 with antilysozyme Gold labelling is detected into cylindrical channels (a)thick) andthick) onofthe surface of SBAFigure 5. TEM immunogold labelling of ultrathin sections (~60–80 nm of SBA-15 with lysozyme antibody. Gold labelling is detected into cylindrical channels (a) and on the surface of SBA15 (b). Reprinted with permission from [50]. Copyright Royal channels Society of(a) Chemistry. anti-lysozyme antibody. Gold labelling is detected into 2011, cylindrical and on the surface of 15 (b). Reprinted with permission from [50]. Copyright 2011, Royal Society of Chemistry. SBA-15 (b). Reprinted with permission from [50]. Copyright 2011, Royal Society of Chemistry.
The selection of an appropriate antibody in immunolocalization assays is extremely crucial to selection of an antibody in immunolocalization assays is extremely to gain The reliable results, in appropriate particular, choices have to be made on the crucial basis of The selection of an appropriateconsidering antibody inthat immunolocalization assays is extremely crucial gain reliable results, in particular, considering that choices have to be made on the basis of availability, quality of antibodies and considering specificity towards a given The achievement to gain reliable results, in particular, that choices haveprotein. to be made on the basis of of availability,results qualityin ofimmunolocalization antibodies and specificity towards a given protein. The of achievement of significant investigations deals with the aptitude the primary availability, quality of antibodies and specificity towards a given protein. The achievement of significant results in immunolocalization investigations deals with the aptitude of the primary antibody to results bind specifically proteins through an importantdeals biochemical interaction leads to the significant in immunolocalization investigations with the aptitudethat of the primary antibody to bind specifically proteins through an important biochemical interaction that leads to the the formation of bind the antibody-antigen complex [51].anThe present biochemical method, being characterized by antibody to specifically proteins through important interaction that leads to formation of the antibody-antigen complex [51]. The present method, being characterized by the sequential binding of primary, and secondary antibodies, is generally known as indirect method. the formation of the antibody-antigen complex [51]. The present method, being characterized by sequential of primary, and secondary antibodies, is generallycomplex known as indirect method. Indeed, the binding gold marker associated the primary antibody-antigen linking the the sequential binding ofisprimary, andtosecondary antibodies, is generally knownthrough as indirect method. Indeed, the gold marker is associated to the primary antibody-antigen complex through linking the secondary (Figure 6). Indeed, theantibodies gold marker is associated to the primary antibody-antigen complex through linking the secondary antibodies (Figure 6). secondary antibodies (Figure 6).
Figure 6. 6. Schematic Schematicrepresentation representationof of post post embedding embedding immunogold immunogold staining staining (IGS) (IGS) procedure procedure for for Figure Figure 6. Schematic representation of post embedding immunogold staining (IGS) procedure for protein visualization by conventional TEM. Note that purple and red objects represent additional protein visualization by conventional TEM. Note that purple and red objects represent additional protein visualization byOMS. conventional TEM. that purple and red objects represent additional proteins adsorbedonto onto Reprinted withNote permission from [50]. Copyright 2011, Royal Society proteins adsorbed OMS. Reprinted with permission from [50]. Copyright 2011, Royal Society of proteins adsorbed onto OMS. Reprinted with permission from [50]. Copyright 2011, Royal Society of of Chemistry. Chemistry. Chemistry.
Inorder order to tomeet meetpeculiar peculiarneeds needsof ofimmunochemical immunochemicalassays assaysdifferent differentIGS IGSprocedures procedurescan canbe beused. used. In In order the to meet peculiar needs of immunochemical assays different IGS procedures can be direct used. Remarkably, indirect method in the recent years has gained in popularity compared to the Remarkably, the method the recent years has gained in popularity compared to the direct Remarkably, indirect method in the recent years hastogained in popularity compared toreason thefor direct IGS procedurethe that isisbased on GNPs directly bound thethe primary antibodies. TheThe reason that IGS that based onthe the GNPs directly bound to primary antibodies. for IGS procedure that is based on the GNPs directly bound to the primary antibodies. The reason for maymay be found in thein higher sensitivity of the indirect method method as a result capacity secondary that be found the higher sensitivity of the indirect as of a the result of theofcapacity of that may be found in the higher sensitivity of the indirect method as a result of the capacity of antibodies to interact with multiple epitopes located on primary antibodies [51,52]. Because of secondary antibodies to interact with multiple epitopes located on primary antibodies [51,52]. secondary antibodies to interact with multiple epitopes located on primary antibodies [51,52]. the sequential steps of incubations with primary secondary antibodies, it shouldit be noticed Because of the sequential steps of incubations with and primary and secondary antibodies, should be Because of the sequential of the incubations withof primary and secondary antibodies, it should[51]. be that indirect methods maysteps imply concurrence cross-reactions with with unrelated molecules noticed that indirect methods may imply the concurrence of cross-reactions unrelated molecules noticed that indirect may the concurrence ofofcross-reactions with unrelated molecules It follows that suitable and appropriate control samples utmost In our investigation [51]. It follows that methods suitable and imply appropriate control are samples are importance. of utmost importance. In our [51]. It follows that suitable and appropriate control samples are of utmost importance. In our we performed controls in order to verify thetospecificity lysozyme loaded investigation wespecific performed specific controls in order verify the of specificity of labelling lysozymein labelling investigation we performed specific controls in order to verify the specificity of lysozyme labelling in loaded SBA-15. Indeed, the failure of antibody performance may occur at any stage of the in loaded SBA-15. Indeed, the failure of antibody performance may occur at any stage of the
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SBA-15. Indeed, the failure of antibody performance may occur at any stage of the procedure. Int. J. Mol. Sci. 2018, 19, x 7 of 14and During our investigations unloaded samples of SBA-15 were incubated with both primary secondary antibodies to ascertain antibody specificity. Moreover, additional controls were performed During our investigations unloaded samples of SBA-15 were incubated with both primary byprocedure. incubating human lysozyme loaded samples with non-immune serum or with the secondary and secondary antibodies to ascertain antibody specificity. Moreover, additional controls were antibody only and omitting the primary antibody (negative control). No gold labelling was detected performed by incubating human lysozyme loaded samples with non-immune serum or with the in any of these cases [50]. secondary antibody only and omitting the primary antibody (negative control). No gold labelling Peculiar modifications of the standard IGS protocol regards the use of multiple sequential staining was detected in any of these cases [50]. procedure of two or more proteins in the same sample, using different size gold conjugated antibodies. Peculiar modifications of the standard IGS protocol regards the use of multiple sequential One of the main advantages of the double procedure concerns theconjugated opportunity staining procedure of two or more proteins in immunostaining the same sample, using different size gold toantibodies. study simultaneously several proteins adsorbed in the same material. Choosing different One of the main advantages of the double immunostaining procedure concerns thegold nanosized conjugated antibodies specific for the primary allows toChoosing monitor the opportunity to studysecondary simultaneously several proteins adsorbed in antibodies the same material. different proteins separately. This technique, successfully applied in several histochemical studies, different gold nanosized conjugated secondary antibodies specific for the primary antibodies allows involves the use of different size nanogold conjugated antibodies that can easily be distinguished to monitor the different proteins separately. This technique, successfully applied in several during TEM analysis, allowing to visualize spatial of two or more proteins in the histochemical studies,thus involves the use of different size relationships nanogold conjugated antibodies that can same section. easily be distinguished during TEM analysis, thus allowing to visualize spatial relationships of two or more proteins in the same section.
2.3. Silver Enhancement Technique and Ultra-Small Gold Nanoparticles: The Case of an Antibody Fragment 2.3. Silver Enhancement Technique and Ultra-Small Gold Nanoparticles: The Case of an Antibody Fragment
Small GNPs (gold size ≈ 1–3 nm) were firstly introduced in order to improve the penetration Small GNPs (gold size ≈ 1–3 nm) were cells firstlyand introduced in order to improve the penetration of of gold conjugated antibodies into animal tissues and to enhance labelling efficacy [53,54]. gold conjugated antibodies into animal cells and tissues and to enhance labelling efficacy [53,54]. In In pre-embedding immunocytochemical assays gold conjugated antibody efficiency deals with the size assays conjugated antibody with the size ofpre-embedding gold particles immunocytochemical used. It was shown that thegold antibodies employed in efficiency routinely deals IGS procedures with of gold It was shown that the antibodies employed in routinely IGS procedures with gold size particles particlesused. around 10–30 nm show reduced interaction efficiency. Instead, smaller nanoparticles gold shown size particles around 10–30bynm show degree reducedofinteraction efficiency. Instead, smaller were to be characterized a higher penetration, improved gold conjugated nanoparticles were shown to be characterized by a higher degree of penetration, improved gold of antibody interaction with the targeted antigens and higher resolution of labelling [54,55]. Because conjugated antibody interaction with the targeted antigens and higher resolution of labelling [54,55]. their properties ultra-small GNPs may be used in high resolution labelling for protein detection Because of their properties ultra-small GNPs may be used in high resolution labelling for protein in nanostructured materials and in the development of targeted drug delivery systems. However, detection in nanostructured materials and in the development of targeted drug delivery systems. the ultra-small GNPs cannot be visualized by conventional TEM but need to be amplified and converted However, the ultra-small GNPs cannot be visualized by conventional TEM but need to be amplified in larger size particles through silver enhancement procedure [56]. and converted in larger size particles through silver enhancement procedure [56]. Similarly, we used the silver enhancement procedure to visualize the antibody fragments Similarly, we used the silver enhancement procedure to visualize the antibody fragments F(ab’)GAMIgG to ultra-small ultra-smallgold goldnanoparticles nanoparticles(GNPs (GNPs diameter nm) immobilized F(ab’)GAMIgG conjugated conjugated to diameter 0.80.8 nm) immobilized into functionalizedSBA-15 SBA-15particles particles[57]. [57]. The silver-enhanced GNPs resulted intothe themesopores mesoporesof of amino amino functionalized The silver-enhanced GNPs resulted visible at conventional TEM, thus allowing for an unequivocal imaging of the location of the antibody visible at conventional TEM, thus allowing for an unequivocal imaging of the location of the antibody fragment-GNPs the channels channelsof ofSBA-15 SBA-15particles particles(Figure (Figure fragment-GNPsconjugates conjugates inside inside the 7).7).
Figure 7. (a) Representation of the antibody fragment-gold nanoparticle [F(ab’)GAMIgG-GPNs] Figure 7. (a) Representation of the antibody fragment-gold nanoparticle [F(ab’)GAMIgG-GPNs] conjugate; and (b,c) Graphical representation of the main sequential steps of silver enhancement conjugate; and (b,c) Graphical representation of the main sequential steps of silver enhancement process on ultrathin section of F(ab’)GAMIgG-GPNs loaded SBA15. (a) is adapted with permission process on ultrathin section of F(ab’)GAMIgG-GPNs loaded SBA15. (a) is adapted with permission from [57]. Copyright 2015, American Chemical Society. from [57]. Copyright 2015, American Chemical Society.
The use of sub-nanometer gold increased enormously its diffusion and penetration efficiency usereduced of sub-nanometer golddecreased increasedsteric enormously its This diffusion and penetration efficiency dueThe to the overall size and hindrance. procedure was carried out by due to the reduced overall size and decreased steric hindrance. This procedure was carried embedding both F(ab’)GAMIgG-GNPs loaded and unloaded SBA-15 micro-particles in resin, out byfollowing embedding bothpreparation F(ab’)GAMIgG-GNPs loaded and cutting unloaded SBA-15 micro-particles in resin, standard procedure [58] and finally ultrathin sections (70–90 nm thick) of both embedded samples with ultramicrotome. The ultrathin sections were finally treated through the silver enhancement procedure. In detail, the silver enhancement technique permitted the
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following standard preparation procedure [58] and finally cutting ultrathin sections (70–90 nm thick) of both embedded samples with ultramicrotome. The ultrathin sections were finally treated through the Int. J. Mol. Sci. 2018, 19, x 8 of 14 silver enhancement procedure. In detail, the silver enhancement technique permitted the amplification of the ultra-small GNPs that acted as nucleation sites the deposition ofthe Ag deposition atoms obtained through amplification of the ultra-small GNPs that acted as for nucleation sites for of Ag atoms + ions in all loaded + the reduction of Ag samples of SBA-15. The unloaded samples used as controls obtained through the reduction of Ag ions in all loaded samples of SBA-15. The unloaded samples were of labelling. The TEM observation revealed the presence of unequivocal silver labelling as used devoid as controls were devoid of labelling. The TEM observation revealed the presence of unequivocal 20–30 nm size black dots on the mesoporous inner structure. The black dots highlighted the presence silver labelling as 20–30 nm size black dots on the mesoporous inner structure. The black dots and distribution of antibody F(ab’)GAMIgG either inside the pores or either adsorbed on the highlighted the presence andfragments distribution of antibody fragments F(ab’)GAMIgG inside the surface of SBA-15 microparticles (Figure 8). pores or adsorbed on the surface of SBA-15 microparticles (Figure 8).
Figure 8. Transmission electron micrographs of ultrathin sections of SBA-15 particles loaded with Figure 8. Transmission electron micrographs of ultrathin sections of SBA-15 particles loaded with F(ab’)GAMIgG-GNPs conjugates. Distribution of gold conjugated F(ab’)GAMIgG in SBA-15 particles F(ab’)GAMIgG-GNPs conjugates. Distribution of gold conjugated F(ab’)GAMIgG in SBA-15 particles was revealed by silver enhancement procedure. Black spots correspond to the “silver enhanced” was revealed by silver enhancement procedure. Black spots correspond to the “silver enhanced” GNPs GNPs (silver labelling). (a) Overview of silver localization in loaded SBA-15 samples. Note that SBA(silver labelling). (a) Overview of silver localization in loaded SBA-15 samples. Note that SBA-15 15 particles show variable labelling. Dark area (asterisks) is due to the electron beam passing through particles show variable labelling. Dark area (asterisks) is due to the electron beam passing through a thicker portion of SBA-15 section; (b) Details showi ng at higher magnification the presence of silver a thicker portion of SBA-15 section; (b) Details showi ng at higher magnification the presence of labelling along the parallel channel of loaded SBA-15; and (c) Silver labelling is confined to the silver labelling along the parallel channel of loaded SBA-15; and (c) Silver labelling is confined to the external portion of of SBA-15 particles. Reprinted permission from from [57]. [57]. Copyright external portion SBA-15 particles. Reprinted with with permission Copyright 2015, 2015, American American Chemical Society. Chemical Society.
On the basis of these results, it should be remarked the importance of this powerful procedure On the basis of these results, it should be remarked the importance of this powerful procedure to study and gain site-specific data concerning the functionalization process of specific peptides and to study and gain site-specific data concerning the functionalization process of specific peptides and proteins in OMS materials, and thus in the engineering of new drug delivery systems. Moreover, the proteins in OMS materials, and thus in the engineering of new drug delivery systems. Moreover, use of ultra-small gold particles represents the best choice for molecular labelling, since it does not the use of ultra-small gold particles represents the best choice for molecular labelling, since it does not seem to prevent the penetration of the proteins into the pore channels of SBA-15, considering that the seem to prevent the penetration of the proteins into the pore channels of SBA-15, considering that the silver labelling is highly specific and highly sensitive towards the gold particles [59]. silver labelling is highly specific and highly sensitive towards the gold particles [59]. 3. Exploring the Formation Mechanism of the Protein Corona: The Case of BSA-GNP Conjugates 3. Exploring the Formation Mechanism of the Protein Corona: The Case of BSA-GNP Conjugates Gold nanoparticles can be bound directly to a large variety of biomolecules by using definite Gold nanoparticles can be bound directly to a large variety of biomolecules by using definite chemical methodologies. For instance, hydrophobic interactions or charge pairing represent the main chemical methodologies. For instance, hydrophobic interactions or charge pairing represent the main procedures employed to conjugate GNPs to specific molecules. During our investigations we used procedures employed to conjugate GNPs to specific molecules. During our investigations we used gold complexes to study the adsorption process of biomolecules on the surface of functionalized gold complexes to study the adsorption process of biomolecules on the surface of functionalized mesoporous silica nanoparticles (MSNs) dispersed in biological media. In detail, we investigated the interaction of gold conjugated bovine serum albumin (BSA) with hyaluronic acid (HA) and chitosan (CHIT) functionalized MSNs to provide new insights on the molecular mechanisms that lead to the formation of the surface layer of biomolecules around the MSN, known as the “protein corona” [10]. Protein corona induces significant changes in the physico-chemical properties of MSNs surface. This in turn may affect MSNs cellular interaction and uptake. Protein corona formation is influenced by
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mesoporous silica nanoparticles (MSNs) dispersed in biological media. In detail, we investigated the interaction of gold conjugated bovine serum albumin (BSA) with hyaluronic acid (HA) and chitosan (CHIT) functionalized MSNs to provide new insights on the molecular mechanisms that lead to the formation of the surface layer of biomolecules around the MSN, known as the “protein corona” [10]. Protein Int. J. Mol. Sci. 2018, 19, x corona induces significant changes in the physico-chemical properties of MSNs 9 of 14 surface. This in turn may affect MSNs cellular interaction and uptake. Protein corona formation is influenced bothfeatures chemical MSNs features of and of theItbiological has both chemical by MSNs and composition thecomposition biological fluids. has been fluids. reportedIt that been reported MSNs, that functionalized when injected in characterized biological media, areinteraction characterized by functionalized when injectedMSNs, in biological media, are by the of their the interaction their surface with theThis dispersed processbyisspecific supposed to be surface with theof dispersed biomolecules. processbiomolecules. is supposed to This be initiated physicoinitiated by specific physico-chemical interactions that rely features mainly on theaspeculiar chemical interactions that rely mainly on the peculiar MSNs such surface MSNs chargefeatures [60,61]. such as surface [60,61]. To the behavior BSA interaction with theMSNs differently To this purpose,charge the behavior of this BSApurpose, interaction with the of differently functionalized was functionalized MSNs was investigated by conventional TEM analysis. We used BSA conjugated with investigated by conventional TEM analysis. We used BSA conjugated with gold nanoparticles (BSAgold nanoparticles (BSA-GNPs). gold labelling observed on the surface of both MSN-HA GNPs). Evident gold labelling Evident was observed on the was surface of both MSN-HA and MSN-CHIT and MSN-CHIT that were characterized by patterns. similar BSA-GNPs patterns. Occasionally, samples, that weresamples, characterized by similar BSA-GNPs Occasionally, higher gold labelling higher gold labelling intensity was found in MSN-CHIT than in MSN-HA, pointing out that intensity was found in MSN-CHIT than in MSN-HA, pointing out that BSA interaction dependsBSA on interaction depends on of thethe molecular features of the biopolymers adsorbed on9). MSN surface (Figure 9). the molecular features biopolymers adsorbed on MSN surface (Figure
Figure micrographsofofMSN-HA+BSA-GNPs MSN-HA+BSA-GNPs MSN-CHIT+BSA-GNPs (b). Reprinted Figure 9. 9. TEM TEM micrographs (a)(a) andand MSN-CHIT+BSA-GNPs (b). Reprinted with with permission from [10]. Copyright 2018, Elsevier. permission from [10]. Copyright 2018, Elsevier.
Here, it should be remarked that, at a pH around 7, BSA carries a negative charge (being pI ≈ Here, it should be remarked that, at a pH around 7, BSA carries a negative charge (being pI ≈ 4.7), 4.7), whereas MSN-CHIT and MSN-HA surfaces are positively and negatively charged, respectively. whereas MSN-CHIT and MSN-HA surfaces are positively and negatively charged, respectively. Indeed, Indeed, protein adsorption is mainly addressed by electrostatic interactions according to the order protein adsorption is mainly addressed by electrostatic interactions according to the order MSN-CHIT MSN-CHIT > MSN-HA; however, the relevant BSA adsorption on the negatively charged MSN-HA > MSN-HA; however, the relevant BSA adsorption on the negatively charged MSN-HA implies implies important contributions of van der Waals attractive interactions, as demonstrated by zeta important contributions of van der Waals attractive interactions, as demonstrated by zeta potential potential data (see table 2 in [10]). A similar trend was also observed after the gold labelling data (see Table in [10]). A similar trend was also observedmethodology after the goldfor labelling procedure. The GNP procedure. The2 GNP conjugation provided a powerful the direct visualization of conjugation provided a powerful methodology for the direct visualization of interaction process of BSA interaction process of BSA with functionalized MSN surfaces, correlating surface characterization of with functionalized MSN surfaces, correlating surface characterization MSNsaround with BSA adsorption. MSNs with BSA adsorption. In other words, the formation of a protein of corona functionalized In other words, the formation of a protein corona around functionalized MSN particles, independently MSN particles, independently of the electrostatic forces, was unequivocally proven [10]. of the electrostatic forces, was unequivocally proven [10]. 4. Toward the Realization of Theranostic Nanobioconjugates 4. Toward the Realization of Theranostic Nanobioconjugates In the previous paragraphs we have described the use of GNPs as diagnostic devices for the In the previous paragraphs we have described the use of GNPs as diagnostic devices for the localization of proteins within the internal and the external surface of OMS. Besides diagnostic localization of proteins within the internal and the external surface of OMS. Besides diagnostic imaging, GNPs may find different application in nanomedicine. GNPs are indeed characterized by a imaging, GNPs may find different application in nanomedicine. GNPs are indeed characterized by a surface plasmon frequency in the visible range, which makes them suitable for therapeutic treatments surface plasmon frequency in the visible range, which makes them suitable for therapeutic treatments (i.e., photothermal therapy [62] or, more interestingly, for “theranostics” that is the combination (i.e., photothermal therapy [62] or, more interestingly, for “theranostics” that is the combination between therapy and diagnostics [63]. In a very recent work [64], we assembled multicomponent between therapy and diagnostics [63]. In a very recent work [64], we assembled multicomponent nano-bioconjugates based on mesoporous silica nanoparticles (MSNs), proteins (BSA or lysozyme), and gold nanoparticles (Figure 10). The purpose of the realization of such nano-bioconjugates was for a possible application in nanomedicine as theranostic devices. Indeed, MSNs can act as drug carriers, proteins stabilize MSNs within the bloodstream, or may have therapeutic or targeting functions. Finally, GNPs could either be used as contrast agents for imaging or for photothermal therapy. The synthesized MSNs, functionalized with amino-terminated groups (MSN-NH2), were
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nano-bioconjugates based on mesoporous silica nanoparticles (MSNs), proteins (BSA or lysozyme), and gold nanoparticles (Figure 10). The purpose of the realization of such nano-bioconjugates was for a possible application in nanomedicine as theranostic devices. Indeed, MSNs can act as drug carriers, proteins stabilize MSNs within the bloodstream, or may have therapeutic or targeting functions. Finally, GNPs could either be used as contrast agents for imaging or for photothermal therapy. The synthesized MSNs, functionalized with amino-terminated groups (MSN-NH2 ), were conjugated to BSA or lysozyme on the external surface of MSN-NH2 to obtain MSN-BSA and MSN-Lysozyme bioconjugates, respectively. The MSN-protein samples were dispersed in a GNP solution to obtain Int. J. Mol. Sci. 2018, 19, x 10 of 14 MSN-protein-GNPs nano-bioconjugates. TEM analysis showed the occurrence of GNPs on the MSN-protein surface. Interestingly, we demonstrated, through fluorescence and Raman measurements, the MSN-protein surface. Interestingly, we demonstrated, through fluorescence and Raman that GNP-protein interaction occurs through the involvement of tryptophan amino acid measurements, thatspecific GNP-protein specific interaction occurs through the involvement of tryptophan residues. Hence, the MSN-protein-GNPs nano-bioconjugates formed in the presence of lysozyme amino acid residues. Hence, the MSN-protein-GNPs nano-bioconjugates formed in the presence of display more conjugated GNPs than those formed in the presence of BSA: This was related to the lysozyme display more conjugated GNPs than those formed in the presence of BSA: This was related higher number of tryptophan residues in lysozyme compared to BSA. to the higher number of tryptophan residues in lysozyme compared to BSA.
Figure 10. Assembly of theranostic nanobioconjugates. Schematic representation of the MSN-proteinFigure 10. Assembly of theranostic nanobioconjugates. Schematic representation of the MSN-proteinGNP nanobioconjugate: MSN (orange/blue), protein (red), GNP (yellow) (a), graphical 3D GNP nanobioconjugate: MSN (orange/blue), protein (red), GNP (yellow) (a), graphical 3D reconstruction of the MSN-protein-GNP nanobioconjugate (b), TEM micrographs of the MSNreconstruction of the MSN-protein-GNP nanobioconjugate (b), TEM micrographs of the lysozyme-GNP (c) and MSN-BSA-GNP (d) nanobioconjugates. MSN-lysozyme-GNP (c) and MSN-BSA-GNP (d) nanobioconjugates.
5. Conclusions 5. Conclusions The use of GNPs represents one of the most encouraging strategies to monitor proteins in many The use of GNPs represents one of the most encouraging strategies to monitor proteins in many research fields. They have been suggested as biosensors because of their optical properties and are research fields. They have been suggested as biosensors because of their optical properties and are excellent labels that can be detected through several procedures. GNPs can be conjugated with a large excellent labels that can be detected through several procedures. GNPs can be conjugated with a large variety of biomolecules. Because of their comparable size with proteins, peptides, oligonucleotides, variety of biomolecules. Because of their comparable size with proteins, peptides, oligonucleotides, and other biomolecules, GNPs can be used in many biomedical application fields. They can be used and other biomolecules, GNPs can be used in many biomedical application fields. They can be used as drug carriers to deliver specific therapeutic molecules as a result of their ability to link and load as drug carriers to deliver specific therapeutic molecules as a result of their ability to link and load different molecules such as peptides, proteins, or oligonucleotides [65–67]. The GNPs, when different molecules such as peptides, proteins, or oligonucleotides [65–67]. The GNPs, when conjugated conjugated to specific targeting proteins, can also be used as intrinsic drug agents. Their capacity to to specific targeting proteins, can also be used as intrinsic drug agents. Their capacity to penetrate penetrate cellular compartments and induce cellular damage by cellular oxidative stress, or by cellular compartments and induce cellular damage by cellular oxidative stress, or by photothermal photothermal therapy has been used to ablate specifically cancer cells and tissues. However, even therapy has been used to ablate specifically cancer cells and tissues. However, even though their use though their use as contrast agents remains the main application in imaging and diagnostic medicine, as contrast agents remains the main application in imaging and diagnostic medicine, it should be it should be remarked that GNPs based approaches are becoming a fundamental tool in the remarked that GNPs based approaches are becoming a fundamental tool in the development of new development of new theranostic nanobioconjugates. The main aim of this review was to bring the theranostic nanobioconjugates. The main aim of this review was to bring the attention of the readers attention of the readers to the extraordinary high resolving power of the gold labelling techniques, to the extraordinary high resolving power of the gold labelling techniques, building a bridge with the building a bridge with the other investigative physical and chemical disciplines. This may provide other investigative physical and chemical disciplines. This may provide new eyes to the way how we new eyes to the way how we look at the nanostructured world of the ordered mesoporous silica look at the nanostructured world of the ordered mesoporous silica materials. materials. Author Contributions: Conceptualization, M.P., L.M., M.M., and A.S.; Writing-Original Draft Preparation, M.P.; Author Contributions: Conceptualization, M.P., Writing-Review & Editing, M.P., L.M., M.M., andL.M., A.S. M.M., and A.S.; Writing-Original Draft Preparation, M.P.; Writing-Review & Editing, M.P., L.M., M.M., and A.S. Funding: This research was funded by FIR 2017-2018, Fondazione di Sardegna/Regione Autonoma della Sardegna (CUP F72F16003070002). AS thanks FFABR 2017 (MIUR). Fondazione di Sardegna/Regione Autonoma della Funding: This research was funded by FIR 2017-2018, Sardegna (CUP F72F16003070002). AS thanks FFABR 2017 (MIUR). Conflicts of Interest: The authors declare no conflict of interest.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations OMS
Ordered mesoporous silica
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Abbreviations OMS MSN GNP IGS AMP BSA HA CHIT
Ordered mesoporous silica Mesoporous silica nanoparticle Gold nanoparticles Immunogold staining Antimicrobial peptides Bovine serum albumin Hyaluronic acid Chitosan
References 1. 2. 3.
4. 5.
6.
7.
8. 9. 10.
11.
12. 13. 14. 15. 16. 17.
Kresge, C.T.; Leonowicz, M.E.; Roth, W.J.; Vartuli, J.C.; Beck, J.S. Ordered mesoporous molecular sieves synthesized by a liquid-crystal template mechanism. Nature 1992, 359, 710–712. [CrossRef] Davis, M.E. Ordered porous materials for emerging applications. Nature 2002, 417, 813–821. [CrossRef] [PubMed] Bhattacharyya, M.S.; Hiwale, P.; Piras, M.; Medda, L.; Steri, D.; Piludu, M.; Salis, A.; Monduzzi, M. Lysozyme Adsorption and Release from Ordered Mesoporous Materials. J. Phys. Chem. C 2010, 114, 19928–19934. [CrossRef] Taguchi, A.; Schüth, F. Ordered mesoporous materials in catalysis. Microporous Mesoporous Mater. 2005, 77, 1–45. [CrossRef] Salis, A.; Casula, M.F.; Bhattacharyya, M.S.; Pinna, M.; Solinas, V.; Monduzzi, M. Physical and Chemical Lipase Adsorption on SBA-15: Effect of Different Interactions on Enzyme Loading and Catalytic Performance. ChemCatChem 2010, 2, 322–329. [CrossRef] Pitzalis, F.; Monduzzi, M.; Salis, A. A bienzymatic biocatalyst constituted by glucose oxidase and Horseradish peroxidase immobilized on ordered mesoporous silica. Microporous Mesoporous Mater. 2017, 241, 145–154. [CrossRef] Vallet-Regí, M.; Doadrio, J.C.; Doadrio, A.L.; Izquierdo-Barba, I.; Pérez-Pariente, J. Hexagonal ordered mesoporous material as a matrix for the controlled release of amoxicillin. Solid State Ion. 2004, 172, 435–439. [CrossRef] Vallet-Regi, M.; Rámila, A.; Del Real, R.P.; Pérez-Pariente, J. A new property of MCM-41: Drug delivery system. Chem. Mater. 2001, 13, 308–311. [CrossRef] Gómez-Cerezo, N.; Izquierdo-Barba, I.; Arcos, D.; Vallet-Regí, M. Tailoring the biological response of mesoporous bioactive materials. J. Mater. Chem. B 2015, 3810–3819. [CrossRef] Nairi, V.; Medda, S.; Piludu, M.; Casula, M.F.; Vallet-Regì, M.; Monduzzi, M.; Salis, A. Interactions between bovine serum albumin and mesoporous silica nanoparticles functionalized with biopolymers. Chem. Eng. J. 2018, 340, 42–50. [CrossRef] Castillo, R.R.; Baeza, A.; Vallet-Regí, M. Recent applications of the combination of mesoporous silica nanoparticles with nucleic acids: Development of bioresponsive devices, carriers and sensors. Biomater. Sci. 2017, 5, 353–377. [CrossRef] [PubMed] Castillo, R.R.; Colilla, M.; Vallet-Regí, M. Advances in mesoporous silica-based nanocarriers for co-delivery and combination therapy against cancer. Expert Opin. Drug Deliv. 2017, 14, 229–243. [CrossRef] [PubMed] Tang, F.; Li, L.; Chen, D. Mesoporous Silica Nanoparticles: Synthesis, Biocompatibility and Drug Delivery. Adv. Mater. 2012, 24, 1504–1534. [CrossRef] [PubMed] Wu, S.-H.; Hung, Y.; Mou, C.-Y. Mesoporous silica nanoparticles as nanocarriers. Chem. Commun. (Camb.) 2011, 47, 9972–9985. [CrossRef] [PubMed] Paris, J.L.; Cabanas, M.V.; Manzano, M.; Vallet-Regí, M. Polymer-Grafted Mesoporous Silica Nanoparticles as Ultrasound-Responsive Drug Carriers. ACS Nano 2015, 9, 11023–11033. [CrossRef] [PubMed] Nairi, V.; Medda, L.; Monduzzi, M.; Salis, A. Adsorption and release of ampicillin antibiotic from ordered mesoporous silica. J. Colloid Interface Sci. 2017, 497, 217–225. [CrossRef] [PubMed] Sevimli, F.; Yilmaz, A. Surface functionalization of SBA-15 particles for amoxicillin delivery. Microporous Mesoporous Mater. 2012, 158, 281–291. [CrossRef]
Int. J. Mol. Sci. 2018, 19, 1991
18.
19. 20.
21.
22.
23. 24.
25.
26.
27. 28.
29.
30.
31.
32. 33. 34. 35. 36. 37. 38.
12 of 14
Salis, A.; Fanti, M.; Medda, L.; Nairi, V.; Cugia, F.; Piludu, M.; Sogos, V.; Monduzzi, M. Mesoporous Silica Nanoparticles Functionalized with Hyaluronic Acid and Chitosan Biopolymers. Effect of Functionalization on Cell Internalization. ACS Biomater. Sci. Eng. 2016, 2, 741–751. [CrossRef] Popat, A.; Liu, J.; Lu, G.Q.; Qiao, S.Z. A pH-responsive drug delivery system based on chitosan coated mesoporous silica nanoparticles. J. Mater. Chem. 2012, 22, 11173. [CrossRef] Liu, W.T.; Yang, Y.; Shen, P.H.; Gao, X.J.; He, S.Q.; Liu, H.; Zhu, C.S. Facile and simple preparation of pH-sensitive chitosan-mesoporous silica nanoparticles for future breast cancer treatment. Express Polym. Lett. 2015, 9, 1068–1075. [CrossRef] Huang, D.-M.; Chung, T.-H.; Hung, Y.; Lu, F.; Wu, S.-H.; Mou, C.-Y.; Yao, M.; Chen, Y.-C. Internalization of mesoporous silica nanoparticles induces transient but not sufficient osteogenic signals in human mesenchymal stem cells. Toxicol. Appl. Pharmacol. 2008, 231, 208–215. [CrossRef] [PubMed] Xia, Y.; Li, M.; Peng, T.; Zhang, W.; Xiong, J.; Hu, Q.; Song, Z.; Zheng, Q. In vitro cytotoxicity of fluorescent silica nanoparticles hybridized with aggregation-induced emission luminogens for living cell imaging. Int. J. Mol. Sci. 2013, 14, 1080–1092. [CrossRef] [PubMed] Guo, H.; Qian, H.; Sun, S.; Sun, D.; Yin, H.; Cai, X.; Liu, Z.; Wu, J.; Jiang, T.; Liu, X. Hollow mesoporous silica nanoparticles for intracellular delivery of fluorescent dye. Chem. Cent. J. 2011, 5, 1. [CrossRef] [PubMed] Zhang, J.; Postovit, L.M.; Wang, D.; Gardiner, R.B.; Harris, R.; Abdul, M.M.; Thomas, A.A. In situ loading of basic fibroblast growth factor within porous silica nanoparticles for a prolonged release. Nanoscale Res. Lett. 2009, 4, 1297–1302. [CrossRef] [PubMed] Nairi, V.; Magnolia, S.; Piludu, M.; Nieddu, M.; Caria, C.A.; Sogos, V.; Vallet-Regì, M.; Monduzzi, M.; Salis, A. Mesoporous silica nanoparticles functionalized with hyaluronic acid. Effect of the biopolymer chain length on cell internalization. Colloids Surfaces B Biointerfaces 2018, 168, 50–59. [CrossRef] [PubMed] Nel, A.E.; Mädler, L.; Velegol, D.; Xia, T.; Hoek, E.M.V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Understanding biophysicochemical interactions at the nano-bio interface. Nat. Mater. 2009, 8, 543–557. [CrossRef] [PubMed] Fang, W.; Wang, Z.; Zong, S.; Chen, H.; Zhu, D.; Zhong, Y.; Cui, Y. PH-controllable drug carrier with SERS activity for targeting cancer cells. Biosens. Bioelectron. 2014, 57, 10–15. [CrossRef] [PubMed] Ferris, D.P.; Lu, J.; Gothard, C.; Yanes, R.; Thomas, C.R.; Olsen, J.C.; Stoddart, J.F.; Tamanoi, F.; Zink, J.I. Synthesis of biomolecule-modified mesoporous silica nanoparticles for targeted hydrophobic drug delivery to cancer cells. Small 2011, 7, 1816–1826. [CrossRef] [PubMed] Cheng, K.; Blumen, S.R.; MacPherson, M.B.; Steinbacher, J.L.; Mossman, B.T.; Landry, C.C. Enhanced Uptake of Porous Silica Microparticles by Bifunctional Surface Modification with a Targeting Antibody and a Biocompatible Polymer. ACS Appl. Mater. Interfaces 2010, 2, 2489–2495. [CrossRef] [PubMed] Li, Z.-Y.; Hu, J.-J.; Xu, Q.; Chen, S.; Jia, H.-Z.; Sun, Y.-X.; Zhuo, R.-X.; Zhang, X.-Z. A redox-responsive drug delivery system based on RGD containing peptide-capped mesoporous silica nanoparticles. J. Mater. Chem. B 2015, 3, 39–44. [CrossRef] Zhang, J.; Yuan, Z.F.; Wang, Y.; Chen, W.H.; Luo, G.F.; Cheng, S.X.; Zhuo, R.X.; Zhang, X.Z. Multifunctional envelope-type mesoporous silica nanoparticles for tumor-triggered targeting drug delivery. J. Am. Chem. Soc. 2013, 135, 5068–5073. [CrossRef] [PubMed] Chan, A.C.; Carter, P.J. Therapeutic antibodies for autoimmunity and inflammation. Nat. Rev. Immunol. 2010, 10, 301–316. [CrossRef] [PubMed] Carter, P. Improving the efficacy of antibody-based cancer therapies. Nat. Rev. Cancer 2001, 1, 118–129. [CrossRef] [PubMed] Weiner, L.M.; Surana, R.; Wang, S. Monoclonal antibodies: Versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 2010, 10, 217–327. [CrossRef] [PubMed] Pingarrón, J.M.; Yáñez-Sedeño, P.; González-Cortés, A. Gold nanoparticle-based electrochemical biosensors. Electrochim. Acta 2008, 53, 5848–5866. [CrossRef] Li, Y.; Schluesener, H.J.; Xu, S. Gold nanoparticle-based biosensors. Gold Bull. 2010, 43, 29–41. [CrossRef] Howes, P.D.; Chandrawati, R.; Stevens, M.M. Colloidal nanoparticles as advanced biological sensors. Science 2014, 346, 1247390. [CrossRef] [PubMed] Huang, X.; El-Sayed, M.A. Gold nanoparticles: Optical properties and implementations in cancer diagnosis and photothermal therapy. J. Adv. Res. 2010, 1, 13–28. [CrossRef]
Int. J. Mol. Sci. 2018, 19, 1991
39.
40.
41.
42. 43.
44. 45.
46.
47.
48. 49.
50. 51. 52. 53.
54. 55. 56. 57.
58. 59.
13 of 14
Brown, S.D.; Nativo, P.; Smith, J.-A.; Stirling, D.; Edwards, P.R.; Venugopal, B.; Flint, D.J.; Plumb, J.A.; Graham, D.; Wheate, N.J. Gold Nanoparticles for the Improved Anticancer Drug Delivery of the Active Component of Oxaliplatin. J. Am. Chem. Soc. 2010, 132, 4678–4684. [CrossRef] [PubMed] Sean Norman, R.; Stone, J.W.; Gole, A.; Murphy, C.J.; Sabo-Attwood, T.L. Targeted photothermal lysis of the pathogenic bacteria, pseudomonas aeruginosa, with gold nanorods. Nano Lett. 2008, 8, 302–306. [CrossRef] [PubMed] Dickerson, E.B.; Dreaden, E.C.; Huang, X.; El-Sayed, I.H.; Chu, H.; Pushpanketh, S.; McDonald, J.F.; El-Sayed, M.A. Gold nanorod assisted near-infrared plasmonic photothermal therapy (PPTT) of squamous cell carcinoma in mice. Cancer Lett. 2008, 269, 57–66. [CrossRef] [PubMed] Faulk, W.P.; Taylor, G.M. Immunocolloid method for the electron microscope. Immunochemistry 1971, 8, 1081–1083. [PubMed] Piludu, M.; Lantini, M.S.; Cossu, M.; Piras, M.; Oppenheim, F.G.; Helmerhorst, E.J.; Siqueira, W.; Hand, A.R. Salivary histatins in human deep posterior lingual glands (of von Ebner). Arch. Oral Biol. 2006, 51, 967–973. [CrossRef] [PubMed] Piras, M.; Hand, A.R.; Piludu, M. Electron microscopic immunogold localization of salivary mucin MUC5B in human buccal and palatal glands. Acta Histochem. 2011, 113, 844–847. [CrossRef] [PubMed] Piludu, M.; Piras, M.; Pichiri, G.; Coni, P.; Orrù, G.; Cabras, T.; Messana, I.; Faa, G.; Castagnola, M. Thymosin beta 4 may translocate from the cytoplasm in to the nucleus in HepG2 cells following serum starvation. An ultrastructural study. PLoS ONE 2015, 10, e0119642. [CrossRef] [PubMed] Piras, M.; Tandler, B.; Tomassini Barbarossa, I.; Piludu, M. Immunogold labeling of carbonic anhydrase isozyme (CA-VI) in secretory granules of human parotid glands. Acta Histochem. 2012, 114, 406–408. [CrossRef] [PubMed] Song, S.W.; Hidajat, K.; Kawi, S. Functionalized SBA-15 materials as carriers for controlled drug delivery: Influence of surface properties on matrix-drug interactions. Langmuir 2005, 21, 9568–9575. [CrossRef] [PubMed] Yu, H.; Zhai, Q.Z. Mesoporous SBA-15 molecular sieve as a carrier for controlled release of nimodipine. Microporous Mesoporous Mater. 2009, 123, 298–305. [CrossRef] Medda, L.; Casula, M.F.; Monduzzi, M.; Salis, A. Adsorption of lysozyme on hyaluronic acid functionalized SBA-15 mesoporous silica: A possible bioadhesive depot system. Langmuir 2014, 30, 12996–13004. [CrossRef] [PubMed] Piras, M.; Salis, A.; Piludu, M.; Steri, D.; Monduzzi, M. 3D vision of human lysozyme adsorbed onto a SBA-15 nanostructured matrix. Chem. Commun. 2011, 47, 7338. [CrossRef] [PubMed] Murtey, M.D. Immunogold Techniques in Electron Microscopy. In Modern Electron Microscopy in Physical and Life Sciences; InTech: London, UK, 2016; pp. 143–160. ISBN 978-953-51-2252-4. Bunea, M. New current Aspects on the Immunohistochemical Techniques. Biotechnol. Bioeng. 2001, 6, 177–209. Baschong, W.; Wrigley, N.G. Small colloidal gold conjugated to fab fragments or to immunoglobulin g as highresolution labels for electron microscopy: A technical overview. J. Electron Microsc. Tech. 1990, 14, 313–323. [CrossRef] [PubMed] Vandré, D.D.; Burry, R.W. Immunoelectron microscopic localization of phosphoproteins associated with the mitotic spindle. J. Histochem. Cytochem. 1992, 40, 1837–1847. [CrossRef] [PubMed] Takizawa, T.; Robinson, J.M. Use of 1.4-nm immunogold particles for immunocytochemistry on ultra-thin cryosections. J. Histochem. Cytochem. 1994, 42, 1615–1623. [CrossRef] [PubMed] Holgate, C.S.; Jackson, P.; Cowen, P.N.; Bird, C.C. Immunogold-silver staining: New method of immunostaining with enhanced sensitivity. J. Histochem. Cytochem. 1983, 31, 938–944. [CrossRef] [PubMed] Piludu, M.; Medda, L.; Cugia, F.; Monduzzi, M.; Salis, A. Silver Enhancement for Transmission Electron Microscopy Imaging of Antibody Fragment-Gold Nanoparticles Conjugates Immobilized on Ordered Mesoporous Silica. Langmuir 2015, 31, 9458–9463. [CrossRef] [PubMed] Thorpe, J.R. The application of LR gold resin for immunogold labeling. Methods Mol. Biol. 1999, 117, 99–110. [PubMed] Lah, J.J.; Hayes, D.M.; Burry, R.W. A neutral pH silver development method for the visualization of 1-nanometer gold particles in pre-embedding electron microscopic immunocytochemistry. J. Histochem. Cytochem. 1990, 38, 503–508. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2018, 19, 1991
60.
61. 62. 63. 64.
65.
66. 67.
14 of 14
Aggarwal, P.; Hall, J.B.; McLeland, C.B.; Dobrovolskaia, M.A.; McNeil, S.E. Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy. Adv. Drug Deliv. Rev. 2009, 61, 428–437. [CrossRef] [PubMed] Lynch, I.; Dawson, K.A. Protein-nanoparticle interactions. Nano Today 2008, 3, 40–47. [CrossRef] Huang, X.; El-Sayed, I.H.; Qian, W.; El-Sayed, M.A. Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J. Am. Chem. Soc. 2006. [CrossRef] [PubMed] Xie, J.; Lee, S.; Chen, X. Nanoparticle-based theranostic agents. Adv. Drug Deliv. Rev. 2010, 62, 1064–1079. [CrossRef] [PubMed] Delpiano, G.R.; Casula, M.F.; Piludu, M.; Corpino, R.; Ricci, P.C.; Vallet-Regí, M.; Monduzzi, M.; Salis, A. Assembly of Multicomponent Nano-bioconjugates Composed of Mesoporous Silica Nanoparticles, Proteins and Gold Nanoparticles. Submitted Manuscript. 2018. Rosi, N.L.; Giljohann, D.A.; Thaxton, C.S.; Lytton-Jean, A.K.R.; Han, M.S.; Mirkin, C.A. Oligonucleotide-modified gold nanoparticles for infracellular gene regulation. Science 2006, 312, 1027–1030. [CrossRef] [PubMed] Cheng, Y.; Samia, A.C.; Li, J.; Kenney, M.E.; Resnick, A.; Burda, C. Delivery and efficacy of a cancer drug as a function of the bond to the gold nanoparticle surface. Langmuir 2010, 26, 2248–2255. [CrossRef] [PubMed] Giljohann, D.A.; Seferos, D.S.; Prigodich, A.E.; Patel, P.C.; Mirkin, C.A. Gene regulation with polyvalent siRNA-nanoparticle conjugates. J. Am. Chem. Soc. 2009, 131, 2072–2073. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
