Adjuvant Properties of Gold Nanoparticles - Springer Link

ISSN 19950780, Nanotechnologies in Russia, 2010, Vol. 5, Nos. 11–12, pp. 748–761. © Pleiades Publishing, Ltd., 2010. Original Russian Text © L.A. Dykman, S.A. Staroverov, V.A. Bogatyrev, S.Yu. Shchyogolev, 2010, published in Rossiiskie nanotekhnologii, 2010, Vol. 5, Nos. 11–12.

REVIEWS

Adjuvant Properties of Gold Nanoparticles L. A. Dykman, S. A. Staroverov, V. A. Bogatyrev, and S. Yu. Shchyogolev Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, pr. Entusiastov 13, Saratov, 410049 Russia email: [email protected] Received March 31, 2010

Abstract—This review summarizes known data and the results of our own investigations into the application of gold nanoparticles as an antigen (AG) carrier and as an adjuvant in immunization in the in vivo preparation of antibodies (ABs). We have developed a technology for the production of ABs against various AGs by using colloidal gold as a carrier and as an adjuvant. The repeated injection of colloidal gold (CG)–AG conjugates (with or without the use of Freund’s complete adjuvant) into animals yielded specific hightiter ABs for a variety of AGs with no concomitant ABs. Gold nanoparticles used as an AG carrier activated the phagocytic activity of macrophages and influenced the functioning of lymphocytes, which apparently may be responsible for their immunomodulating effect. Thus, gold nanoparticles can facilitate the synthesis of ABs in rabbits, rats, and mice, in particular, by reducing the amount of required AG when compared to immunization using Freund’s complete adjuvant. DOI: 10.1134/S1995078010110029

INTRODUCTION Gold was a one of the first metals found by humans, and the history of its study goes back a few thousand years. The first book that we have on colloidal gold (CG) was published in 1618 by the philosopher and medical doctor Francisco Antonii [1]. It contained information about obtaining CG and its medical application, including practical advice. Until recently, gold salts (chrisotherapy) [2, 3] have been used for the treatment of autoimmune diseases and solutions of CG have been applied for the treatment of rheumatoid arthritis [4, 5]. The ability of gold nanoparticles to inhibit angiogenesis and the growth of tumor cells was found recently [6]. Gold nanoparticles with different sizes and shapes are applied for the targeted delivery of drugs [7, 8] and the photothermolysis of tumor cells [9–11]. A lot of information about CG and the impor tance of its application can be found in books and reviews [12–20]. In the 1920s, the immunogenic properties of col loidal metals and, in particular, gold attracted much interest from researchers. This was related, first of all, to the physical–chemical immunity theory of J. Bor det, who postulated that immunogenicity, like AG specificity, depends on the physical–chemical proper ties of substances and, first and foremost, on their col loidal state. L.A. Zil’ber successfully attempted to obtain agglutinating serums for CG [21]. Some studies have shown that injecting a complete antigen com bined with colloidal metals stimulates the production of antibodies (ABs) [22]. Moreover, it was found that some haptens can cause AT production, being adsorbed on colloidal particles [23]. The numerous

data on how CG influences nonspecific immune reac tions are described in the distinguished review by G. Pacheco [24]. It is noted that a considerable increase in the total amount of leukocytes in 1 ml of blood (from 9900 to 19800) on the background of an insufficient decrease of mononuclear forms (from 5200 to 4900) and a considerable increase of polynu clear (from 4700 to 14900) was observed 2 h after the intravenous injection of 5 ml of CG to rabbits. The injection of other colloidal metals did not cause any similar reactions. Unfortunately, interest in the immunological prop erties of colloids has decreased with the development of immunology and the denial of many statements of Bordet’s theory. In spite of this, data on the accelera tion of the immune response to the effect of AGs adsorbed on colloidal particles were used for creation of various adjuvants [25]. This review briefly describes two interrelated prob lems of modern immunology that have attracted the attention of numerous researchers. First is the produc tion of ABs for lowmolecular substances (haptens); second is the creation of a new generation of vaccines based on natural (microbial) or synthetic peptides [26]. It is known that the biosynthesis of ABs in an organism induces substances with sufficiently devel oped structures (immunogenicity). They include pro teins, polysaccharides, and some synthetic polymers [27]. A significant portion of active substances (neuro mediators, hormones, vitamins, antibiotics, and oth ers) has a relatively small molecular weight. Low molecular ABs belong to socalled weak AGs; i.e., no distinct response develops against them. Studies on the creation of socalled complex ABs, which are syn

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thesized macromolecular complexes with the required AG determinants and carriers and/or adjuvants, have been carrying out over last few years. In particular, the synthetic polyelectrolytes (polyLlysine, polyacrylic acid, polyvinyl pyridine, polystyrene sulfonate, ficoll, etc.) have been suggested as adjuvants [28]. These polymeric compounds are produced by the radical polymerization of the appropriate monomers. These carriers–adjuvants are capable of introducing AGs, accelerating the AG presentation to immunocompe tent cells, and inducing the production of the required cytokines. However, the low immunogenicity of these complexes is stipulated by the small epitope density and impels the researchers to search for new nontoxic and effective carriers with adjuvant properties. In our opinion, corpuscular carriers of nanosizes, such as polymeric nanoparticles (for instance polyme thylmethacrylate, polyalkylcyanoacrylate, polylac tidecoglycolide, and others) [29], liposomes, proteo somes and microcapsules [30–32], fullerenes [33, 34], carbonic nanotubes [35], dendrimers [36], paramag netic particles [37], and others are prospective in this regard. Their application changes the manner of immunogenicity of a certain substance in the immune system of the host organism. Antibodies adsorbed and entrapped by nanoparticles can be used as adjuvants for the optimization of organism’s response to vacci nation. In their pioneering work done in 1986 [38], Japa nese researchers reported on their successful attempt at producing ABs for glutaminic acid using CG parti cles as the carrier. After that, there were few studies published in which the authors applied and developed this method for the production of ABs to the following haptens: amino acids [39, 40], the plateletactivating factor [41, 42], quinolinic acid [43], recombinant pep tides [44–46], lysophosphatide acid [47], endostatin [48], αamidated peptides [49], peptides of capsid of hepatitis C viruses [50], azobenzene [51], Aβ peptides [52], and the surface AGs of Yersinia pestis [53]. In all the abovementioned studies, the hapten was directly conjugated with CG particles, mixed with Freund’s complete adjuvant (FCA), and injected into animals. As the result, high titer antiserums, which did not require further purification from ballast ABs, were obtained. Research was published in 1993 [54] in which the authors suggested attaching hapten (gammaaminobutyric acid) to a protein carrier before conjugation with CG. This suggestion was sup ported in the studies devoted to the production of ABs for some peptides [55–59], amino acids [60–63] and phenylβDthioglucuronide [64]. FCA and muramil dipeptides have been used in these studies as adjuvants. Antibodies obtained by this method had a high speci ficity to the studied AGs and higher titer (according to the authors [54] it was “extremely high”) from 1 : 250 000 to 1 : 1 000 000 in comparison with ABs obtained by the routine method. ImmunoSolution NANOTECHNOLOGIES IN RUSSIA

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offers ABs to some neurotransmitters and amino acids that were obtained according to Pow et al. [54]. Paper [65], which was published in 1996, first showed the possibility of applying CG particles as part of an antivirus vaccine as carriers of the AG protein of the capsid of tickborn encephalitis. According to the authors, in spite of fact that the vaccine did not con tain adjuvants, the suggested experimental vaccine possessed higher protective properties than commer cial analogues. Numerous researches have been published devoted to using CG particles for the creation of DNA vaccines [66, 67]. In the first studies, immunization was carried out by the subcutaneous or intramuscular injection of “naked” DNA. However, in order to obtain this, the “biolistic” transfection using CG particles was applied immediately, which was more efficient probably due to the multiplicity of interaction places of the transgene with the tissues and the direct penetration into the cells and nuclei [68]. The method of gene immuniza tion or socalled DNAvaccination developed in experiments with animals has shown the high effi ciency, especially in regard to the infections of tick born encephalitis, HIV infection, hepatitis B, and some others, in detail [69]. “Naked” DNA intramuscular injections are not in use for DNA vaccination. Nanoparticles have begun to be used as carriers of genetic material, and vaccine material is injected subcutaneously, intracutaneously, cutaneously, and intranasaly [70–72]. CG particles are the most popular nanoparticle carriers of DNA [73–75]. Polysaccharides and peptides are also used alongside DNA as the vector for similar vaccines [76, 77]. Thus, although gold used to be used only as a carrier, the authors of [78] noted that “although the mechanism behind this is not well understood, it appears that gold cartridges might enhance immune responses in vivo.” THE PRODUCTION OF ANTIBODIES USING GOLD NANOPARTICLES Since we have not found data on the mechanism of creating ABs during immunization using gold nano particles as carriers in the available literature, the aim of our study was to evaluate the efficiency of CG as a tool for the production of ABs for possibly a greater number of AGs of different natures and collect data on the mechanisms of their biosynthesis. Biotin (vitamin H) was chosen as the first AG to be used to develop the method [79]. It is known that biotin interacts highly specifically with the glycoprotein of whiteegg protein (avidin) or with the membrane pro tein of Streptomyces avidini (streptavidin (Kd = 10–15 M)). This interaction requires only a ureide ring of vitamin, and the carboxylic group of valeric acid residue in biotin can be modified, which makes it possible to obtain active biotinylated protein derivatives and nucleic acids [80]. Biotin related with macromolecule

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Table 1. Indexes of AB titers during the immunization of rabbits with AGs of Yersinia Compound

Immuni zation 1

CG + AG (1 mg) FCA + AG (100 mg) Physiological saline + AG (100 mg)

1 : 32 1 : 32 1:2

Immuni Reimmu zation 2 nization 1 : 256 1 : 256 1 : 16

1 : 10240 1 : 10240 1 : 512

keeps the ability to interact with an active center of avidin. The avidin–biotin system is widely applied in studies devoted to determining the localization and purification of proteins, investigating genetic struc tures using oligo and polynucleotide samples, in immune analysis, etc. Antibiotin ABs can be used as supplements to the application of avidin and streptavi din [81, 82]. Our previous experiments have shown the ineffi ciency of the traditional approach to the immuniza tion of rabbits using preparations of biotin conjugate with BSA. Therefore, according to the protocol described by Shiosaka et al. [38], particles of CG were used as the carrier for the immunization of animals with biotin. One milliliter of solution of a stable com plex of CGbiotin was mixed with 1.0 ml FCA. The obtained emulsion (1.0 ml) was introduced partially subcutaneously to a rabbit with a dose of 0.1 ml per dot. This procedure was repeated four times with an interval of two weeks. A similar scheme (with some modifica tions) was used for the production of ABs for other AGs. The ABs were verified by the methods of precipita tion rings, double immunodiffusion, and dot analysis using the indirect method of identifying coupled ABs with CGlabeled protein A [83]. Biotinylated BSA obtained according to Bayer et al. [80] was used as the model system. The reaction of immunoprecipitation has shown a clear visible ring with biotinylated BSA. The method of double immunodiffusion identified the precipitates at the dilution of the initial serum 1 : 128. Dot analysis with antibiotin ABs has shown to be more sensitive to the identification of biotin when compared with streptavidin, which agrees with the literature data [81, 82]. The following step of the study included the immu nization of rabbits using highmolecular complete

AGs (the surface protein of Yersinia pseudotuberculosis bacteria [84]). The data characterizing the changes of the AB titer upon immunization according to various schemes are shown in Table 1. The first group of rab bits were immunized with 100 μl of conjugate AGs with CG (the injection dose was 1 μg AGs). The sec ond group was immunized with 100 μg AGs diluted in a physiological saline and emulsificated in 100 μl of FCA. The third control group was immunized with 100 μg AGs diluted in a physiological saline. For a week after the first immunization, the AB titer was 1 : 32 in the test group (using FCA or CG) and 1 : 2 in the control group (without the application of adjuvants). After the second immunization, the titers of ABs in all groups were 1 : 256 for the first and second groups and 1 : 16 for the third group. The general results were obtained after reimmunization. In the second group, where FCA was used as the adjuvant, the AB titer increased to 1 : 10240. In the experiment where CG was used as the adjuvant, the titer was also 1 : 10240. However, the immunization dose was two orders of magnitude lower. The titer was 1 : 512 upon the injec tion of AGs with a physiological saline. A study of the phagocytic activity of lymphoid cells was carried out taking into account the ability of the cells to reduce the grains of formazan in the test with nitrotetrazolium blue (MTT test) and on the basis of the formation of cationic protein in the cytoplasm of lymphocytes (Tab. 2). The parameters of serum of nonimmunized rabbits are shown as the control in Table 2. The study results are evidence of the ability of CG to stimulate AB production during immunization with AGs from the cell wall of microbial cells. This is indicated by an increase in activity both of cationic proteins of the cells of the neutrophilic group and the higher activity of these cells in the reduction of forma zan grains during the MTT test. In this case, FCA causes a similar or even lower reaction. In our opinion, the intensified phagocytosis of the particles by mac rophages can be responsible for the adjuvant effect of CG. The resulting data show the efficiency of applying CG as an adjuvant for the production of an immune serum against complete AGs. At the next stage, we obtained ABs for a weak immunogenic highmolecular AG (BSA) and evalu ated the factors of the natural cellular and humoral

Table 2. Indexes of the alteration of activity of cation protein (CP) and the reduction of formazan in the MTT test during immunization Immunization 1

Immunization 2

Immunization 3

Compound CG + AG FCA + AG Physiological saline + AG Control

CP

MTT

CP

MTT

CP

MTT

255 ± 1.4 153 ± 1 120 ± 0.9 110 ± 1

92 ± 0.5 62 ± 0.6 50 ± 0.6 50 ± 1

291 ± 0.9 175 ± 0.9 175 ± 1.2 115 ± 1

118 ± 0.4 113 ± 0.4 100 ± 0.5 50 ± 1

300 ± 1.2 160 ± 1.2 130 ± 1.2 115 ± 1

120 ± 0.6 113 ± 0.6 110 ± 0.4 49 ± 1

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resistance of the organism of rats. The animals were immunized with an AG conjugated with CG, and an AG in a mixture with FCA and AG were diluted in a physiological saline. The growth of titer of serums dur ing the immunization of rats with BSA according to the schemes described above is shown in Table 3. The fact that the maximal titer after immunization was in a serum obtained after the injection of the BSA + CG conjugate without FCA is noteworthy. An analysis of the alterations of nonspecific factors of cellular and humoral resistance during the immuni zation of rats with BSA according to different schemes has shown that CG used as an AG carrier activates the phagocytic activity of lymphoid cells, which probably determines its immunomodulating effect. The task of the following stage of our research was to evaluate the efficiency of applying CG as the means for producing ABs in vivo with a greater amount of AGs of different natures in order to unify the methods and identify the adjuvant properties of CG [85, 86]. Highmolecular AGs such as bacteriorhodopsin, chicken smoothmuscular actin, the surface AG of Salmonella typhimurium, and the transmissive gastro enteritis virus have been used as complete AGs. Some antibiotics with different chemical compositions like levomicetin, gentamycin, neomycin, lincomycin, kana mycin, clindamycin, and tilmicosin; antiparasitic prep arations like ivermectin and diminazene; β2adrenos timulator clenbuterol; the antagonist of β2adrenergic receptors xylazine; and steroid hormone nortestoster one have been used as haptens for immunization. In addition, we used three synthetic peptides of bacteri orhodopsin, two synthetic peptides aquaporins, two synthetic troponin peptides, synthetic peptide from human protooncogene cmyc (further “cmyc peptide”), indole3acetic acid phytohormone, and tuberculin. The most interesting results and observa tions obtained during the study are introduced further. ABs with a sufficiently high titer, which varied for different AGs from 1 : 256 to 1 : 51 200, were obtained for all AGs. The greatest difficulties were related to the

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Table 3. Alteration of titers of serums during the immuni zation of rats with BSA Compound

Immuniza tion 2

Immuniza tion 3

Immuniza tion 4

BSA + CG BSA + FCA BSA

1 : 128 1 : 128 1 : 128

1 : 2560 1 : 2560 1 : 1280

1 : 6400 1 : 3200 1 : 2560

“joining” of haptens and the gold particles. In partic ular, since ivermectin and tilmicosin can be dissolved in water, the conjugation CG with these substances was carried out in a solution of polypropylene glycol or dimethylacetamide [87]. Not all haptens stabilized the gold sol that appeared; the gold number (the minimal protective amount of substance preventing the salt aggregation of CG) could not be determined. In this case we have mixed 0.1–0.5% solutions of haptens with a CG in ratio 1 : 1. Thus, the amount of the AG used per each injection varied within the range of from 0.2 to 125 μg. Nevertheless, these amounts were always less than those used for the conjugates of haptens with proteins or complete AGs. Using the application of diminazene as the AG, we could not obtain the stable conjugates of diminazene with CG. Therefore, first we obtained a conjugate of diminazene with BSA, then the diminazeneprotein complex was conjugated with CG [88]. This considerably decreased the dose for immunization. The production of ABs for actins is still difficult due to the high level of identity of their amino acid sequences in the members of various taxonomic groups and their correspondingly low immunogenic ity. One case has been described when one rabbit out of fifteen had a positive immune reaction to chicken smoothmuscular actin with AB titer in serum 1 : 250 [89]. We have obtained ABs for the conjugate of actin with CG with titer 1 : 1024 [90, 91]. The result of a dot analysis of chicken Gactin with conjugate CG with antiactin ABs is shown in Fig. 1. ABs were obtained by

1

2

3

4 Fig. 1. Results of a dot analysis of chicken Gactin by conjugate CG with antiactin ABs. Ab obtained by the immunization of mice with native actin (1), actin with FCA (2), a conjugate of actin with CG (3), and a conjugate of actin with CG using FCA (4). NANOTECHNOLOGIES IN RUSSIA

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(b) (2) (a)

(b) (3) (a)

(b)

(c)

Fig. 2. Results of a dot analysis of cmyc peptide (a) a conjugate of cmyc peptide with BSA (b), and BSA (c). Revealing serums obtained upon the immunization of mice with a conjugate of cmyc peptide with CG without FCA (1), with FCA (2), and a con jugate cmyc peptide with BSA using FCA (3).

the immunization of mice with native actin, actin using FCA, the conjugate of actin with CG, and the conjugate of actin with CG using FCA. Test results of the ABs that were produced have shown that the minimal detectable amount of ABs (both haptens and complete AGs) in dot analysis using serums that were obtained using CG is not lower than when using the serums obtained by the routine method. The specificity of ABs obtained by the method described above is often higher than in ABs obtained upon immunization with the conjugate of hapten with protein. It was found for clenbuterol that serum obtained from a rabbit immunized with the conjugate clenbuterol–CG is more specific because it interacts only with terbutaline. In contrast, the serum obtained from rabbits immunized with conjugate clen buterol + BSA has shown crosses with all used ana logues of clenbuterol (terbutaline, phenoterol, and salbutamol) [92]. In the immunization of mice with the cmyc peptide with and without FCA, no positive reaction was observed (Fig. 2). Attention is drawn to the fact that the response was determined using AG and CG conjugates without FCA. Thus, the experimental data allow the following conclusions:

(1) ABs for haptens, in particular, antibiotics, vita mins, and nonimmune peptides, in which ABs that are difficult to be obtained using the traditional method can be obtained by the method of “gold immunization”; (2) The amount of ABs used in this case for immu nization is sufficiently less than in the traditional method, even if the immune response can be obtained with their application; (3) The immune reaction without the usage of other adjuvants was obtained in the experiments with haptens and complete AGs; (4) The gold nanoparticles used as the AG carrier activate phagocytic activity of lymphoid cells. In our opinion, all the abovementioned factors significantly testify to the adjuvant properties of CG. However, a question appeared in regard to the action mechanism of gold nanoparticles. It is reasonable to begin studies in this field from an evaluation of the interaction between gold nanoparticles and the cells of the immune system ex vivo. STUDY OF THE ADJUVANT PROPERTIES OF GOLD NANOPARTICLES The direct influence of CG particles (without AGs) on the culture of lymphoid cells has been studied in


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the reaction of blastcell transformation. This reaction indicates the total immune reactivity of the organism and indirectly evidences the potential ability of the immune system to react to an AG [27]. The induced AG activation and differentiation of T and B cells usu ally occurs in lymphoid tissues and can be repeated in vitro upon the cultivation of lymphocytes in the pres ence of the activating agent. Bacterial lipopolysaccha rides (LPSs) and lectins (more often phytohemagglu tinin (PHA) and concanavalin A) in particular can serve as these agents. Lectins stimulate Tlymphocytes and LPSs stimulate B cells. We have evaluated the influence that CG particles with diameters of 15 and 60 nm have on the reaction of the blast cell transformation of lymphocytes in the presence of PHA and LPSs from Pseudomonas aerug inosa. These results are shown in Tab. 4. As follows from Table 4, there is an increase in the percentage of blasttransformed lymphocytes after the addition of CG. The application of PHA with the addition of CG with a particle size of 15 nm resulted in an 11% increase in blasttransformed lymphocytes and, with the addition of CG with a particle size of 60 nm, there was 20% increase when compared to application of PHA without CG in control. The amount of blast transformed lymphocytes increased by 8% when using LPSs with CG with a particle size of 15 nm and it increased by 5% when CG with a particle size of 60 nm was added when compared with the application of LPSs without CG. Thus, it can be concluded that, besides stimulating phagocytic activity, particles of CG are able to activate lymphocytes (mostly T cells). In relation with this, the first task is to search for an answer to the following question: with which immu nocompetent cells do conjugates of CG with AGs interact at the initial stages of immunization? We have obtained peritoneal macrophages and lymphocytes of peripheral blood. Conjugates of CG with hapten (iver mectin) were incubated for 3 h with suspensions of these cells. After that, the cells were washed three times by a cold physiological saline and lysed by 0.1% Tween 80. The amount of hapten in cell lysate was determined by highperformance liquid chromatogra phy. As a result of this analysis, we determined that about 45% of hapten added to the cells is contained in the lysate of macrophages, while the lysate of lympho cytes contained about 5%. Thus it can be concluded with a greater probability that conjugates of an AG with CG at the first stages of immunization are included in the cytoplasm of microphages. This result was confirmed by immunocytochemical analysis using fluorescent microscopy. A conjugate of ivermectin with CG was incubated with a suspension of peritoneal microphages. After that, the hapten accumulated in them was identified using phage miniantibodies to ivermectin labeled with fluorescein isothiocyanate (FITC). Immunocytochemical analysis has demon NANOTECHNOLOGIES IN RUSSIA

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Table 4. Indexes of blasttransformation of lymphocytes (%) upon stimulation by various mitogens Compound CG15 CG60 Control with mitogen Control without mitogen

PHA as mitogen LPS as mitogen 70.3 ± 0.4 80 ± 0.4 59.3 ± 0.4 12.6 ± 0.4

74.6 ± 0.4 71.6 ± 0.4 66.6 ± 0.4 14.3 ± 0.4

strated the penetration of a conjugate of hapten with CG into the cytoplasm of macrophages [93]. The next experiment was carried out in collabora tion with M.V. Sumaroka, a colleague at the Univer sity of Pennsylvania (United States). The scheme of the experiment was that a synthetic HIV peptide and a conjugate of this peptide with CG in a twofold dilution was added to B cells (targets), which were restricted on this peptide and further added with the cytotoxic T cells. A specific peptide must cause the proliferation of these cells. The experiments showed that the peptide “sitting” on gold accelerated proliferation 10 times (!) when compared with the native peptide. Thus there was direct proof on the influence that CG particles have on the functional activity of immunocompetent cells. This fact shows the principal opportunity for the purposeful activation of the T cell (for instance, AGs of the mycobacteria of tuberculosis, HIV, etc.) with the further activation of macrophages by them and the destruction of a pathogen, which is obviously a pro spective approach for the creation of a new generation of vaccines. The following part of our study was devoted to investigating the interaction of CG with phagocytes using an evaluation of the alterations of the activity of cell respiration and the activity of mitochondrial dehydrogenases of peritoneal macrophages of rats and mice upon their interaction with conjugates of gold nanoparticles with high and lowmolecular AGs. The protein complexes isolated from S. typhimurium by the extraction of cells with dimethyl sulfoxide (DMSO) were used as complete AGs. Ivermectin conjugated with gold nanoparticles of the same size was used as lowmolecular AGs. The incubation of rat peritoneal macrophages, which were isolated according to the standard method, with conjugates was carried out for 48 h at 37°C. First, the influence that conjugates of highmolec ular AGs with CG have on the respiratory activity of peritoneal rat cells in vitro has been evaluated. The highest level of reduction of formazan was determined upon the cultivation of cells with conjugate DMSO AG + CG and with CG itself and was 0.26 ± 0.05 mg/ml and 0.25 ± 0.05 mg/ml, respectively (Fig. 3). The amount of reduced formazan in the cells cultivated in the presence of an AG and without it (the control) was 0.15 ± 0.03 and 0.06 ± 0.01 mg/ml, respectively. An analysis of the interaction between conjugates of lowmolecular AG (ivermectin) and CG (Fig. 4)

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Colloidal gold

0.07

Colloidal gold + cells

Ivermectin + colloidal gold

0.06

AG + cells

0.07 Formazan concentration, mg/ml

Formazan concentration, mg/ml

754

0.05 Cells

0.04 0.03 0.02 0.01

Ivermectin

0.06 Dimethylacetamide

0.05 Control

0.04 0.03 0.02 0.01

0 0

has shown similar results. The concentration of reduced formazan was 0.063 ± 0.003 mg/ml upon an interaction of the cells with CG that twice exceeds the control, where the concentration of formazan was 0.037 ± 0.003 mg/ml. The addition of either ivermec tin or solvent DMSO to the cell population results in the inhibition of cell respiration, which is confirmed by the obtained values for the concentration of forma zan of 0.01 ± 0.002 mg/ml and 0.009 ± 0.003 mg/ml, respectively. However, the presence of CG in the con jugate with ivermectin caused a significant decrease in the toxic effect, and the amount of reduced formazan in the sample was 0.018 ± 0.003 mg/ml. An analysis of the influence that CG has on the activity of mitochondrial enzymes (succinate dehy drogenase and glycerophosphate dehydrogenase) yielded the following results. In experiments with suc cinate dehydrogenase, the concentration of reduced formazan in the samples with cells was 0.0256 ± 0.006 mg/ml, upon the cultivation of cells with conju gate ivermectin–CG it was 0.0385 ± 0.004 mg/ml, upon the cultivation of cells with ivermectin it was 0.0119 ± 0.002 mg/ml, and in the control it was 0.0095 ± 0.006 mg/ml (Fig. 5). The concentration of reduced formazan under the influence of CG on alphaglycerophosphate dehydro genase in samples of the cells cultivated with CG was 0.017 ± 0.004 mg/ml, in samples with the cells culti vated with conjugate CG–ivermectin it was 0.016 ± 0.004 mg/ml, in samples with the cells cultivated with ivermectin it was 0.007 ± 0.001 mg/ml, and in the con trol it was 0.015 ± 0.004 mg/ml (Fig. 6). Thus, evaluations of the activity of oxidation reduction enzymes located in the mitochondria of the cytoplasm of immunesystem cells have shown that

Fig. 4. Results of MTT test on peritoneal rat macrophages after their incubation with the conjugate of CG with iver mectin.

conjugate ivermectin–CG increases mitochondrial activity. It was determined that CG also increases the mitochondrial activity of the cells. These observations, which evidence an increase in the respiratory activity of the cells of the reticuloendothelial system affected by CG in the experiments in vitro, supplement the data on the possible mechanisms of the immunomod ulating properties of gold nanoparticles. The following step of our research was to study the influence that gold nanoconjugates have on the cells of the reticuloendothelial system in the organism of white nonlinear mice. It was found that the addition of an AG conjugated with CG caused the highest activity of macrophages and the concentration of reduced for 0.045 Formazan concentration, mg/ml

Fig. 3. Alteration of concentration of reduced formazan depending on the cultivation conditions of DMSOAG with peritoneal rat macrophages

Colloidal gold

0.040 0.035

Ivermectin + colloidal gold

0.030

Ivermectin

0.025 Control

0.020 0.015 0.010 0.005

Fig. 5. Influence that ivermectin and its conjugate with CG have on the activity of succinate dehydrogenase.


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ADJUVANT PROPERTIES OF GOLD NANOPARTICLES Colloidal gold Ivermectin + colloidal gold


0.025

Ivermectin

0.020

Control

0.015 0.010 0.005 0

Fig. 6. Influence that ivermectin and its conjugate with CG have on the activity of alphaglycerophosphate dehydroge nase.

mazan was 0.26 ± 0.03 mg/ml (Fig. 7). The injection of a nonconjugated AG into mice resulted in an insuf ficient decrease of macrophage activity, which corre sponded to a concentration of 0.23 ± 0.02 mg/ml of reduced formazan. The concentration of formazan in the control group was 0.15 ± 0.03 mg/ml. It was shown that the bacterial activity of the serum was on average 20% higher than in the control if a conjugate AG with CG was used for immunization. These data evidence an absence of any toxic effect of CG in regard to the peritoneal cells possessing phagocytic activity and the smoothing of the activity of a toxic AG conjugated with CG [94].

In order to verify this supposition, we have solved the task of AB production to conjugates of tuberculin with CG and studied the interaction mechanisms between this complex and the phagocytes of the immune system. Tuberculin is used for the diagnostics of tuberculo sis, which is extracted from mycobacteria consisting of thermostable peptides and fatty acids. Tuberculin is a diagnostic preparation widely applied to diagnose tuberculosis as a skin test (the Mantoux test) [95]. Numerous questions related with the mechanisms of the influence that tuberculin has on the organism of animals and people are still unsolved. It is known that, upon intradermal injection, ABs for tuberculin do not form and the immune response is cellular. Therefore, producing ABs for tuberculin and studying the inter action between tuberculin and the immunocompetent cells using them are important tasks. The production of specific ABs for tuberculin is complex because its composition includes a range of proteins with molecular weights of from 9 to 65 kDa [96]. Major polypeptide 9.7 kDa in the composition of tuberculin is capable of inhibiting the migration of mac rophages and partially inhibiting blasttransformation in guinea pigs, thus inhibiting AB production [97]. Tuberculin purifiedprotein derivative (PPD) has been used as an AG. Tuberculin was conjugated with CG with an average particle diameter of 15 nm. This conjugate was intramuscularly injected into three chinchilla rabbits four times with an interval of 14 days. The blood of the animals was sampled for the presence of ABs 10 days after the fourth injection. The control group obtained tuberculin nonconjugated with gold in a similar dose. The titer of the serums was determined by ELISA using antirabbit ABs labeled with the peroxidase of horseradish. The sensitivity was determined by dot analysis. As a result of the immunization of rabbits with con jugates of tuberculin with CG, the titer of the serums DMSOAG + colloidal gold


0.35 0.30

Native control

0.25 Control 0.20 0.15 0.10 0.05 0

Fig. 7. Result of MTT test with mouse macrophages after the injection of an AG and its conjugate with CG into the animals. NANOTECHNOLOGIES IN RUSSIA

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(a)

(b)

Fig. 8. Dark field image of peritoneal rat cells cultivated with a conjugate of CG with (a) tuberculin and (b) native tuberculin.

in all three rabbits was 1 : 512. No ABs for tuberculin were found in the blood serum sampled in the control group of animals injected with nonconjugated prepa ration. A test of the serum by dot analysis determined that the sensitivity of serums was, on average, 0.78 nm. The following step of our investigation included a study of the mechanisms of the interaction between the conjugates and the phagocytes of the immune sys tem. The dynamics of the alteration of the respiratory activity of the peritoneal rat cells cultivated in the presence of tuberculin and the conjugate of tuberculin with CG in the MTT test has been studied. It was determined that tuberculin possesses toxicity in regard to peritoneal cells, and their respiratory activity decreases 37% every 4 h and 40% every 6 h when com pared with cell respiration in the control group. The respiratory activity of the cells cultivated with the con jugate of tuberculin with CG decreases 17% in 4 h; however, after that, the activity of the cells is gradually reduced and the deviation between the sample and control becomes 8% after 6 h. There was one last unsolved question on the local ization of the conjugate of tuberculin with CG in phagocytes. Therefore, we conducted a microscopy

Fig. 9. Permeation of conjugate of CG with tuberculin + FITC into peritoneal macrophages (confocal micros copy).

analysis of the preparations of the cells cultivated in the presence of tuberculin and its conjugates with CG. The localization of colloidal gold particles in the cells and their membranes is shown on Fig. 8a. The colloi dal gold in the cells glowed red upon overhead illumi nation. This luminescence was not observed during the cultivation of cells with nonconjugated tuberculin (Fig. 8b). In order to clarify the localization of the conjugate (if it permeates inside macrophages or is localized on the membrane), we used confocal laser microscopy. One of the imaged Z stacks is shown in Fig. 9. The optical section demonstrates the perme ation of gold particles into the cytoplasm of macroph ages. It should be underlined as a result of these experi ments that CG partially relieves the toxic effect of tuberculin in peritoneal rat macrophages due to pene tration into intracellular space. It promotes the more active development of the humoral reaction and the production of ABs for tuberculin. Therefore, CG can be used as a carrier for the production of ABs to the substances that possess a toxic effect. Further, the ABs will be used for the creation of diagnosticum using Mycobacterium tuberculosis. ABs obtained upon the immunization of the ani mals with the transmissive gastroenteritis virus that were conjugated with gold nanoparticles had a higher titer (1 : 1365) than ABs for the native virus (1 : 170). Immunization with a virus–CG complex resulted in the reliable acceleration of the respiratory activity of peritoneal macrophages and an increase in the level of γ interferon in the blood plasma of immunized ani mals. Thus, we have shown the variants of AB production in vivo to complete AGs and haptens of different natures using CG as the carrier. The production of ABs for the conjugates of some haptens and complete AGs with CG without FCA can evidence the adjuvant properties of CG, which is prospective, e.g., in researches devoted to a new generation of vaccines. It is shown that CG used as a carrier of AG activates the phagocytic activity of macrophages (probably due to the acceleration of mitochondrial respiration) and


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influences the functions of lymphocytes, which prob ably determines its immunomodulating effect. The most interesting aspect of the immunogenic properties of haptens upon their immobilization on CG is that the gold nanoparticles play the role of carrier and, thus, deliver hapten to T cells. It is a fact that substances of different natures that have different molecular weights (69 000 Da in BSA and about 700–1000 Da in the antibiotics and pep tides used in our study), chemical natures (polypep tides and polyheterocyclic compounds), and physi cal–chemical properties have been used as an AG fixed to CG particles. The fixation of the molecules used by us on the solid surfaces occurs as a result of socalled weak inter actions, i.e., Van der Waals, Coulomb, and hydropho bic. The amount of AGs adsorbed on the gold nano particles and preventing colloid from aggregation is usually 5–15 μg per 1 ml of sol. The surplus of uncou pled AGs can be removed by centrifugation. Thus, a dose of AGs for immunization becomes considerably less than that required for the application of AGs with FCA (in particular, that shown above in the case of Yersinia AGs; see Table 1). It is noted that the absorp tion of biomolecules is a dynamical process and sub stances very similar to goldlike proteins of blood serum can displace biomolecules from the surface of gold particles. However the nature of the absorbent proba bly does not affect the adjuvant properties of CG. The presence of dissociating and polar groups in the structure of biotin yields its strong interaction with the surface of metal colloid particles. However, the rel atively small molecular weight does not stabilize met alcolloids against salt aggregation. In spite of their considerable larger molecular weight when compared with biotin, the peptides of bacteriorhodopsin do not stabilize CG because their composition does not have amino acids with base groups in the side chain. The c myc peptide is of great interest from the point of view of absorption on CG and protective activity. The com position of this peptide includes five residues with dis sociating groups in the side chains like lysine, glutaminic, and asparaginic acids. The N terminal end of peptide brings cysteine residue (which is absent in the native sequence) with tertbutyl protective thiolic group. In spite of the smaller sequence length and respective molecular weight, the cmyc peptide totally stabilized CG, in contrast to peptides 1 and 2 of bacte riorhodopsin. The markers obtained by conjugation with an AG specifically identified the homologue serums. The cross reactions were observed only in the case of iver mectin and tilmicosin during this experiment. How ever, in our opinion, these results are evidence in favor of the conclusion that the molecules with molecular weights lower than 1000 Da are strongly fixed on the surface of gold particles to be delivered to immuno competent cells. This fact can be confirmed by vibra tional spectroscopy [98]. NANOTECHNOLOGIES IN RUSSIA

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There is currently no answer to the question about the origin of the adjuvant properties of gold nanopar ticles, but the final chapter of this review describes some suppositions. INTERACTION BETWEEN GOLD NANOPARTICLES AND CELLS OF THE IMMUNE SYSTEM What is known today about the interaction between gold nanoparticles and immunocompetent cells which makes it possible to come to a conclusion about the mechanisms of the immune reaction upon the injec tion of an AG conjugated with CG? These data are meager and contradictive. In our opinion, the discus sion in the research work of Pow et al. [54] on the pre ferred macrophage response to corpuscular AGs as opposed to soluble is correct. This fact is confirmed by researchers studying the mechanisms of DNAvaccine action which use gold particles for the transport of genetic material into the cell [73, 78]. These studies show the role of Kupffer and Langerhans cells in the formation of the immune response. The influence that dendritic cells have on the formation of the immune response upon the injection of an AG conjugated with gold nanoparticles is discussed in [99, 100]. The secre tion level of cytokines has increased, which indicated the activation of the immune response. The authors of [99] noted that, during the application of nanoparti cles in medical practice, it is necessary to make sure that there are no LPSs on their surface. Gold nanoparticles were used by some researchers to study endocytosis in macrophages [101], Kupffer cells [102], neutrophils [103], and tumor cells [104, 105]. The gold particles included into the lysosomes of phagocytes were called aurosomes [106]. The influ ence that the size and shape of nanoparticles and the size and nature of the molecules adsorbed on them (in particular, peptide CALNN) have on the ability to penetrate conjugates inside the cell [107–109] and cytotoxicity [110, 111] are being discussed. It is noted that gold nanoparticles are not recognized by immu nocompetent cells during their conjugation with inert polymers (mostly with polyethylene glycol) [112]. Studies [113, 114] on the microscopic level have shown that the permeation of gold nanoparticles con jugated with peptides into the cytoplasm of macroph ages caused their activation. These authors deter mined that the permeation of nanoparticles into the cell, entailing the excretion of proinflammatory cytokines TNFα, IL1β and IL6, and the inhibition of macrophage proliferation occurs after conjugates interact with the TLR4 receptors of macrophages. The review by Dobrovolskaya et al. [115] suggests another (noninflammatory) approach to the perme ation of gold nanoparticles into macrophages by inter action with scavenger receptors. However these data do not yield an answer to the further mechanisms of how an AG is presented to

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T helpers. If, according to the modern assumptions [27], the processing preceded the presentation of AGs to Tcells, i.e., its splitting to peptide fragments, and then the formation of a connection with molecules of the main major histocompatibility complex which transport a fragment of an AG to the surface of AG presenting cell, then the question arises as to how this process can occur with hapten. The hypothesis on multivalent AGs, i.e., AGs created by a high local con centration of a monovalent AG on the surface of a gold particle, does not provide an answer to this question. Studies [4, 5] reported on the successful therapy of rheumatoid arthritis with a colloid solution of gold. According to the study by Graham [2], the effect of CG in this case is the result of the inhibition of mono cyteinduced proliferation of lymphocytes. Merchant’s study [116] discusses the transformation of Au (0) to Au (I) in the cells of an immune system affected by some sulfur amino acids. Contradicting data have been found in studies [117, 118] devoted to investigating the sensitivity of mammalian organisms to the injection of metal gold. In particular, the study by Eisler [117] noted that injecting CG into laboratory animals can result in inflammatory reactions, the accumulation of gold in the reticular cells of the lymphoid tissue, and the acti vation of cellular and humoral immunity. At the same time, in [118], the authors studied the influence of gold nanoparticles on the cells of the immune system and concluded that CG particles are not cytotoxic or immunogenic and are biocompatible material for potential application in various fields of nanoimmu nology, nanomedicine, and nanobiotechnology. In spite of that, researchers have found gold nanoparti cles in the lysosomes and perinuclear space of mac rophages using atomic absorption analysis. The influ ence that nonconjugated CG have on the immuno competent cells in vivo was studied in [119, 120]. The authors showed that injecting CG into mice causes the proliferation of lymphocytes and cytotoxic Tcells to accelerate and the production of IL2 to increase. It was mentioned above that synthetic and natural polymeric biodegradable nanomaterials (polymethyl methacrylate, poly(lactidcoglycolide), chitosan, gelatin, and others) can serve as AG carriers [121]. The advantages of nanoparticles of this type is that they are utilized well in the organism, the target substance is highly efficiently involved, there is a higher capability to overcome different physiological barriers, and there are less systematic side effects. The action mecha nisms of biodegradable nanoparticles and CG as carri ers of AGs in the immune system are probably similar. These two types of nanoobjects can compete in the development of a new generation of vaccines [123], taking into account data on bioinertness, low toxicity, and the good excretion of gold nanoparticles from the organism with the involvement of a hepatobiliary sys tem [19, 122].

Numerous studies have appeared recently (for instance, [7, 8]) where questions on the application of gold nanoparticles for the delivery of drugs are consid ered. It should be mentioned that, in our opinion, this problem should be carefully approached while taking into account the opportunity for the production of ABs in the organisms of animals and people to the injected drug adsorbed on CG particles. It is noted in conclusion that it is probably time to speak not only about biochemistry, but also about the biophysics of immune response, because the unique biophysical properties of metal particles, in particular, the surface charge and electrostatic field of a particle influencing the charge, orientation, and polarization of adsorbed AG molecules on the particles, consider ably influence the immuneresponse process. ACKNOWLEDGMENTS The authors thank M.V. Sumaroka, N.G. Khlebt sov, O.I. Sokolov, and V.A. Nesmeyanov (deceased) for their collaboration in the studies discussed in this review. REFERENCES 1. F. Antonii, Panacea AureaAuro Potabile (Ex Bibliopo lio Frobeniano, Hamburg, 1618). 2. G. Graham, Agents Actions Suppl. 44, 209 (1993). 3. N. R. Panyala, E. M. PeñaMéndez, and J. Havel, J. Appl. Biomed. 7, 75 (2009). 4. G. E. Abraham and P. B. Himmel, J. Nutr. Environ. Med. 7, 295 (1997). 5. C. L. Brown, G. Bushell, M. W. Whitehouse, D. S. Agrawal, S. G. Tupe, K. M. Paknikar, and E. R. T. Tiekink, Gold Bull. (London) 40, 245 (2007). 6. R. Bhattacharya and P. Mukherjee, Adv. Drug Delivery Rev. 60, 1289 (2008). 7. G. F. Paciotti, L. Myer, D. Weinreich, D. Goia, N. Pavel, R. E. McLaughlin, and L. Tamarkin, Drug Delivery 11, 169 (2004). 8. P. Ghosh, G. Han, M. De, C. K. Kim, and V. M. Rotello, Adv. Drug Delivery Rev. 60, 1307 (2008). 9. C. M. Pitsillides, E. K. Joe, X. Wei, R. R. Anderson, and C. P. Lin, Biophys. J. 84, 4023 (2003). 10. X. Huang, P. K. Jain, I. H. ElSayed, and M. A. El Sayed, Lasers Med. Sci. 23, 217 (2008). 11. S. Lal, S. E. Clare, and N. J. Halas, Acc. Chem. Res. 41, 1842 (2008). 12. Colloidal Gold: Principles, Methods, and Applications, Ed. by M. A. Hayat (Academic, San Diego, California, United States, 1989). 13. L. A. Dykman, V. A. Bogatyrev, S. Yu. Shchegolev, and N. G. Khlebtsov, Gold Nanoparticles: Synthesis, Prop erties, and Biomedical Applications (Nauka, Moscow, 2008) [in Russian]. 14. Gold Nanoparticles: Properties, Characterization, and Fabrication, Ed. by P.E. Chow (Nova Science, New York, 2010).


Vol. 5

Nos. 11–12

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ADJUVANT PROPERTIES OF GOLD NANOPARTICLES 15. J. A. Edgar and M. B. Cortie, in Gold: Science and Applications, Ed. by C. Corti and R. Holliday (CRC, Boca Raton, Florida, United States, 2010), p. 369. 16. L. A. Dykman and V. A. Bogatyrev, Usp. Khim. 76, 199 (2007) [Russ. Chem. Rev. 76, 181 (2007)]. 17. C. L. Brown, M. W. Whitehouse, E. R. T. Tiekink, and G. R. Bushell, Inflammopharmacology 16, 133 (2008). 18. P. C. Chen, S. C. Mwakwari, and A. K. Oyelere, Nan otechnol. Sci. Appl. 1, 45 (2008). 19. E. Boisselier and D. Astruc, Chem. Soc. Rev. 38, 1759 (2009). 20. V. Sharma, K. Park, and M. Srinivasarao, Mater. Sci. Eng., A 65, 1 (2009). 21. L. A. Zil’ber and V. V. Frize, Zh. Eksp. Biol. 11, 128 (1929). 22. D. B. Steabben, Br. J. Exp. Pathol. 6, 1 (1925). 23. J. Zozaya and J. Clark, J. Exp. Med. 57, 21 (1933). 24. G. Pacheco, Mem. Inst. Oswaldo Cruz 18, 119 (1925). 25. H. F. Stills, Jr., ILAR J. 46, 280 (2005). 26. I. E. Kovalev and O. Yu. Polevaya, Biochemical Funda mentals of Immunity to LowMolecularWeight Chemi cal Compounds (Nauka, Moscow, 1985) [in Russian]. 27. I. Roitt, J. Brostoff, and D. Male, Immunology (Mosby, London, 1998). 28. R. V. Petrov and R. M. Khaitov, Immunologiya, No. 1, 4 (1998). 29. J. Kreuter, Pharm. Biotechnol. 6, 463 (1995). 30. G. Gregoriadis, Immunol. Today 11, 89 (1990). 31. M. Fukasawa, FEBS Lett. 441, 353 (1998). 32. W. Morris, Vaccine 12, 5 (1994). 33. O. V. Masalova, A. V. Shepelev, S. N. Atanadze, Z. N. Parnes, V. S. Romanova, O. M. Vol’pina, Yu. A. Semiletov, and A. A. Kushch, Dokl. Akad. Nauk 369, 411 (1999) [Dokl. Biochem. 369, 180 (1999)]. 34. S. M. Andreev, A. A. Babakhin, A. O. Petrukhina, V. S. Romanova, Z. N. Parnes, and R. V. Petrov, Dokl. Akad. Nauk 370, 261 (2000) [Dokl. Biochem. 370, 4 (2000)]. 35. D. Pantarotto, C. D. Partidos, J. Hoebeke, F. Brown, E. Kramer, J.P. Briand, S. Muller, M. Prato, and A. Bianco, Chem. Biol. 10, 961 (2003). 36. N. A. Balenga, F. Zahedifard, R. Weiss, M. N. Sar bolouki, J. Thalhamer, and S. Rafati, J. Biotechnol. 124, 602 (2006). 37. K. Müller, J. N. Skepper, M. Posfai, R. Trivedi, S. Howarth, C. Corot, E. Lancelot, P. W. Thompson, A. P. Brown, and J. H. Gillard, Biomaterials 28, 1629 (2007). 38. S. Shiosaka, H. Kiyama, A. Wanaka, and M. Tohyama, Brain Res. 382, 399 (1986). 39. A. Wanaka, Y. Shiotani, H. Kiyama, T. Matsuyama, T. Kamada, S. Shiosaka, and M. Tohyama, Exp. Brain Res. 65, 691 (1987). 40. O. P. Ottersen and J. StormMathisen, Trends Neuro sci. 10, 250 (1987). 41. A. Tomii and F. Masugi, Jpn. J. Med. Sci. Biol. 44, 75 (1991). NANOTECHNOLOGIES IN RUSSIA

Vol. 5

Nos. 11–12

759

42. N. Tatsumi, Y. Terano, K. Hashimoto, M. Hiyoshi, and S. Matsuura, Osaka City Med. J. 39, 167 (1993). 43. J. R. Moffett, M. G. Espey, and M. A. A. Namboodiri, Cell Tissue Res. 278, 461 (1994). 44. L. D. Walensky, P. Gascard, M. E. Fields, S. Black shaw, J. G. Conboy, N. Mohandas, and S. H. Snyder, J. Cell Biol. 141, 143 (1998). 45. L. D. Walensky, T. M. Dawson, J. P. Steiner, D. M. Sabatini, J. D. Saurez, G. R. Klinefelter, and S. H. Snyder, Mol. Med. (Manhasset, NY, USA) 4, 502 (1998). 46. Y.S. Chen, Y.C. Hung, I. Liau, and G. S. Huang, Nanoscale Res. Lett. 4, 858 (2009). 47. J. Chen, F. Zou, N. Wang, S. Xie, and X. Zhang, Bioorg. Med. Chem. Lett. 10, 1691 (2000). 48. A. L. Feldman, L. Tamarkin, G. F. Paciotti, B. W. Sim pson, W. M. Linehan, J. C. Yang, W. E. Fogler, E. M. Turner, H. R. Alexander, and S. K. Libutti, Clin. Cancer Res. 6, 4628 (2000). 49. G. P. Mueller and W. J. Driscoll, in Posttranslational Modification of Proteins: Tools for Functional Proteom ics, Ed. by C. Kannicht (Humana Press, Totowa, New Jersey, United States, 2002), p. 241. 50. L. V. Olenina, E. F. Kolesanova, Yu. V. Gervaziev, I. S. Zaitseva, T. E. Kuraeva, B. N. Sobolev, and A. I. Archakov, Med. Immunol. 3, 231 (2001). 51. N. Ishii, F. Fitrilawati, A. Manna, H. Akiyama, Y. Tamada, and K. Tamada, Boisci., Biotechnol., Bio chem. 72, 124 (2008). 52. R. Kayed, E. Head, J. L. Thompson, T. M. McIntire, S. C. Milton, C. W. Cotman, and C. G. Glabe, Science (Washington) 300, 486 (2003). 53. M. N. Kireev, T. A. Polunina, N. P. Guseva, N. A. Pod bornova, Ya. M. Krasnov, and T. M. Taranenko, Probl. Osobo Opasnykh Infekts., No. 96, 43 (2008). 54. D. V. Pow and D. K. Crook, J. Neurosci. Methods 48, 51 (1993). 55. A. Baude, Z. Nusser, E. Molnar, R. A. J. McIlhinney, and P. Somogyi, Neuroscience 69, 1031 (1995). 56. D. P. Harris, H.M. Vordermeier, A. Arya, K. Bogdan, C. Moreno, and J. Ivanyi, Immunology 88, 348 (1996). 57. L. Pickard, J. Noël, J. M. Henley, G. L. Collingridge, and E. Molnar, J. Neurosci. 20, 7922 (2000). 58. M. K.H. Schäfer, H. Varoqui, N. Defamie, E. Weihe, and J. D. Erickson, J. Biol. Chem. 277, 50 734 (2002). 59. S. Holmseth, Y. Dehnes, L. P. Bjørnsen, J.L. Boul land, D. N. Furness, D. Bergles, and N. C. Danbolt, Neuroscience, 136, 649 (2005). 60. M. J. Schell, M. E. Molliver, and S. H. Snyder, Proc. Natl. Acad. Sci. USA 92, 3948 (1995). 61. M. J. Schell, O. B. Cooper, and S. H. Snyder, Proc. Natl. Acad. Sci. USA 94, 2013 (1997). 62. M. J. L. Eliasson, S. Blackshaw, M. J. Schell, and S. H. Snyder, Proc. Natl. Acad. Sci. USA 94, 3396 (1997). 63. D. Huster, O. P. Hjelle, F.M. Haug, E. A. Nagelhus, W. Reichelt, and O. P. Ottersen, Anat. Embryol. 198, 277 (1998). 64. N. Staimer, S. J. Gee, and B. D. Hammock, Fresenius J. Anal. Chem. 369, 273 (2001). 2010

760

DYKMAN et al.

65. V. A. Demenev, M. A. Shchinova, L. I. Ivanov, R. N. Vorob’eva, N. I. Zdanovskaya, and N. V. Nebai kina, Vopr. Virusol. 41, 107 (1996). 66. D. W. Kowalczyk and H. C. J. Ertl, Cell. Mol. Life Sci. 55, 751 (1999). 67. U. A. Hasan, A. M. Abai, D. R. Harper, B. W. Wren, and W. J. W. Morrow, J. Immunol. Methods 229, 1 (1999). 68. N. S. Yang and P. Christou, Particle Bombardment Technology for Gene Transfer (Oxford University Press, Oxford, 1994). 69. J. J. Donnelly, B. Wahren, and M. A. Liu, J. Immunol. 175, 633 (2005). 70. P. Sundaram, W. Xiao, and J. L. Brandsma, Nucleic Acid Res. 24, 1375 (1996). 71. Z. Cui and R. J. Mumper, Eur. J. Pharm. Biopharm. 5, 11 (2003). 72. L. Zhang, G. Widera, and D. Rabussay, Bioelectro chemistry 63, 369 (2004). 73. D. Chen and L. G. Payne, Cell Res. 12, 97 (2002). 74. M. Thomas and A. M. Klibanov, Proc. Natl. Acad. Sci. USA 100, 9138 (2003). 75. A. K. Salem, C. F. Hung, T. W. Kim, T. C. Wu, P. C. Searson, and K. W. Leong, Nanotechnology 16, 484 (2005). 76. R. Ojeda, J. L. de Paz, A. G. Barrientos, M. Martín Lomas, and S. Penadés, Carbohydr. Res. 342, 448 (2007). 77. W.H. Cheung, V. S.F. Chan, H.W. Pang, M.K. Wong, Z.H. Guo, P. K.H. Tam, C.M. Che, C.L. Lin, and W.Y Yu, Bioconjugate Chem. 20, 24 (2009). 78. Z. Zhao, T. Wakita, and K. Yasui, J. Virol. 77, 4248 (2003). 79. L. A. Dykman, L. Yu. Matora, and V. A. Bogatyrev, J. Microbiol. Methods 24, 247 (1996). 80. E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980). 81. S. Tomlinson, A. Luga, E. Huguenel, and N. Datt agupta, Anal. Biochem. 171, 217 (1988). 82. J. MüllerHöcker, S. Schäfer, A. Sendelhofert, and S. Weis, Histochem. Cell Biol. 109, 119 (1998). 83. L. A. Dykman and V. A. Bogatyrev, Biokhimiya (Mos cow) 62 (4), 411 (1997) [Biochemistry (Moscow) 62 (4), 350 (1997)]. 84. S. A. Staroverov, D. N. Ermilov, A. A. Shcherbakov, S. V. Semenov, S. Yu. Shchegolev, and L. A. Dykman, Zh. Mikrobiol., Epidemiol. Immunobiol., No. 3, 54 (2003). 85. L. A. Dykman, M. V. Sumaroka, S. A. Staroverov, I. S. Zaitseva, and V. A. Bogatyrev, Izv. Akad. Nauk, Ser. Biol. 31 (1), 86 (2004) [Biol. Bull. (Moscow) 31 (1), 75 (2004)]. 86. L. A. Dykman, V. A. Bogatyrev, S. A. Staroverov, D. V. Pristensky, S. Yu. Shchyogolev, and N. G. Khlebtsov, Proc. SPIE—Int. Soc. Opt. Eng. 6164, 616 404 (2006). 87. S. A. Staroverov, D. V. Pristenskii, D. N. Ermilov, S. V. Semenov, N. M. Aksinenko, S. Yu. Shchegolev, and L. A. Dykman, Biotekhnologiya, No. 6, 76 (2007) [Biotechnology in Russia, No. 6, 100 (2007)].

88. S. A. Staroverov, O. A. Vasilenko, K. P. Gabalov, D. V. Pristensky, D. N. Yermilov, N. M. Aksinenko, S. Y. Shchyogolev, and L. A. Dykman, Int. Immunop harmacol. 8, 1418 (2008). 89. S. W. Craig, S. K. Sanders, and J. V. Pardo, Methods Enzymol. 134, 460 (1986). 90. L. A. Dykman, V. A. Bogatyrev, I. S. Zaitseva, M. K. Sokolova, V. V. Ivanov, and O. I. Sokolov, Biofizika 47 (4), 632 (2002) [Biophysics 47 (4), 587 (2002)]. 91. N. V. Kostesha, A. G. Laman, A. O. Shepelyak ovskaya, I. S. Zaitseva, V. P. Orlov, L. A. Dykman, F. A. Brovko, and O. I. Sokolov, Biokhimiya (Mos cow) 70 (8), 1070 (2005) [Biochemistry (Moscow), 70 (8), 884 (2005)]. 92. O. A. Vasilenko, S. A. Staroverov, D. N. Yermilov, D. V. Pristensky, S. Yu. Shchyogolev, and L. A. Dyk man, Immunopharmacol. Immunotoxicol. 29, 563 (2007). 93. D. V. Pristenskii, S. A. Staroverov, D. N. Ermilov, S. Yu. Shchegolev, and L. A. Dykman, Biomed. Khim. 53, 57 (2007) [Biochemistry (Moscow) Suppl. Ser. B: Biomed. Chem. 1, 249 (2007)]. 94. S. A. Staroverov, N. M. Aksinenko, K. P. Gabalov, O. A. Vasilenko, I. V. Vidyasheva, S. Yu. Shchyogolev, and L. A. Dykman, Gold Bull. (London) 42, 153 (2009). 95. A. Lalvani, Chest 131, 1898 (2007). 96. T. Ozekinci, E. Ozbek, and Y. Celik, J. Int. Med. Res. 35, 696 (2007). 97. S. Kuwabara, J. Biol. Chem. 250, 2543 (1975). 98. A. A. Kamnev, L. A. Dykman, P. A. Tarantilis, and M. G. Polissiou, Biosci. Rep. 22, 541 (2002). 99. H. Vallhov, J. Qin, S. M. Johansson, N. Ahlborg, M. A. Muhammed, A. Scheynius, and S. Gabrielsson, Nano Lett. 6, 1682 (2006). 100. C. L. Villiers, H. Freitas, R. Couderc, M.B. Villiers, and P. N. Marche, J. Nanopart. Res. 12, 55 (2010). 101. H. S. Kruth, J. Chang, I. Ifrim, and W. Y. Zhang, Eur. J. Cell Biol. 78, 91 (1999). 102. E. Sadauskas, H. Wallin, M. Stoltenberg, U. Vogel, P. Doering, A. Larsen, and G. Danscher, Part. Fibre Toxicol., 4, article 10 (2007); www.particleandfibre toxicology.com/content/4/1/10. 103. M. Bartneck, H. A. Keul, G. ZwadloKlarwasser, and J. Groll, Nano Lett. 10, 59 (2010). 104. E. J. Andreu, J. J. M. de Llano, I. Moreno, and E. Knecht, J. Histochem. Cytochem. 46, 1199 (1998). 105. J. A. Khan, B. Pillai, T. K. Das, Y. Singh, and S. Maiti, ChemBioChem 8, 1237 (2007). 106. F. N. Ghadially, J. Rheumatol. 6 (Suppl. 5), 45 (1979). 107. B. D. Chithrani, A. A. Ghazani, and W. C. W. Chan, Nano Lett. 6, 662 (2006). 108. J. A. Ryan, K. W. Overton, M. E. Speight, C. N. Old enburg, L. N. Loo, W. Robarge, S. Franzen, and D. L. Feldheim, Anal. Chem. 79, 9150 (2007). 109. P. Nativo, I. A. Prior, and M. Brust, ACS Nano 2, 1639 (2008). 110. Y. Pan, S. Neuss, A. Leifert, M. Fischler, F. Wen, U. Simon, G. Schmid, W. Brandau, and W. Jahnen Dechent, Small 3, 1941 (2007).


Vol. 5

Nos. 11–12

2010

ADJUVANT PROPERTIES OF GOLD NANOPARTICLES 111. A. M. Alkilany, P. K. Nagaria, C. R. Hexel, T. J. Shaw, C. J. Murphy, and M. D. Wyatt, Small 5, 701 (2009). 112. J. C. Kah, K. Y. Wong, K. G. Neoh, J. H. Song, J. W. Fu, S. Mhaisalkar, M. Olivo, and C. J. Sheppard, J. Drug Targeting 17, 181 (2009). 113. N. G. Bastús, E. SánchezTilló, S. Pujals, C. Farrera, M. J. Kogan, E. Giralt, A. Celada, J. Lloberas, and V. Puntes, Mol. Immunol. 46, 743 (2009). 114. N. G. Bastús, E. SánchezTilló, S. Pujals, C. Farrera, C. López, E. Giralt, A. Celada, J. Lloberas, and V. Puntes, ACS Nano 3, 1335 (2009). 115. M. A. Dobrovolskaia and S. E. McNeil, Nat. Nano technol. 2, 469 (2007). 116. B. Merchant, Biologicals 26, 49 (1998). 117. R. Eisler, Biol. Trace Elem. Res. 100, 1 (2004).


Vol. 5

761

118. R. Shukla, V. Bansal, M. Chaudhari, A. Basu, R. R. Bhonde, and M. Sastry, Langmuir 21, 10 644 (2005). 119. Y. Tian, Y. Cui, H. Lou, J. Li, and P. Yan, Chin. Agric. Sci. Bull. 23, 7 (2007). 120. H. Lou, Y. Tian, J.Q. Gao, S.Y. Deng, and J.L. Li, J. Foshan Sci. Tech. Inst., Nat. Sci. Ed. (Foshan Kexue Jishu Xueyuan Xuebao, Ziran Kexueban) 25, 24 (2007). 121. Nanotechnology in Biology and Medicine, Ed. by E. V. Shlyakhto (St. Petersburg, 2009) [in Russian]. 122. N. Lewinski, V. Colvin, and R. Drezek, Small 4, 26 (2008). 123. Y.S. Chen, Y.C. Hung, W.H. Lin, and G. S. Huang, Nanotechnology 21, 195 101 (2010).

Nos. 11–12

2010