Molecular Recognition and Transport of Active Pharmaceutical Ingredients on Anionic Calix[4]arene-Capped Silver Nanoparticles

1 ICBMS-UMR 5246, Université Claude Bernard Lyon 1, 69622 Villeurbanne, France 2 LMI-UMR 5615, CNRS, Université Claude Bernard Lyon 1, 69622 Villeurbanne, France 3 LIMMS/CNRS-IIS (UMI 2820), University of Tokyo, Tokyo, Japan 4 Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01 224 Warszawa, Poland 5 Faculty of Biology and Environmental Sciences, Cardinal Stefan Wyszynski University in Warsaw, Wóycickiego 1/3, 01 938 Warszawa, Poland CIRMM, Institute of Industrial Science, University of Tokyo, Tokyo, Japan


Introduction
e noble metal nanoparticles are formed by the reduction of a suitable metal salt, for example, chloroauric acid or silver nitrate to the zero oxidation state in the presence of a suitable capping molecule or polymer to stabilize them [1].ey are well known for their ability to act as sensors for molecular interactions by means of shis in the plasmon resonance absorption [2].A great amount of work exists concerning their biological properties [3], including biological imaging [4], of particular interest are the antibacterial effects of both the silver and gold nanoparticles [5,6].Indeed the silver nanoparticles have seen commercial usage in areas ranging from hygiene (disinfection of socks) [7] to treatment of computer keyboards [8].
e calix [n]arenes are a group of supramolecular systems [9] widely studied for their ability to complex a very wide range of molecules and ions [10].e relative ease with which the calix[n]arenes can be modi�ed at either aromatic paraposition or at the phenolic face, [11] has made them perhaps the most attractive of organic hosts.Interest in the biochemistry of the calix[n]arenes has grown dramatically with emergence of a wide range of water soluble derivatives [12].eir direct behavior to act as Active Pharmaceutical Ingredients (APIs) is now emerging, [13] with activities ranging from anti-viral, [14] anti-bacterial, [15] enzyme activators [16] or blockers [17] anti-coagulant, [18] through anti-cancer [19] to detoxi�cation [20].As with other supramolecular systems the calix[n]arenes provide means to transport APIs in the solidstate as co-crystals, [21], as solubilizing agents, [22], or as colloidal suspensions, via solid lipid nanoparticle formation [23] or via the formation of cocolloidal complexes with highly hydrophobic partners [24].eir possible pharmaceutical applications have been made more attractive by a lack of   hemolytic effects [25], an absence of an immune response [26], and a clear lack of in vivo toxicity [27].
e combination of the useful biopharmaceutical properties of the calix[n]arenes with known biological properties thus makes them attractive as biosensors [28] or as transporters or more interestingly by using the calix[n]arenes to act capping stabilisers for nanoparticles and the using their complexation capacities towards APIs to form multifunction APU cocktails.[4]arene, 1 SO 3 H , was synthesized as per the literature method and physical characteristics correspond to the literature values [27].e two suspensions were characterized by UV-visible absorption assays.We monitored the change in absorbance between 340 nm and 650 nm, using a 96-titer well-visible spectrometer, (BioTek Power Wave 340).e stability over the time of the different calix[n]arenecapped silver nanoparticles (Ag_NP_Calix[n]arene) solutions as characterized using UV-visible spectroscopy 1 hour and 24 hours aer their preparation (Figure 1).Even if the intensity of absorbance decreases for some silver nanoparticle solutions, in particular with Ag_NP_3 SO 3 H , Table 1 shows that the maximum wavelength of all Ag_NP_Calix[n]arene remains stable.is shows that silver nanoparticles are still present aer 24 hours of their preparation.For all but Ag_NP_3 SO 3 H , the plasmon resonance absorption peak is sharp and centered at 390 nm; however in the case of Ag_NP_3 SO 3 H , the plasmonic peak is quite broad and centred at 420 nm suggesting a different assembly mode and also some aggregation.

Results and Discussion
Titration experiments were carried out between the six different types of calix [4]arene-capped nanoparticles and three APIs, chlorhexidine, chloramphenicol, and gentamycine.
Chlorhexidine is a clinically important antiseptic, disinfectant, and preservative.It is a potent membrane-active  agent against bacteria and inhibits outgrowth, but not germination, of bacterial spores.Chloramphenicol is considered a prototypical broad-spectrum antibiotic, alongside the tetracyclines.e most serious adverse effect associated with chloramphenicol treatment is bone marrow toxicity, which is rare (0.1% of the cases), unpredictable, unrelated to dose and in general fatal.As a consequence, it is no longer a �rst-line agent for any infection in developed nations, although it is sometimes used topically for eye infections.Nevertheless, the global problem of advancing bacterial resistance to newer drugs has led to renewed interest in its use.
Gentamycine sulfate is an aminoglycoside antibiotic, used to treat many types of bacterial infections, particularly those caused by Gram-negative organisms.Its bactericidal effect involves binding the 30S subunit of the bacterial ribosome, interrupting protein synthesis.Like all aminoglycosides, when gentamicin is given orally, it is not systemically active.is is because it is not absorbed to any appreciable extent from the small intestine.It is administered intravenously, intramuscularly, or topically to treat infections.e choice of the APIs was based on the facts that chlorhexidine is known to complex with all three types of calix [4]arene derivatives; the conformational �exibility of the molecule led to the presence of three distinct isomers in the solid state [34].In the case of gentamycine it has already been shown that aminoglycosides complex with 1 SO 3 H [10]; however for streptomycin the complexation was associated over several weeks with glycosidic bond cutting [35].With regard to chloramphenicol, preliminary studies had shown a total lack of interaction and the molecule was chosen as a negative control.
Representative visible spectratitration curves at 1H and 24H are shown in Figures 2 and 3; for both gentamycine and chlorhexidine there are strong shis in the plasmon resonance and intensity variations.For gentamycine in particular the changes in wavelength clearly arise from complexation on the nanoparticles by the API [28].Given the large shis in wavelength observed for the absorption associated with the plasmon resonance, the change in wavelength was plotted as a function of API concentration; see Figures 4 and 5.
For chloramphenicol, no change in the wavelength is observed; this is expected in view of the lack of complexation.For chlorhexidine and gentamycine, the curves show a typical concentration-dependent absorption associated with saturation.ese re�ect the di�erent binding affinities of the various calix [4]arene derivatives with regard to the two APIs.
Given the incertitude in the exact concentration of complexation sites on the surfaces of the nanoparticles we consider it unwise to calculate association constants.
However comparative values can be extracted from the initial slopes of the curves and these are summarized in Tables 2 and  3.
ere are clear variations in the affinity, while in general the hybrid silver nanoparticles capped with Ag_NP_2 SO 3 H show the highest affinity with chlorhexidine; aer 1H the system is fourth in respect to affinity.Of the systems studied only Ag_NP_4 COOH shows little or no affinity for these two APIs.
us we have observed selectivity in the interactions between the calix [4]arene capped nanoparticles and the APIs studied.is variation is dependent on the nature of the calix[n]arene, the API, and the kinetics of the interaction.

Conclusion
A series of novel calix[n]arene-capped silver nanoparticles have been prepared and their stability demonstrated.We have been able to couple calix [4]arenes having sulfonate, carboxylate, and phosphonate functions onto the silver nanoparticles.Tests with the APIs, chlorohexidine, chloramphenicol, and gentamycine sulfate, show strong affinity towards chlorohexidine and gentamycine, whereas for chloramphenicol little or no affinity exists.e affinities for the APIs depend on the nature of the calix [4]arene and on the nature of the API, and the kinetics of interaction vary as a function of both of the above.
e chemical structures of the calix[n]arene derivatives are given in Scheme 1. e chemical structures of the APIs are given in Scheme 2 below.
silver colloidal suspensions was monitored with UV-visible absorption spectra aer mixing for 1 hour and 24 hours.