Optical Characterization of Physicochemical Interactions in Multicomponent Doxorubicin-BSA-Gold Nanoparticle System
Gold Nanoparticles as a Factor of Influence on Doxorubicin-BSA Complex
The effectiveness of chemotherapy for cancer treatment when using traditional antitumor drugs is insufficient, as it is accompanied by a number of adverse side effects. In the first place, all drug products of this direction are characterized by high total toxicity. Doxorubicin (Dox) belongs to the anthracycline group of antibiotics due to its wide range of chemotherapeutic effects on malignant tumors and oncological diseases of the blood [50-52]. To overcome the mechanisms of development of Dox resistance, to increase the selectivity of its action, and to ensure the detoxification associated with radical forms of oxygen, a variety of methods are used for the creation of modified drugs (prodrugs) through the attachment of chemical fragments to antitumor agents, synthesis of conjugates with metallic, semiconductor, carbon nanoparticles, and metal ions [53-56].
A special place among doxorubicin-based prodrugs is taken by its conjugates with the noble metal nanoparticles, primarily Au. A high surface-to-volume ratio inherent to nanoparticles enables rapid response kinetics and provides improved drugloading capabilities. In addition, AuNPs provide a high degree of biocompatibility and controlled synthesis. Since the LSPR spectra of AuNPs are very sensitive to changes in the dielectric permittivity of their environment, they can react to processes of electron- conformational transformations in the molecules of drugs, including their complexation with antioxidants. In addition, nanoparticles can heat up due to light absorption, providing thermal effects on tumor cells, and serve as containers for transport and release of drugs in malignant tumors [57-59]. An important factor in the expediency of using gold nanoparticles as nanocontainers for medicinal products is the stability of a nanoparticle-drug system, which eliminates the effects of aggregation with the subsequent withdrawal of aggregates by a macrophage system leading to a reduction or complete cancellation of the therapeutic effect of drugs.
Antioxidant drugs administered to the body to reduce the harmful effect of Dox include BSA, one of the transport proteins . It is known that a free sulfide group, which occurs in 70% of albumin molecules, participates in disulfide exchange with the formation of intermolecular complexes and has a Dox effect .
It is known that BSA also forms complexes with Dox [61, 62]. It is obvious that physical interaction in BSA complexes with doxorubicin is accompanied by electron-conformational transformations in the molecules of the drug itself. Similar formation of complexes takes place in conjugates of BSA and Dox with AuNPs. At the same time, the influence of gold nanoparticles on the processes of complexation between molecules of antitumor and antioxidant drugs has not been sufficiently studied . This section presents a relevant study of the AuNPs effect on Dox-BSA complexation .
Concentration-Dependent Evolution of Light Absorbance in Doxorubicin-BSA GoldNanoparticle System
The pharmaceutical form of doxorubicin containing lactose as the most often used form for such type of antibiotic was used to investigate the physicochemical interaction between doxorubicin and the BSA complex in the presence of gold nanoparticles. Deionized water titrated to a pH of 6.9 and chemically synthesized citrate- stabilized gold nanoparticles with an average size of 13 nm and an absorption peak in the vicinity of 520 nm were used to prepare the multicomponent doxorubicin-BSA-gold nanoparticles solutions with different content of protein molecules.
Figure 8.9 shows the optical density spectra for solutions of pure Dox and Dox-BSA conjugates with gold nanoparticles. Upon the addition of AuNPs, a new band at about 498 nm appears, which corresponds to the Dox-AuNPs conjugate. With the addition of the lowest concentration of BSA (1.33 x Ю'5 M), upon the creation of gold nanoparticle conjugates with Dox and albumin solutions, the spectral reconstruction of optical absorption is observed with preserving a doxorubicin-specific band near Я = 481 nm and sharp increase in optical density. The band at 498 nm shifts with growing BSA concentration to a maximum at about Я = 500 nm followed by a slight optical density decrease (Fig. 8.9a, inset). Changes in this part of the spectrum are more likely to be the result of conformational changes in the Dox molecule. Creating conjugates of protein and antibiotic with gold nanoparticles leads to a substantial visible rearrangement of the right side of the optical density spectrum of Dox molecule, which may be due to both the change in the Dox conformation and the aggregation of gold nanoparticles. Since the stabilization of gold nanoparticles in an aqueous solution is provided by a layer of citrate, which leads to the appearance of negative charges on their surface, the coating of AuNPs with Dox molecules in monocationic prototropic form is considered a result of electrostatic interaction. The magnitude of such binding essentially depends on the pH of the medium that surrounds the resulting conjugates and is manifested as the red shift of the resonant band characteristic to AuNPs at about 520 nm and the emergence of new optical absorption bands in the range up to 700 nm, if aggregation of nanoparticles is observed .
Figure 8.9 (a) Optical density spectra of solutions of Dox (1.33 x 10~4 M) (1),
Dox conjugates with AuNPs (2) and Dox with 1.33 x 10~5 M (3), 2.66 x 10'5 M (4), 3.98 x 1СГ5 M (5), 5.31 x Ю'5 M (6), 6.64 x Ю’5 M (7), 9.29 x Ю"5 M (8), 13.3 x 10'5 M bovine serum albumin (9) with gold nanoparticles at a concentration of 1.25 nM. Inset: parts of spectra in the wavelength range near A = 481 nm. (b) Parts of the same normalized spectra in the wavelength range near A = 481 nm. Adapted by permission from Springer Customer Service Centre GmbH: Springer Applied Nanoscience, Ref. , Copyright 2018.
Aggregation of gold nanoparticles with their subsequent precipitation is most likely due to the loss of stabilizing properties of citrate coating as a result of the transfer of protons with a change in the prototropic form of Dox. Indeed, in accordance with Ref. , only compounds with highly protonated amino groups and at high concentrations can neutralize the negative charge of citrate- stabilized AuNPs with subsequent aggregation. The Dox molecule has an amino group, but under conditions of neutral pH, its presence cannot be a key factor in destabilizing the colloidal solution and initiating the aggregation process. However, the addition of colloidal gold with pH = 5 changes the acidity of a neutral solution of Dox, which leads to the protonation of the Dox molecule and the aggregation of gold nanoparticles. The indicated protonation is also evidenced as the displacement of the peak of the vibrational mode of the amino group at a frequency of 3777 cm'1 to the 3373 cm"1 position in the infrared absorption spectrum . That is, the aggregation process in the present case may be due to the charge transfer process in the Dox-AuNPs conjugate, which is accompanied by spectral changes in the Dox molecule, characteristic of the anionic prototropic form of doxorubicin. The calculation of the difference between the optical densities obtained for Dox solutions with AuNPs (ADox+AuNPs] and the sum of the optical densities of constituents ADox + AAuNPs reveals an intense absorption band located near A = 600 nm (Fig. 8.10). It can be assumed that this band is characteristic to the anionic proto tropic form of doxorubicin , the occurrence of which is due to the transfer of charges in the conjugate with subsequent aggregation of nanoparticles and their precipitation, which depends on the concentration of BSA in solutions.
In the presence of BSA in solution, this band gradually disappears and a new band near 550 nm appears (Fig. 8.10), which belongs to the cationic double-charged form of doxorubicin, that is, when the initial concentration of BSA increases, an anionic prototropic form of doxorubicin is transformed into its cationic double-charged form. At significant concentrations of protein in the presence of AuNPs, the most probable is the transition of Dox from a single-cationic prototropic form to a double-cationic form. Thus, protein molecules suspend the proton transfer process and play a stabilizing role in conjugates with AuNPs, forming not only C, but also D forms of antibiotic molecules, which contributes to the preservation of negative citrate-induced charges on their surfaces, and thus keeps AuNPs from aggregation. With increasing concentration of BSA, the precipitate of aggregated gold nanoparticles gradually disappears and, at the highest content of 1.33 x 10~4 M, is not observed (Fig. 8.10, inset).
Figure 8.10 Spectra of the optical densities difference ADox+AuNPs-(ADox+AAuNPs)
- (1) and ADox+bsa+AuNPs-(ADox+bsa+Aaunps) at BSA concentrations of 1.33 x 1СГ5 M
- (2) , 2.66 x 1СГ5 M (3), 3.98 x Ю"5 M (4), S.31 x Ю"5 M (S), 6.64 x Ю'5 M (6), 9.29 x 1СГ5 M (7), 13.3 x 10‘5 M (8), 1.33 x 1СГ4 M Dox and 1.2S nM AuNPs. Inset: photograph of samples 1, 2, 3, and 8, where differences in precipitation of AuNP-Dox-BSA aggregates for samples with different BSA concentration are observed. Reprinted by permission from Springer Customer Service Centre GmbH: Springer Applied Nanoscience, Ref. , Copyright 2018.
The results presented not only evidence the substantial binding between BSA and Dox with the formation of the complex, but also imply the significant effect of gold nanoparticles on the mechanism of complex formation and conformational changes in the protein- antibiotic system at LSPR conditions. That is, the presence of AuNPs enhances the regulatory capabilities of the Dox-BSA complex and can be used to create prodrugs.
Specific studies described in this chapter demonstrate the versatility of plasmonic nanoparticles, namely AuNPs, for a range of applications from molecular sensing to drug development. These include the high-sensitive LSPR-based sensing of small molecules and biomolecules based on AuNPs light extinction spectral analysis in different optical response formation modes and AuNPs-assisted characterization of interactions in antibiotic-protein system with tunable complexation properties.
In one of the studies, the validity of the proposed methods of measuring the LSPR response #t+op and Pright in the LSPR biosensor based on colloidal gold in the mode of surface modification of AuNPs was confirmed. Using the H*op LSPR response measurement mode on real samples ofbiomolecules improved the detectivity of the LSPR biosensor by 4.5-48 times, depending on the type and concentration of biomolecules, which even exceeds the theoretically derived values (see Section 2.3.3). The magnitude of response Pright on a real biomolecular sample was shown to be close to the maximum value of the "vertical” LSPR response 7max, which confirms the results of theoretical calculations.
Drastic effect of cooperative functionalities in a single molecular conjugate on the AuNP aggregation has been investigated in another study, explaining the optimal route for building a high-sensitive LSPR sensor based on the aggregation of colloidal AuNPs. Namely, the degree of aggregation of AuNPs was found to be dependent on the chemical structure and charge of the analyte molecule. Thus, the presence in the molecules of the analyte of atoms that interact with gold atoms on the surface of nanoparticles in a covalent manner, in conjunction with a positive charge of the molecule, caused rapid aggregation and precipitation of AuNPs detectable by a naked eye at an analyte concentration starting from 5 pmol/L. A theoretical model based on the FDTD method was proposed for consideration of evolution of the light extinction properties of the AuNP system during the aggregation process. It was shown that the optical response of an aggregated Au colloid should be formed both by close to spherical and chain-like nanoparticle aggregates present in solution. The theoretical approach applied was demonstrated to be useful for the estimation of the distribution of aggregate shapes corresponding to the experimental light extinction spectrum.
The last considered study applied light extinction spectroscopy to study the interactions within the multicomponent Dox-BSA- AuNPs system. Dox conjugate with citrate-stabilized AuNPs was characterized by the appearance of a new intense absorption band located near A = 600 nm, which was assumed to belong to the anionic prototropic form of Dox as a result of the observed AuNPs aggregation. In the presence of BSA in solution and with an increase in its concentration, this band disappeared with a transformation in a band near 550 nm, which may belong to the cationic double- charged form of Dox due to the absence of AuNP aggregation. When adding AuNPs to Dox solutions with different albumin content, the optical absorption spectra underwent significant changes due to a decrease in the interaction of an antibiotic with nanoparticles, a significant association of albumin molecules with AuNPs, and, as a consequence, the rearrangement of the Dox-BSA-AuNPs system to a more stable state, which is important in the drug-delivery process.
The results of the study indicate the possibility of creating effective prodrugs comprising Dox-BSA-AuNPs with regulated properties of antibiotic and protein complexation due to the presence of AuNPs.