The Mechanism of Dynamic Interaction between Doxorubicin and Calf Thymus DNA at the Single-Molecule Level Based on Confocal Raman Spectroscopy

It is of great fundamental significance and practical application to understand the binding sites and dynamic process of the interaction between doxorubicin (DOX) and DNA molecules. Based on the Confocal Raman spectroscopy, the interaction between DOX and calf thymus DNA has been systemically investigated, and some meaningful findings have been found. DOX molecules can not only interact with all four bases of DNA molecules, i.e., adenine, thymine, cytosine, guanine, and phosphate, but also affect the DNA conformation. Meanwhile, the binding site of DOX and its derivatives such as daunorubicin and epirubicin is certain. Furthermore, the interaction between DOX and DNA molecules is a dynamic process since the intensities of each characteristic peaks of the base, e.g., adenine, cytosine, and phosphate, are all regularly changed with the interaction time. Finally, a dynamic mechanism model of the interaction between DOX and DNA molecules is proposed; that is, there are two kinds of interaction between DOX and DNA molecules: DOX-DNA acts to form a complex, and DOX-DOX acts to form a multimer. The two effects are competitive, as the former compresses DNA molecules, and the latter decompresses these DNA molecules. This work is helpful for accurately understanding and developing new drugs and pathways to improve and treat DOX-induced cytotoxicity and cardiotoxicity.


Introduction
The research on the therapeutic mechanism and developments of anticancer drugs has always been a frontier hot topic. Many drugs for the prevention and treatment of cancer are designed with DNA molecules as the target [1][2][3]. At present, it is generally believed that the interaction between drug molecules and DNA molecules is mostly noncovalent bonding force, including electrostatic force, groove binding, and intercalation. At the single molecular level, understanding the action mode of anticancer drugs and DNA molecules and clarifying the action mechanism of anticancer drugs have scientific and practical significance for evaluating medicine efficacy and guiding drug development.
Doxorubicin (DOX) is a highly efficient and broad-spectrum anthraquinone anticancer drug which can be used to treat various diseases, such as leukemia, lymphoma, breast cancer, liver cancer, etc. [4][5][6]. However, cytotoxicity and cardiotoxicity limited its clinical application; it was found that DOX could cause cardiomyocyte damage and severe cardiotoxicity which led to arrhythmias and even fatal heart failure in patients [7][8][9][10]. In recent decades, thoroughly and incubated at 4 • C for a period time; as a result, the DOX-DNA, DAU-DNA, and EPI-DNA solutions were obtained. Raman spectra were acquired by an Alpha 500R Confocal Raman microscopy system (Alpha 500R, WITec GmbH, Ulm, Germany). Briefly, a fiber-coupled 785 nm semiconductor laser was collimated into a 20× objective lens (NA = 0.2, N-A chroplan, Zeiss, Oberkochen, Germany) for Raman excitation and spectral measurements. During the experiment, the power of the laser was 10 mW, the spot diameter of 20× objective was 1 µm, and the power density was 1.3 × 10 7 mW/mm 2 . The spectral signal was recorded by a spectrometer (UHTS300, WITec GmbH, Germany) incorporated with a 600 gmm −1 grating blazed at 500 nm, with a back-illuminated deep-depletion charge-coupled device (CCD, Du401A-BR-DD-352, Andor Technology, Belfast, UK) camera working at −60 • C. When measuring Raman spectra, each spectrum was measured three times and the acquisition time was approximately 20 s.
Before observing and recording the Raman spectral signals, a 10 µL droplet of solution (ctDNA, DOX, DAU, EPI, DOX-DNA, DAU-DNA, and EPI-DNA) was dripped onto the gold-plated substrate and dried at room temperature. The spectral data were firstly preprocessed with the data analysis software of WITec Project 4, including removing cosmic rays from the original spectrum. All Raman spectra were clipped to the fingerprint region (400-1800 cm −1 ), and the background interference was removed by 9-order polynomial fitting. The spectrum was smoothed using 5-order Savitzky-Golay (SG).
Furthermore, the absorption spectra of the DOX-DNA compounded solution were detected by the ultraviolet-visible (UV-vis) spectroscopy (DS-11). The light source of UV-vis spectrophotometer is long-life pulsed Xenon lamp, whose detector is a silicified CCD array with 2048 elements, and the detection accuracy and resolution reach to 1 nm. Figure 1a shows the diagrammatic sketch of ctDNA molecular structure; Figure 1b shows the average Raman spectra of ctDNA fiber (blue line) and ctDNA solution (black line) with a concentration of 9.8 × 10 −2 M. experimental solution was prepared with deionized water, in which the concentrations of DOX, DAU, and EPI were 5 10 M, and the concentration of ctDNA was 9.8 10 M. During experiments, an appropriate amount of DOX, DAU, EPI, and ctDNA solutions were measured respectively, and then put them into three different reagent bottles that contained the same amount of DNA solution by a pipette gun. Subsequently, they were mixed them thoroughly and incubated at 4 °C for a period time; as a result, the DOX-DNA, DAU-DNA, and EPI-DNA solutions were obtained. Raman spectra were acquired by an Alpha 500R Confocal Raman microscopy system (Alpha 500R, WITec GmbH, Ulm, Germany). Briefly, a fiber-coupled 785 nm semiconductor laser was collimated into a 20× objective lens (NA = 0.2, N-A chroplan, Zeiss, Oberkochen, Germany) for Raman excitation and spectral measurements. During the experiment, the power of the laser was 10 mW, the spot diameter of 20× objective was 1 μm, and the power density was 1.3 × 10 7 mW/mm 2 . The spectral signal was recorded by a spectrometer (UHTS300, WITec GmbH, Germany) incorporated with a 600 gmm grating blazed at 500 nm, with a back-illuminated deep-depletion charge-coupled device (CCD, Du401A-BR-DD-352, Andor Technology, Belfast, UK) camera working at −60 °C. When measuring Raman spectra, each spectrum was measured three times and the acquisition time was approximately 20 s.

Raman Spectra of ctDNA Molecule
Before observing and recording the Raman spectral signals, a 10 μL droplet of solution (ctDNA, DOX, DAU, EPI, DOX-DNA, DAU-DNA, and EPI-DNA) was dripped onto the gold-plated substrate and dried at room temperature. The spectral data were firstly preprocessed with the data analysis software of WITec Project 4, including removing cosmic rays from the original spectrum. All Raman spectra were clipped to the fingerprint region (400-1800 cm ), and the background interference was removed by 9-order polynomial fitting. The spectrum was smoothed using 5-order Savitzky-Golay (SG).
Furthermore, the absorption spectra of the DOX-DNA compounded solution were detected by the ultraviolet-visible (UV-vis) spectroscopy (DS-11). The light source of UVvis spectrophotometer is long-life pulsed Xenon lamp, whose detector is a silicified CCD array with 2048 elements, and the detection accuracy and resolution reach to 1 nm.   From Figure 1b, it can be observed clearly that the main characteristic Raman peaks of ctDNA are at 733, 785, 877, 1006, 1100, 1378, 1484, 1574, 1624, and 1660 cm −1 . These characteristic peaks reveal the structural information of ctDNA. The bands at 733, 785, 1378, 1484, 1574, and 1660 cm −1 are the characteristic peaks of base pairs, 1006 cm −1 is the characteristic peak of deoxyribose, and 1100 cm −1 is the characteristic peak of the phosphate group. Table 1 lists the detailed attribution information of the main Raman peaks of ctDNA [20,[30][31][32][33][34][35][36]. The 733 cm −1 is assigned to the symmetric stretching vibration of adenine (A). The 785 cm −1 is mainly related to the symmetric stretching vibration of cytosine (C). The 877 cm −1 is related to the C of DNA conformation. The 1006 cm −1 is the contribution of C-O stretching vibration of deoxyribose. The 1100 cm −1 is attributed to symmetric stretching vibration of O=P=O in PO 2 − . The 1378 cm −1 is attributed to the thymine (T). The 1484 cm −1 is attributed to the guanine (G). The 1574 cm −1 is attributed to the adenine (A). The 1624 cm −1 is attributed to the N7C5 + C8N7 vibration of A. The 1660 cm −1 is the contribution of C2=O stretching vibration of C.  Figure 2a shows the molecular structure of DOX; Figure 2b shows the average characteristic Raman spectra of DOX solution with a concentration of 5 × 10 −2 M.

Raman Spectra of DOX
From Figure 1b, it can be observed clearly that the main characteristic Raman peaks of ctDNA are at 733, 785, 877, 1006, 1100, 1378, 1484, 1574, 1624, and 1660 cm . These characteristic peaks reveal the structural information of ctDNA. The bands at 733, 785, 1378, 1484, 1574, and 1660 cm are the characteristic peaks of base pairs, 1006 cm is the characteristic peak of deoxyribose, and 1100 cm is the characteristic peak of the phosphate group. Table 1 lists the detailed attribution information of the main Raman peaks of ctDNA [20,[30][31][32][33][34][35][36]. The 733 cm is assigned to the symmetric stretching vibration of adenine (A). The 785 cm is mainly related to the symmetric stretching vibration of cytosine (C). The 877 cm is related to the C of DNA conformation. The 1006 cm is the contribution of C-O stretching vibration of deoxyribose. The 1100 cm is attributed to symmetric stretching vibration of O=P=O in PO . The 1378 cm is attributed to the thymine (T). The 1484 cm is attributed to the guanine (G). The 1574 cm is attributed to the adenine (A). The 1624 cm is attributed to the N7C5 + C8N7 vibration of A. The 1660 cm is the contribution of C2=O stretching vibration of C.  Figure 2a shows the molecular structure of DOX; Figure 2b shows the average characteristic Raman spectra of DOX solution with a concentration of 5 10 M.   mainly at 440, 462, 989, 1082, 1209, 1241, 1299, 1448, 1578, and 1637 cm . These characteristic peaks reveal abundant structural information of DOX molecules [37,38]. The bands at 440 and 462 cm are assigned to C-C-O and C-O respectively. The band at 989 cm is attributed to the vibration of the C-C of ring A (as shown in Figure 2a). The bands at 1082, 1209, and 1241 cm relate to C-O, C-O-H, and C-H, respectively. The 1578 cm is attributed to ring stretching and the band at 1637 cm is attributed to C=O. Generally these characteristic peaks (440, 462, 989, 1082, 1209, 1241, 1578, and 1637 cm −1 ) are the crucial criteria to identify whether the solute is DOX (DAU/EPI), while 1299 cm −1 and 1448 cm −1 are not the crucial criteria. The band at 1299 cm −1 is assigned to the vibration involving in-plane C-O, C-O-H, and C-H bending modes; the band at 1448 cm −1 is assigned to the C=C and the C-C stretching of the aromatic hydrocarbons.   Table 1 lists the detailed attribution information of Raman spectral characteristic peaks of DOX-DNA. Comparing the characteristic peaks of ctDNA with DOX-DNA complex, some distinguishing features can be found, e.g., the characteristic peak a 877 cm in the Raman spectrum of the original ctDNA solution, indicating that the DNA may be C-DNA under this experimental condition [30][31][32][33]. When DOX interacts with DNA for 24 h, Raman peaks at 841 and 857 cm appear, indicating that DNA may coexist with B-DNA and C-DNA after DOX interaction with DNA. The Raman spectrum of the DOX-DNA complex has both redshift and blueshift. The redshift may be due to the influence of the hydrogen bond [34]. For example, the characteristic peak at 1100 cm belongs to the symmetrical stretching vibration of phosphate ion PO , and the Raman peak at 1100 cm moves to 1089 cm due to the hydrogen bond between phosphate ion PO of DNA molecule with NH and OH of DOX. The characteristic peak at 1660 cm belongs to the C2=O stretching vibration of cytosine. The redshift from 1660 to 1657 cm is caused by the deformation of the hydrogen bond between C2=O of cytosine and NH of DOX Similarly, the characteristic peak at 1624 cm is mainly attributed to the vibration of N7C5 + C8N7, and the redshift from 1624 to 1600 cm may also be due to the formation of the hydrogen bond between N7 of A and DOX. Due to the influence of DOX, both bands  Table 1 lists the detailed attribution information of Raman spectral characteristic peaks of DOX-DNA. Comparing the characteristic peaks of ctDNA with DOX-DNA complex, some distinguishing features can be found, e.g., the characteristic peak at 877 cm −1 in the Raman spectrum of the original ctDNA solution, indicating that the DNA may be C-DNA under this experimental condition [30][31][32][33]. When DOX interacts with DNA for 24 h, Raman peaks at 841 and 857 cm −1 appear, indicating that DNA may coexist with B-DNA and C-DNA after DOX interaction with DNA. The Raman spectrum of the DOX-DNA complex has both redshift and blueshift. The redshift may be due to the influence of the hydrogen bond [34]. For example, the characteristic peak at 1100 cm −1 belongs to the symmetrical stretching vibration of phosphate ion PO − 2 , and the Raman peak at 1100 cm −1 moves to 1089 cm −1 due to the hydrogen bond between phosphate ion PO − 2 of DNA molecule with NH 2 and OH of DOX. The characteristic peak at 1660 cm −1 belongs to the C2=O stretching vibration of cytosine. The redshift from 1660 to 1657 cm −1 is caused by the deformation of the hydrogen bond between C2=O of cytosine and NH 2 of DOX. Similarly, the characteristic peak at 1624 cm −1 is mainly attributed to the vibration of N7C5 + C8N7, and the redshift from 1624 to 1600 cm −1 may also be due to the formation of the hydrogen bond between N7 of A and DOX. Due to the influence of DOX, both bands at 1484 cm −1 (attribute to G) and 1378 cm −1 (attribute to T) have blueshifts. Evidently, the DOX not only interacts with adenine, thymine, cytosine, guanine, and phosphate ion PO − 2 , but may also affect the DNA conformation.

Raman Spectra of DAU, EPI, DAU-DNA, and EPI-DNA
In order to further confirm the binding sites of DOX with DNA, we also analyzed Raman spectra of DOX derivatives, i.e., DAU and EPI, and their complexes with DNA. Figure 4 shows the chemical structure diagram of (a) DAU and (b) EPI, respectively. The red circle in the figure indicates their structural differences from DOX. Comparing Figure 4 to Figure 2a, it is found that the structures of the three anticancer drugs are very similar, but they also have differences. For example, the hydroxyl at position 14 of DOX is changed into hydrogen in DAU, and the hydroxyl at amino sugar part of EPI is changed from cis to trans.
Micromachines 2022, 13, x FOR PEER REVIEW 6 of 16 at 1484 cm (attribute to G) and 1378 cm (attribute to T) have blueshifts. Evidently, the DOX not only interacts with adenine, thymine, cytosine, guanine, and phosphate ion PO , but may also affect the DNA conformation.

Raman Spectra of DAU, EPI, DAU-DNA, and EPI-DNA
In order to further confirm the binding sites of DOX with DNA, we also analyzed Raman spectra of DOX derivatives, i.e., DAU and EPI, and their complexes with DNA. Figure 4 shows the chemical structure diagram of (a) DAU and (b) EPI, respectively. The red circle in the figure indicates their structural differences from DOX. Comparing Figure 4 to Figure 2a, it is found that the structures of the three anticancer drugs are very similar, but they also have differences. For example, the hydroxyl at position 14 of DOX is changed into hydrogen in DAU, and the hydroxyl at amino sugar part of EPI is changed from cis to trans. Figure 5a-c are the Raman spectra of DOX, DAU and EPI, respectively. In Figure 5, the black curve represents the average Raman spectrum of DOX solution; the red curve represents the average Raman spectrum of DAU solution; the blue curve represents the average Raman spectrum of EPI solution.
Because the DOX, DAU, and EPI have similar structures, here DAU and EPI were mainly used to verify the binding sites between DOX and DNA; that is, the adenine, thymine, cytosine, guanine, and phosphate. In the process of normalizing the ordinate of the Raman spectrum as shown in Figure 5, the characteristic peaks corresponding to the maximum value of all spectra are consistent, and the different spectra are comparable.
Due to their similar chemical structures and Raman spectra [39], the main characteristic Raman peaks such as 440, 462, 1082, 1209, and 1241 cm are found in DOX, DAU, and EPI solutions. Raman peaks at 440 and 462 cm belong to C-C-O and C-O, respectively. The bands at 1082, 1209, and 1241 cm respectively relate to C-O, C-O-H, and C-H. The Raman spectra of DOX, DAU, and EPI showed strong similarity, with all major characteristic peaks being the same, and only a small difference was found at 1300 cm −1 . This characteristic peak was more significant in DOX and EPI, but less significant in DAU.
It should be noted that Figure 5 was only used to illustrate the structural similarity of DOX, DAU, and EPI. Their differences are not the focus of this study. Therefore, there are no in-depth analyses on the Raman spectra of the different drugs in Figure 5.  In Figure 6, the black curve represents the average Raman spectrum of ctDNA solu tion; the red curve represents the average Raman spectrum of DOX-DNA complex ob tained by DOX interaction with DNA for 24 h; the blue curve represents the average Ra Because the DOX, DAU, and EPI have similar structures, here DAU and EPI were mainly used to verify the binding sites between DOX and DNA; that is, the adenine, thymine, cytosine, guanine, and phosphate. In the process of normalizing the ordinate of the Raman spectrum as shown in Figure 5, the characteristic peaks corresponding to the maximum value of all spectra are consistent, and the different spectra are comparable.
Due to their similar chemical structures and Raman spectra [39], the main characteristic Raman peaks such as 440, 462, 1082, 1209, and 1241 cm −1 are found in DOX, DAU, and EPI solutions. Raman peaks at 440 and 462 cm −1 belong to C-C-O and C-O, respectively. The bands at 1082, 1209, and 1241 cm −1 respectively relate to C-O, C-O-H, and C-H. The Raman spectra of DOX, DAU, and EPI showed strong similarity, with all major characteristic peaks being the same, and only a small difference was found at 1300 cm −1 . This characteristic peak was more significant in DOX and EPI, but less significant in DAU.
It should be noted that Figure 5 was only used to illustrate the structural similarity of DOX, DAU, and EPI. Their differences are not the focus of this study. Therefore, there are no in-depth analyses on the Raman spectra of the different drugs in Figure 5.
In Figure 6, the black curve represents the average Raman spectrum of ctDNA solution; the red curve represents the average Raman spectrum of DOX-DNA complex obtained by DOX interaction with DNA for 24 h; the blue curve represents the average Raman spectrum of DAU-DNA complex obtained by DAU interaction with DNA for 24 h; the green curve represents the average Raman spectrum of EPI-DNA complex obtained by EPI interaction with DNA for 24 h. In Figure 6, the black curve represents the average Raman spectrum of ctDNA solution; the red curve represents the average Raman spectrum of DOX-DNA complex obtained by DOX interaction with DNA for 24 h; the blue curve represents the average Raman spectrum of DAU-DNA complex obtained by DAU interaction with DNA for 24 h; the green curve represents the average Raman spectrum of EPI-DNA complex obtained by EPI interaction with DNA for 24 h.  By comparing the average Raman spectra of DOX, DAU, and EPI interacting with DNA, it is found that there are also the same peak shifts at 1100, 1378, 1484, and 1660 cm −1 . The only difference is the Raman peak at 1624 cm −1 . The Raman peak at 1624 cm −1 will be shifted to about 1600 cm −1 by DOX and EPI interacting with DNA, but the Raman peak shift at 1624 cm −1 by DAU interacting with DNA is very small. The main reason may be attributed to that both DOX and EPI are hydroxyl at position 14, while DAU is hydrogen. The interaction sites of DOX and DNA are shown in Figure 7.
DNA, it is found that there are also the same peak shifts at 1100, 1378, 1484, and 1660 cm . The only difference is the Raman peak at 1624 cm . The Raman peak at 1624 cm will be shifted to about 1600 cm by DOX and EPI interacting with DNA, but the Raman peak shift at 1624 cm by DAU interacting with DNA is very small. The main reason may be attributed to that both DOX and EPI are hydroxyl at position 14, while DAU is hydrogen. The interaction sites of DOX and DNA are shown in Figure 7.

Raman Spectra of Dynamic Process of DOX Interaction with ctDNA
In order to understand and analyze the dynamic process of the interaction between DOX and DNA, an appropriate amount of DOX with DNA solution is mixed and incu bated, and the characteristic Raman spectra are detected after a series of periods. Figure 8 displays the Raman spectra of DOX interaction with DNA for various times (0, 1, 2, 5, 7 10, 24, 48, and 72 h).
From Figure 8, it can be obviously found that the intensities and peak positions o Raman spectra changed significantly at 726, 785, 1089, 1450, 1578, and 1657 cm . These characteristic peaks do reveal the structural information of DNA molecules [30][31][32][33][34][35][36] Among them, the spectral peak at 726 cm belongs to A, the characteristic peak at 785 cm mainly belongs to C, the characteristic peak at 1089 cm belongs to the symmet rical stretching vibration of phosphate ion PO , the band at 1450 cm belongs to the de oxyribose, the spectral peak at 1578 cm mainly belongs to A, and the characteristic peak at 1657 cm mainly belongs to C.

Raman Spectra of Dynamic Process of DOX Interaction with ctDNA
In order to understand and analyze the dynamic process of the interaction between DOX and DNA, an appropriate amount of DOX with DNA solution is mixed and incubated, and the characteristic Raman spectra are detected after a series of periods. Figure 8 displays the Raman spectra of DOX interaction with DNA for various times (0, 1, 2, 5, 7, 10, 24, 48, and 72 h).
From Figure 8, it can be obviously found that the intensities and peak positions of Raman spectra changed significantly at 726, 785, 1089, 1450, 1578, and 1657 cm −1 . These characteristic peaks do reveal the structural information of DNA molecules [30][31][32][33][34][35][36]. Among them, the spectral peak at 726 cm −1 belongs to A, the characteristic peak at 785 cm −1 mainly belongs to C, the characteristic peak at 1089 cm −1 belongs to the symmetrical stretching vibration of phosphate ion PO − 2 , the band at 1450 cm −1 belongs to the deoxyribose, the spectral peak at 1578 cm −1 mainly belongs to A, and the characteristic peak at 1657 cm −1 mainly belongs to C.
In order to clearly express the changing trend of Raman intensity with incubation time, Figure 9 shows the line charts of Raman intensities with incubation time at 726, 785, 1089, 1450, 1578, and 1657 cm −1 , in which the horizontal axis is the logarithmic time and the vertical axis represents the Raman intensity.
From Figure 9, it can be obviously found that the intensity of Raman peaks at 726, 785, 1089, 1450, 1578, and 1657 cm −1 and has periodic increases and decreases with the increase of incubation time. The detailed changes of Raman intensity can be simply described as the following: the intensities of these Raman peak values decrease in 0-1 h, increase in 1-2 h, decrease again in 2-5 h, and increase again in 5-7 h. However, the degree of increase or decrease each time is different for different characteristic peaks. After approximately 10 h, the change trends are no longer regular. It should be point out that in order to ensure the reliability of the measurement data, the experiment was repeated 6 times under each different working condition, and then the average value was taken as the final result.
The reason for the periodic decrease of Raman intensity may be attributed to DOX acting on adenine, cytosine, phosphate, and deoxyribose of DNA molecules, and reducing the contents of adenine, cytosine, phosphate, and deoxyribose, while the reason for the periodic increase of Raman intensity may be complicated.
The increase of Raman spectral intensity reflects the increase in the number of counterparts in the mixing solution. Without increasing the number of DNA molecules, the main reason for this result may be that the DNA molecule combined with DOX exposes some bases such as adenine and cytosine, phosphate and deoxyribose, where the characteristic Raman peaks can be detected. In other words, the DOX molecule that has been bound to DNA molecule and formed a DOX-DNA complex may detach from the DNA molecule; therefore, those original acting sites, e.g., the bases, phosphate, or deoxyribose, are uncovered and will be detected and contribute to the Raman intensity value. The key question in this process is how DOX molecule leaves the DOX-DNA complex. Some research groups have reported that DOX molecules can combine with DOX molecules to form a multimer, such as dimer, trimer, or tetramer, when the number of DOX molecules is relatively large; for instance, C DOX /C DNA > 0.3 [19,22,40]. C DOX and C DNA is the concentration of DOX and DNA molecules, respectively. In this way, it is possible that the DOX molecule on the DOX-DNA complex combines with free DOX molecules to form a multimer, and then leaves the DNA molecule. In order to clearly express the changing trend of Raman intensity with incubation time, Figure 9 shows the line charts of Raman intensities with incubation time at 726, 785, 1089, 1450, 1578, and 1657 cm , in which the horizontal axis is the logarithmic time and the vertical axis represents the Raman intensity.  From Figure 9, it can be obviously found that the intensity of Raman peaks at 726, 785, 1089, 1450, 1578, and 1657 cm and has periodic increases and decreases with the increase of incubation time. The detailed changes of Raman intensity can be simply described as the following: the intensities of these Raman peak values decrease in 0-1 h, increase in 1-2 h, decrease again in 2-5 h, and increase again in 5-7 h. However, the degree of increase or decrease each time is different for different characteristic peaks. After approximately 10 h, the change trends are no longer regular. It should be point out that in order to ensure the reliability of the measurement data, the experiment was repeated 6 times under each different working condition, and then the average value was taken as the final result.
The reason for the periodic decrease of Raman intensity may be attributed to DOX acting on adenine, cytosine, phosphate, and deoxyribose of DNA molecules, and reducing the contents of adenine, cytosine, phosphate, and deoxyribose, while the reason for the periodic increase of Raman intensity may be complicated.
The increase of Raman spectral intensity reflects the increase in the number of counterparts in the mixing solution. Without increasing the number of DNA molecules, the main reason for this result may be that the DNA molecule combined with DOX exposes

Absorption Spectra of the Culture Medium of DOX and DNA Molecules
In order to determine the changes in the amount of DNA molecules in the solution after culturing DOX and ctDNA for a period of time, the absorption spectra of DOX-DNA culture medium at different times are also systematically measured.
UV-vis absorption spectrum can be used to analyze, determine, and infer the composition, content, and structure of substances of interest. The shape of UV-vis absorption spectrum curve and its maximum absorption wavelength reflect the composition of the substance, and the absorbance reflects the content of the substance. Figure 10a shows the absorption spectrum of DNA molecules when DOX and ctDNA are mixed and cultured for various times, e.g., 0, 1, 2, 5, 7, 10, 24, 48, and 72 h. Figure 10b is the absorbance value of mixed solution of DOX-DNA around 258 nm at different incubating times, in which the horizontal axis is the logarithmic time and the vertical axis represents the Raman intensity. spectrum curve and its maximum absorption wavelength reflect the composition of the substance, and the absorbance reflects the content of the substance. Figure 10a shows the absorption spectrum of DNA molecules when DOX and ctDNA are mixed and cultured for various times, e.g., 0, 1, 2, 5, 7, 10, 24, 48, and 72 h. Figure 10b is the absorbance value of mixed solution of DOX-DNA around 258 nm at different incubating times, in which the horizontal axis is the logarithmic time and the vertical axis represents the Raman intensity. From Figure 10, it can be clearly found that the intensity of the absorption spectrum of the DOX-DNA complex at 258 nm changes regularly with the increase of administration time. The change in the absorbance of DOX-DNA at 258 nm can be simply described as: the absorption peak decreases within 0-1 h, then increases within 1-2 h, and then decreases again within 2-5 h; however, the decrease is smaller. Afterwards, it gradually increases until 24 h; from 24 h to 48 h, the change is not obvious.
The decrease in absorbance intensity reflects a decrease in the amount and area of detectable ctDNA in the mixed solution, while the increase in absorption intensity indicates the increased number and area of DNA molecules exposed. Therefore, it is reasonable to speculate that during the period of experiments, the interaction between DOX and DNA brings about the change of the state and configuration of DNA constantly. From Figure 10, it can be clearly found that the intensity of the absorption spectrum of the DOX-DNA complex at 258 nm changes regularly with the increase of administration time. The change in the absorbance of DOX-DNA at 258 nm can be simply described as: the absorption peak decreases within 0-1 h, then increases within 1-2 h, and then decreases again within 2-5 h; however, the decrease is smaller. Afterwards, it gradually increases until 24 h; from 24 h to 48 h, the change is not obvious.
The decrease in absorbance intensity reflects a decrease in the amount and area of detectable ctDNA in the mixed solution, while the increase in absorption intensity indicates the increased number and area of DNA molecules exposed. Therefore, it is reasonable to speculate that during the period of experiments, the interaction between DOX and DNA brings about the change of the state and configuration of DNA constantly. Figure 11 is a schematic diagram of the dynamic process during the interaction between DOX and DNA, wherein (a-e) represent the changes about DNA morphology at different times after DOX administration, corresponding to the spectra at different times of 0, 1, 2, 5, and 7 h, as shown in Figure 9.

Molecular Mechanism of Interaction between DOX and DNA
It can be seen from Figure 11, after the DNA molecule interacts with DOX, that its configuration exhibited a change process, namely, compaction, decompaction, recompraction, and redecompaction [41]. Meanwhile, the inset in the middle is an example showing the competitive process through which two DOX molecules attract the DOX molecule in the DOX-DNA complex to form a trimer (dimers or tetramers might also be formed with different amounts of DOX molecules) and leaving the complex. That is, if new DOX molecules continuously entered the solution or the concentration of DOX was greater than a certain value, during the process of interaction between DOX and DNA molecules, accompanied by the interaction among DOX and DOX molecules, the two interactions between DOX and DOX molecules or DOX and DNA molecules were in competition, and the interaction between DOX and DNA molecules was constantly changing and unstable. The competition between the two mechanisms made the interaction between DOX and DNA molecules more complicated.
Concretely speaking, the dynamic process of the interaction between DOX and DNA molecules can be described as the following: as shown in Figure 11a, the DNA molecules are in a free self-coiled state in solution before DOX molecules were added into the solution. When DOX molecules are dropped into the DNA solution, DOX molecules act on adenine, cytosine, phosphate radicals, and/or deoxyribose of DNA molecules, and result in the ctDNA strands bending, shrinking, and compressing, as shown in Figure 11b. After a period of action of approximately 2 h, as shown in Figure 11c, compared to the state of the DNA molecule shown in Figure 11b the DNA molecule is in a relatively relaxed state again, i.e., decompaction. At this time, the concentration of DOX is relatively high (C DOX /C DNA > 0.3), and those free DOX molecules in solution can combine with the DOX which is in the DNA-DOX complex to form a multimer, e.g., dimer, trimer, and/or tetramer, and then these multimers detach from the DNA molecule, thus causing the DNA molecule to be stretched or loosened, i.e., in a decompressed state. It should be pointed out that the state and conformation of the DNA molecule here are not exactly the same as that of the DNA molecule shown in Figure 10a. As shown in Figure 11d, after administration of approximately 5 h, those free DOX molecules act on adenine, cytosine, phosphate, and/or deoxyribose of DNA molecules, and cause the decompacted DNA strands to be bent or contracted until compressed again, i.e., recompaction. It is also worth emphasizing that the state and conformation of the DNA molecule at this time are not exactly the same as that of the DNA molecule shown in Figure 11b; the sites of interaction between DOX and DNA molecules may not be the same. Subsequently, free DOX molecules in the solution can form a multimer with the DOX in the DOX-DNA complex, and then the multimer drags away the DOX molecule from the complex and meanwhile a possible action site on the DNA chain is exposed. The separation of the DOX molecule from the complex brings about the recompacted DNA molecule to stretch and relax again, and be in a decompressed state again, i.e., redecompaction, as shown in Figure 11e. For a similar reason, the DNA state and configuration are different from that shown in Figure 11a,c.
Micromachines 2022, 13, x FOR PEER REVIEW 12 of 16 Figure 11 is a schematic diagram of the dynamic process during the interaction between DOX and DNA, wherein (a-e) represent the changes about DNA morphology at different times after DOX administration, corresponding to the spectra at different times of 0, 1, 2, 5, and 7 h, as shown in Figure 9. It can be seen from Figure 11, after the DNA molecule interacts with DOX, that its configuration exhibited a change process, namely, compaction, decompaction, recompraction, and redecompaction [41]. Meanwhile, the inset in the middle is an example showing the competitive process through which two DOX molecules attract the DOX molecule in the DOX-DNA complex to form a trimer (dimers or tetramers might also be formed with different amounts of DOX molecules) and leaving the complex. That is, if new DOX molecules continuously entered the solution or the concentration of DOX was greater than a certain value, during the process of interaction between DOX and DNA molecules, accompanied by the interaction among DOX and DOX molecules, the two interactions between DOX and DOX molecules or DOX and DNA molecules were in competition, and the interaction between DOX and DNA molecules was constantly changing and unstable. The competition between the two mechanisms made the interaction be- The inset surrounded by dashed lines in the middle mainly shows the detailed process of multimer formation and leaving the DOX-DNA complex, which can be divided into three main steps: (1) multiple free DOX molecules in the solution are moving close to the DOX molecule that has been bound to the DNA strand. (2) The DOX trimer is formed with two free DOX and the DOX of the complex, and then the trimer breaks away from the DNA chain; the trimer is only an example. (3) The multimer leaves and moves into the solution and the binding site on the DNA molecule is exposed, and new free DOX molecules will draw near and bind to the DNA molecule again.

Molecular Mechanism of Interaction between DOX and DNA
It can be found that some phenomena in the experiments have been explained more credibly and reasonably by the mechanism model of interactions between DOX and DNA illustrated in Figure 11, which we propose based on our experimental results and those of other peer research groups [19,22,40]. We are conducting further investigations on this model, e.g., by using Dynamic Light Scattering (DLS), Atomic Force Microscopy (AFM), and so on, to determine the magnitude and type of force at the action site, the influence of DOX on the size, and morphology of DNA molecules.

Conclusions
Based on Confocal Raman spectroscopy, the binding sites of DNA molecules and DOX molecules and the dynamic process of their interaction were systematically studied, and some meaningful findings were found: Firstly, the characteristic peaks of the adenine, thymine, cytosine, guanine, and phosphate in the DOX-DNA complex have redshift or blueshift, which means that all four bases A, T, C, G, and PO − 2 of DNA molecules can become the action site when DOX interacts with the DNA molecule and the DNA state and conformation can also be affected by the DOX molecule. Then, combined with the Raman spectral characteristics of DOX derivatives such as DAU and EPI, and the complex DAU-DNA, EPI-DNA, the binding site of DOX was confirmed when it interacts with ctDNA molecule. Moreover, the characteristic Raman spectra of DOX-DNA complexes in different periods were carefully compared and analyzed, and the intensities of each characteristic peaks representing adenine, cytosine, and phosphate were all regularly changed with the interaction time, which indicated that the interaction between DOX and DNA was a dynamic process. Finally, the dynamic mechanism of the interaction between DOX and DNA molecules is proposed as the following: some free DOX molecules bind with four bases and/or phosphates of DNA molecules, resulting in a DNA molecule compression; then, new free DOX molecules bind with the DOX molecule of the DOX-DNA complex and forms a multimer; subsequently, the multimer drags away the DOX and leaves the DNA molecule, thus the DNA molecule loosens and exposes a binding site (some previously shielded binding sites may also be exposed because the state and configuration of DNA molecules in solution are constantly changing), decompressing the DNA molecule; then, some new free DOX molecules bind to DNA molecule, causing the DNA molecule compression again, i.e., recompaction; and afterwards, some new free DOX molecules bind to the DOX molecule in the DOX-DNA complex and form a new multimer and leave, bringing about the DNA molecule decompression again, i.e., redecompaction; and so on.
To investigate and elucidate the binding sites and dynamic process of the interaction between DOX and DNA molecules, it is not only of fundamental significance for molecular pharmacology, but also has practical application for understanding the biological processes at a single molecule level, for the design of hybrid nanomaterials for medicine delivery based on DNA architecture, and for the development of new drugs and pathways to improve and perfect DOX cytotoxicity and cardiotoxicity.