Peptide-Carbon Quantum Dots conjugate, Derived from Human Retinoic Acid Receptor Responder Protein 2, against Antibiotic-Resistant Gram Positive and Gram Negative Pathogenic Bacteria

Antibiotic-resistant bacterial infections have become global issues for public health, which increases the utter need to develop alternatives to antibiotics. Here, the HSER (Homo sapiens retinoic acid receptor) peptide was designed from retinoic acid receptor responder protein 2 of Homo sapiens, and was conjugated with synthesized CQDs (carbon quantum dots) for enhanced antibacterial activity in combination, as individually they are not highly effective. The HSER–CQDs were characterized using spectrophotometer, HPLC coupled with electrospray-ionization quadrupole time-of-flight mass spectrometer (ESI–qTOF) mass spectrometer, zeta potential, zeta size, and FTIR. Thereafter, the antibacterial activity against Vancomycin-Resistant Staphylococcus aureus (VRSA) and Escherichia coli (carbapenem resistant) was studied using growth curve analysis, further supported by microscopic images showing the presence of cell debris and dead bacterial cells. The antibacterial mechanism of HSER–CQDs was observed to be via cell wall disruption and also interaction with gDNA (genomic DNA). Finally, toxicity test against normal human epithelial cells showed no toxicity, confirmed by microscopic analysis. Thus, the HSER–CQDs conjugate, having high stability and low toxicity with prominent antibacterial activity, can be used as a potential antibacterial agent.


Introduction
The evolution of bacteria with every generation to acquire resistance towards new antibiotics have made it a challenge to treat pathogenic bacteria, as their pattern of resistance differs [1]. Thus, bacterial infections due to the rapid increase in antibiotic resistance have become a major threat to global health. Antibiotic resistance occurs due to incomplete course of antibiotic dose and its misuse [2], exposure to constant stress, changes in genomic level, and horizontal gene transfer. The number of multidrug-resistant pathogenic bacteria is increasing every day, creating worse situations for the clinical settings to deal with it [3,4].
The World Health Organization in the year 2017 listed the antibiotic-resistant bacteria in three different categories depending on the threat level priorities: critical, high, and medium [5]. The Vancomycin-Resistant

Cultivation of Bacteria
Bacterial strain, vancomycin-resistant Staphylococcus aureus (VRSA) CCM 1767, was obtained from the Czech Collection of Microorganisms, Faculty of Science, Masaryk University, Brno, Czech Republic, and Escherichia coli ATCC BAA 2340 was obtained from American Type Culture Collection (ATCC), United States. The media used for the growth of the bacteria was Mueller Hinton (MH) media of 15 mL in Erlenmeyer flasks and the bacteria was inoculated and cultures were kept in shaker at 37 • C at 130 rpm for 24 h. Further, dilution of the culture was done using the MH broth to obtain 0.1 Absorbance (0.5 MacFarland standards) at OD 600 nm and used for consecutive experiments [26].

Minimum Inhibitory Concentration (MIC) Determination
The standard broth micro-dilution method (European Committee on Antimicrobial Susceptibility Testing) was used to determine the susceptibility of bacterial cultures against HSER-CQDs and its individual component, with the detection done by the unaided eye. MIC was calculated using the solutions from high to low concentration gradually. The different concentration of HSER-CQDs (250, 125, 75, 50, 25, and 10 µg/mL) was added to the microplate wells and mixed with the bacterial cultures (0.5 MacFarland with final dilution 1:100 using MH medium). Then the plate was incubated at 37 • C for 24 h. The well with lowest concentration of antibacterial agent with almost no bacterial growth was considered the MIC and the control was bacteria without any treatment [26].

Growth Curves and Viability Percentage
The growth curve was obtained using the Multiskan EX (Thermo Fisher Scientific, Waltham, MA, USA) by measuring the absorbance to determine the antibacterial activity. Different concentrations of the HSER-CQDs, Nanomaterials 2020, 10, 325 5 of 19 HSER, and CQDs were used to check the antibacterial effects on gram positive and negative bacteria. The positive control was only bacteria without any treatment. To compare the activity of HSER-CQDs, the bacteria was treated with different concentration of HSER and CQDs, respectively. Further, the viability percentage was calculated from the absorbance value after 24 h, comparing it with the absorbance value of the positive control [27].

Microscopy of Antibacterial Agents against Bacteria in Ambient Light and Live/Dead Cell Assay
Initially the samples were incubated with bacteria and HSER-CQDs conjugate (respective MIC) for 4 h at 37 • C in shaking incubator. The optical Olympus BX51 fluorescence microscope equipped with a 40× phase contrast lens was used to study the antibacterial activity of the HSER-CQDs against VRSA and E. coli. The number of cells observed per sample was observed from 10 randomized microscopic grid fields.
The live/dead cell assay is based on two fluorescent dyes, namely, SYTO9, which permeates both damaged and intact cell membranes, and propidium iodide (PI), to stain only the cells with damaged cell membranes [16]. A total of 1 µL of both the stains were added after the treatment of the bacterial cells for fluorescence microscopy. An inverted Olympus IX 71S8F-3 fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with Olympus UIS2 series objective LUCPlanFLN 40× (N.A. 0.6, WD 2.7-4 mm, F.N. 22), a mercury arc lamp X-cite 12 (120 W; Lumen Dynamics, Mississauga, ON, Canada), and a Camera Olympus DP73 was used to obtain the microscopic image to study the outcomes of live/dead bacterial cells assay. The number of cells observed per sample was observed from 10 randomized microscopic grid fields. The images were processed using the Stream Basic 1.7 Software.

Cell Membrane Break and Leakage Assay and Interaction with DNA
Initially, VRSA was treated with HSER-CQDs (25 µg/mL) and incubated at 37 • C for 4 h in shaking condition. Further, the bacterial cell membrane damage and cell cytoplasmic leakage after the treatment by HSER-CQDs was studied by centrifuging the samples, and the supernatant was used as the template for PCR and the amplification was done using 16S rRNA gene primers (16S Forward-ACTGGGATAACTTCGGGAAAC, and 16S reverse-CAGCGCGGATCCATCTATAA), and confirmation was done using agarose gel electrophoresis. The control was the supernatant of VRSA culture without treatment and the 100 bp ladder was used. The gels were visualized and documented under Azure c600 (Azure Biosystems Inc., USA).
Similarly, the genomic DNA (gDNA) from VRSA was isolated and then incubated with HSER-CQDs and kept for 1 h. Then, the samples were used as the template for PCR reaction and the amplification was done using 16S rRNA gene primers, and confirmation was done using agarose gel electrophoresis with respect to the 100 bp ladder. The control was the gDNA of VRSA culture without treatment. The gels were visualized and documented under Azure c600 (Azure Biosystems Inc., Dublin, CA, USA).
Further, fresh human blood was collected from the volunteer, with signed informed consent, by a venipuncture from an antecubital vein. RBCs were isolated from blood and the suspensions were washed with 150 mM NaCl solution three to five times. RBCs was incubated with the HSER-CQDs for 1 h at 37 • C. The positive and the negative control was 0.1% Triton X-100 and PBS, respectively. The degree of hemolysis was determined by measuring the absorbance of the supernatant at 540 nm, after centrifugation, and calculated according to the following equation: where h is percentage of hemolysis; A c is the absorbance of the supernatant from negative control (PBS, pH 7.4); A t is the absorbance of the supernatant from the samples incubated with the HSER-CQDs; and A 100% is the absorbance of the supernatant of positive control (0.1% Triton X-100), which causes complete lysis of RBCs [26].

Endogenous Reactive Oxygen Species (ROS) Production Assay
Bacterial endogenous ROS production was measured by using DCFDA Cellular Reactive Oxygen Species Detection Kit (Abcam Ab113851) according to the given protocol and instructions for bacterial cells [29,30]. To the cell suspension of VRSA, 10 µM of 2 ,7 -dichlorofluorescein diacetate (DCFDA) was added and kept at 37 • C for 30 min. in the dark condition followed by a wash using 1× washing buffer. Then, VRSA cells were treated with 75 µg/mL of HSER, HSER-CQDs, and CQDs for 3 h. The fluorescence intensity was measured by Tecan infinite m200 pro (wavelength of excitation at 485 nm and emission at 535 nm. The ROS was determined by comparing with the control, which was treated with H 2 O 2 showing positive ROS production [31].

Microscopic Analysis of HSER-CQDs Interaction against Eukaryotic Cells
The MDA-MB-468 (mammary gland adenocarcinoma cells), Du-145 (prostate adenocarcinoma cells), PNT1A (prostate epithelial cells), and HBL-100 (mammary gland epithelial cells) human cell line were cultured and used. Briefly, the suspension of 5000 cells in 300 µL medium supplemented with growth factors was added to each well of microtiter plates, followed by incubation for 24 h at 37 • C with 5% CO 2 to ensure cell growth. After 24 h treatment with HSER-CQDs, the cells were observed under an inverted Olympus IX 71S8F-3 fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with Olympus UIS2 series objective LUCPlanFLN 40× (N.A. 0.6, WD 2.7-4 mm, F.N. 22), a mercury arc lamp X-cite 12 (120 W; Lumen Dynamics, Mississauga, ON, Canada), and a Camera Olympus DP73 to obtain the bright field microscopic image using Stream Basic 1.7 Software. The number of cells observed per sample was observed from 10 randomized microscopic grid fields.

Results and Discussion
The synthesis and characterization of HSER-CQDs, along with its antibacterial efficacy against VRSA and E. coli, was studied and the MIC was determined. Further the toxicity test showed it has no toxicity against eukaryotic cell lines (prostate and mammary gland epithelial cell lines (HBL-100, and PNT1A). The mechanism of action was based on cell wall damage and leakage of cellular content. Some interaction of the HSER-CQDs with gDNA was observed, and assay showed negligible ROS generation. VRSA and E. coli was used as model organism to test the antibacterial effect of the HSER-CQDs due to its pathogenicity [26].

Peptide Structure
The largest protein conformation cluster was reached after~550 ns and represented a nearly continuous duration of 21.85% of entire simulation (437 of 2000 frames) ( Figure 1A). The representative frame ( Figure 1B) for the top cluster was 81,251 (of total 100,000). The video for the last 300 nanoseconds (ns) of simulation, where the peptide has stable conformation, is shown in ESI (Electronic Supplementary Information)  In both the average structure of largest cluster and the representative frame, hydrogen bonds were observed rather in the side-chains than the backbone. Namely, Glu6 and Asp18 side-chains provided acceptor oxygens to the amides of arginines and glutamines (Arg4, Arg15, and Arg17 in addition to Gln10 and Gln11). This observation is important for interpretation of FTIR vibrational spectra. Additionally, indicating that possible change in protonation states (due to pH change) will be detrimental for stable folding.
The ime evolution graph of the secondary structure ( Figure 1C) shows a combination of alpha helix (residues 5-10) bends and turns in the first half of the simulation. In the second half of the simulation, a stable conformation was formed in the middle of peptide. It was featured by turns (residues 12-14) surrounded by bends and finally a beta bridge connecting residues 7 and 17. The rest of the peptide endings were dominated by random coil ( Figure 1C). RMSD analysis confirmed these observations ( Figure 1D). Overall, RMSD were higher by ~1 Å in all atoms compared to backbone-only atoms. With reference to the first frame (α-helix), the The RMSD for the backbone of the top cluster was below 4 Å, while for all atoms was below 6 Å.
In both the average structure of largest cluster and the representative frame, hydrogen bonds were observed rather in the side-chains than the backbone. Namely, Glu6 and Asp18 side-chains provided acceptor oxygens to the amides of arginines and glutamines (Arg4, Arg15, and Arg17 in addition to Gln10 and Gln11). This observation is important for interpretation of FTIR vibrational spectra. Additionally, indicating that possible change in protonation states (due to pH change) will be detrimental for stable folding.
The ime evolution graph of the secondary structure ( Figure 1C) shows a combination of alpha helix (residues 5-10) bends and turns in the first half of the simulation. In the second half of the simulation, a stable conformation was formed in the middle of peptide. It was featured by turns (residues 12-14) surrounded by bends and finally a beta bridge connecting residues 7 and 17. The rest of the peptide endings were dominated by random coil ( Figure 1C). RMSD analysis confirmed these observations ( Figure 1D). Overall, RMSD were higher by~1 Å in all atoms compared to backbone-only atoms. With reference to the first frame (α-helix), the deviations in the backbone and all atoms were in the range of 4-11 Å during all simulation and 7-11 Å in the second half. With reference to the representative frame of the top cluster, RMSD dropped from 4-12 Å in the first half to 1-6 Å in the second half of the simulation for both the backbone and all atoms.

Synthesis and Characterization of the HSER-CQD Conjugate
The HSER peptide and CQDs in 1:1 ratio helps in the formation of the HSER-CQDs conjugate, which was dialyzed and observed under UV-transilluminator and showed a bluish fluorescence. The spectrophotometric analysis was done and the absorbance was detected in the spectral range from 230 to 1000 nm and the maxima absorption peak for HSER and the HSER-CQDs conjugate was obtained at 280 nm ( Figure 2A). Our experimental results clearly indicate the absence of CQDs absorption peak in visible spectral range. However, the lower absorbance peak of HSER detected at 280 nm belongs to transition of diffused pi electrons of the aromatic ring of tyrosine or tryptophan [32]. On the contrary, the higher absorbance peak of HSER-CQDs was slightly shifted and detected at 270 nm. Such a peak probably belongs to the π-π* transition of aromatic -C=C-and -C-C-bonds in the sp2 hybridized domain of CQDs core [33], which indicates that the interaction between HSER and CQDs has occurred. The fluorescence properties of HSER and CQDs can be observed by two fluorescence peaks at 370 and 445 nm from the respective spectra, as shown in Figure 2B. The CQDs emission peak was found at 445 nm, which is ascribed to surface or molecule center of CQDs [34]. Such fluorescence behavior of CQDs is common and usually is attributed to the diversity of particle size as well as the surface state of CQDs [35]. The lower emission peak of HSER was detected at 370 nm, while the higher emission peak of the HSER-CQDs conjugate was shifted and shows the presence of both peaks at 350 and 425 nm, confirming the interaction between HSER and CQDs. A peak detected at 350 nm usually comes from the presence of carboxyl (-COOH) or amine (-NH2) groups on CQDs surface, which are attributed to n-π* transition of -C=O, C-N or -C-OH bonds in the sp3 hybridized domains [33]. This probably indicates the possible interaction between CQDs surface with carboxyl or amine group from HSER. Xiaohui Gao and co-workers reported that shifts in the emission peak contribute to the strong interaction between water molecules and excited dipole moments in CQDs in solution [36]. Shifting of peaks confirm the structural change in HSER due to interaction with CQDs [37]. The dialyzed HSER CQDs were further characterized using HPLC coupled with ESI-qTOF-MS to obtain the individual peaks for HSER-CQD components, but, due to the small size of CQDs, they were not spotted in HPLC; however, sharp peaks for the presence of HSER were obtained, as shown in the Figure 2C.
The FTIR analysis ( Figure 3A) of the synthesized CQDs showed two major bands in the amide area, namely at 1550 and 1385 cm −1 , which are characteristic for secondary amines and carboxylates, respectively [38]. The broad range band near 3000s cm −1 ( Figure 3B) indicates formation of hydrogen-bonded hydroxy groups (-OH) as compared with equivalent CA vibrations [38]. The intensities of these three bands were visibly decreased following the coating by HSER peptide. In peptides and proteins, the amide I band (in the 1600s cm −1 ) is often used as an indicator of secondary structure. This is based on the contributions of C=O stretching and C-N stretching, which are influenced by the strengths of hydrogen bonds involving them [39]. Accordingly, the α-helix structure is correlated with frequency range 1660-1648 cm −1 , whereas the β-sheet structure is correlated with frequency range 1640-1625 cm −1 . Fortunately, due to available data from MD simulations, it is clear that such interpretation of structure can be biased in this situation. Predicted HSER structure appeared to be stabilized by amide-based electrostatic interactions of side-chains (Hydrogen bonds). In a more precise description, the vibrations of the backbone do not involve many hydrogen bonds and thus they can be assigned to the disordered secondary structure with frequency range 1657-1642 cm −1 [40]. The 1625 cm −1 in HSER-CQDs is an indicator of short range backbone aggregation, while the decrease in 1650 cm −1 correlates with loss of random backbone and interrupted side-chain interactions in HSER-CQDs. The contribution of amide bands is dominated by the backbone peptide bond, nevertheless, several side-chain vibrations fall in the amide I and II range [41]. Their extension coefficient (M −1 cm −1 ) contributions in the amide I are in the following range: Arg (ε = 300-490), Gln (ε = 360-380), and Lys (ε = 60-130). As for the amide II region (in the 1500s cm −1 ) they are in the following range: Gln (ε = 220-240), Asp (ε = 290-380), Glu (ε = 450-470) and Lys (ε = 60-130). Furthermore, the Cys thiol (-SH) stretching peak at 2551 cm −1 [41] was absent in both HSER and HSER-CQDs spectra, suggesting that sulfide bridges were formed in both cases. Overall, FTIR vibrational spectra confirmed the coating of CQDs with HSER peptide, mostly via strong non-covalent aggregation. This was characterized by amide-based short-range hydrogen bonds (1625 cm −1 ) between peptide backbone and secondary amines, carboxylates, and hydroxyl groups of the CQDs surface. Nanomaterials 2020, 10, x FOR PEER REVIEW 9 of 21 = 360-380), and Lys (ε = 60-130). As for the amide II region (in the 1500s cm −1 ) they are in the following range: Gln (ε = 220-240), Asp (ε = 290-380), Glu (ε = 450-470) and Lys (ε = 60-130). Furthermore, the Cys thiol (-SH) stretching peak at 2551 cm −1 [41] was absent in both HSER and HSER-CQDs spectra, suggesting that sulfide bridges were formed in both cases. Overall, FTIR vibrational spectra confirmed the coating of CQDs with HSER peptide, mostly via strong non-covalent aggregation. This was characterized by amide-based short-range hydrogen bonds (1625 cm −1 ) between peptide backbone and secondary amines, carboxylates, and hydroxyl groups of the CQDs surface.  Prior to injection the samples were diluted with mobile phase A to get a final concentration 10 µg/mL. HPLC was coupled with ESI-QqTOF mass spectrometer Bruker Maxis Impact (Bruker Daltonik GmbH, Bremen, Germany). The following parameters of mass spectrometer were used: End-plate offset potential, 500 V; capillary potential, 4500 V; nebulizer gas (N 2 ) pressure, 4 bar; drying gas (N 2 ) flow rate, 6 L/min −1 ; drying temperature, 300 • C. Mass range was set from 50 to 3000 m/z. Positive ion mode was used. Prior to HPLC-MS analysis a mass spectrometer was calibrated using ESI-TOF tuning mix.
However, the particle size distribution showing average particle size and the zeta potential of the prepared HSER-CQDs were analyzed using DLS after purification, using dialysis in D-Tube maxi dialyzer tubes against Mili-Q ( Figure 3C,D). Thereafter, the formation of nanoparticles was also confirmed by zeta size and potential measuring. Zeta size results obtained indicates that the size of CQDs is 4 ± 2 nm, while the size of HSER-CQDs was found to be 23 ± 3 nm ( Figure 3D). Furthermore, the value of the zeta potential showed that the lowest zeta potential was found in case of CQDs at −3 mV, and the highest zeta potential show HSER with 33 mV. Finally, the HSER-CQDs conjugate reveal the change of zeta potential after interaction to be 23 mV, showing the tendencies to aggregate which can be connected with peptide aggregation on the surface of nanoparticles. Thus, after the characterization of HSER-CQDs was completed, it was used to further study the antibacterial activity against pathogenic bacterial strains. However, the particle size distribution showing average particle size and the zeta potential of the prepared HSER-CQDs were analyzed using DLS after purification, using dialysis in D-Tube maxi dialyzer tubes against Mili-Q ( Figure 3C,D). Thereafter, the formation of nanoparticles was also confirmed by zeta size and potential measuring. Zeta size results obtained indicates that the size of CQDs is 4 ± 2 nm, while the size of HSER-CQDs was found to be 23 ± 3 nm ( Figure 3D). Furthermore, the value of the zeta potential showed that the lowest zeta potential was found in case of CQDs at −3 mV, and the highest zeta potential show HSER with 33 mV. Finally, the HSER-CQDs conjugate reveal the change of zeta potential after interaction to be 23 mV, showing the tendencies to aggregate which can be connected with peptide aggregation on the surface of nanoparticles. Thus, after the

Effect of HSER-CQDs on Different Bacterial Strains
The antibacterial activity of HSER-CQDs against VRSA and E. coli was initiated by determining the MIC using broth micro-dilution method. The MIC value of HSER-CQD against VRSA and E. coli was seen to be 25 and 50 µg/mL, respectively, and the concentrations below showed turbid solutions proving the presence of the bacteria, whereas the HSER and CQDs were found to have no prominent antibacterial effects or a higher concentration was needed to be applied (>125 µg/mL) (shown in Table 1). Thereafter, the growth curve analysis was performed to understand the stages of the growth of VRSA and E. coli for 24 h in the presence of the HSER-CQDs. The 24 h growth curve showed the concentration from 75 µg/mL and below, showed a rise in viability of E. coli cells, as seen in Figure 4C, but the inhibition was still more than 70%, whereas in case of VRSA the concentration below 25 µg/mL showed very low antibacterial activity, as shown in Figure 4A. Thus, the constant inhibitory effects of HSER-CQD for 24 h against VRSA was observed with the less concentration of 25 µg/mL, but in case of E. coli the concentration of 75 µg/mL showed prominent inhibitory effects compared to lower concentrations. Whereas, individually HSER and CQDs did not show any sign of prominent inhibition against VRSA and E. coli, as shown in Figures S1 and S2. Thus, the HSER-CQDs showed prominent inhibitory effects and high antibacterial activity compared to the individual components. antibacterial effects or a higher concentration was needed to be applied (>125 µg/mL) (shown in Table  1). Bacterial Strains HSER-CQDs (µg/mL) HSER (µg/mL) CQDs (µg/mL) Thereafter, the growth curve analysis was performed to understand the stages of the growth of VRSA and E. coli for 24 h in the presence of the HSER-CQDs. The 24 h growth curve showed the concentration from 75 µg/mL and below, showed a rise in viability of E. coli cells, as seen in Figure  4C, but the inhibition was still more than 70%, whereas in case of VRSA the concentration below 25 µg/mL showed very low antibacterial activity, as shown in Figure 4A. Thus, the constant inhibitory effects of HSER-CQD for 24 h against VRSA was observed with the less concentration of 25 µg/mL, but in case of E. coli the concentration of 75 µg/mL showed prominent inhibitory effects compared to lower concentrations. Whereas, individually HSER and CQDs did not show any sign of prominent inhibition against VRSA and E. coli, as shown in Figures S1 and S2. Thus, the HSER-CQDs showed prominent inhibitory effects and high antibacterial activity compared to the individual components.  Lastly, the percentage of viability after 24 h treatment with HSER-CQDs against VRSA for concentration 125 µg/mL was seen to be less than 1.5%, but lower concentrations, like 75, 50, and 25 µg/mL, showed viability below 18%. Further decrease in HSER-CQDs concentration had no such high inhibitory effects (10 µg/mL (>57%)), as shown in Figure 4B. In case of E. coli, the percentage of viability was below 1% for 125 µg/mL, but for lower concentration, like 75, 50, and 25 µg/mL, showed viability below 27%, as shown in Figure 4D. Therefore, viability percentage of inhibition for VRSA was prominent until 25 µg/mL, with the inhibition of more than 82%, but in case of E. coli, inhibition was best at 125 µg/mL (>99%), but at 25 µg/mL the inhibition was more than 73%. Whereas, the individual components of HSER showed no prominent activity against VRSA, with an inhibition of 32.32% at 125 µg/mL, and in case of E. coli the inhibition was around 22.23% at 125 µg/mL, but CQDs too showed no prominent antibacterial activity against VRSA (32.67%) and E. coli at 125 µg/mL ( Supplementary Figures S1 and S2). Thus, the HSER-CQDs work better as antibacterial agents against the VRSA and E. coli in lower concentrations compared to the individual components.

Microscopic Estimation of Live/Dead Cells and Damage of DNA (Bacterial Cells)
The phase contrast condition in the optical Olympus BX51 fluorescence microscope was used for further confirmation about the antibacterial activity of the HSER-CQDs against VRSA and E. coli. Decrease in the viable bacterial cell numbers and disruption of the cell with loss of cell integrity due to rupture of the cell wall cause a huge change in bacterial morphology, cytoplasmic leakage, and the presence of cell debris, which were seen under the microscope for both VRSA and E. coli after the treatment with HSER-CQDs, whereas the control bacterial samples with no treatments showed high number of viable cells with full cell integrity, no change in morphology, and absence of cell debris, as shown in Figure 5A.  Furthermore, to validate the results, the viability of the VRSA and E. coli after treatment with HSER-CQDs was observed by the live/dead assay using an inverted fluorescence microscopy. The bacterial samples treated with the HSER-CQDs showed a considerable decrease in the viability of the bacterial cells, showing almost negligible numbers of green fluorescent cells (SYTO9 stained), but a significant increase in number of dead cells with red fluorescence (PI stained). On the contrary, in case of controls cells, the number of live green fluorescent cells were high with almost no dead cells were observed, as shown in Figure 5B. Similarly, optical microscopic image (as shown in Figure S4) further validates the antimicrobial activity of the HSER-CQDs against the VRSA and E. coli.
Thus, the results showed prominent reduction in the viability of bacterial cells due to the presence of HSER-CQDs causing cell death by disrupting the cell wall, causing loss of cell morphology and cell death, proving its high-antimicrobial activity against both bacterial strains [42]. Thus, the probable mechanism of action for HSER-CQDs can be the cell wall disruption of bacterial cells.

Mechanism of Action by Interaction with Bacterial Cells, DNA, Changes in Secondary Structure, and Membrane Insertion
The previous studies under microscope showed the loss in cell integrity and disruption of the cells. To further confirm the interaction of HSER-CQDs with the bacterial cell wall causing disruption and leakage of the cellular content, the HSER-CQDs were treated with VRSA cells, and the supernatant after 4 h of treatment was used to study the presence of DNA. The treated VRSA cells supernatant on PCR amplification, using the 16S rRNA primers, confirmed the presence of the band of DNA at 106 bp, whereas the control without any treatment showed no band, as shown in Figure 6A. Thus, it can be concluded that the HSER-CQDs treated cell samples were indeed having cell wall damage and leakage of DNA. Further, the interaction of HSER-CQDs in presence of gDNA was performed separately. Then the PCR amplification of gDNA after treatment with HSER-CQDs for 16S rRNA showed no band, but for the control (gDNA without treatment) a prominent band at 106 bp was obtained, as shown in Figure 6B. Thus, it was seen that the HSER-CQDs not only interacted with the cell wall, which caused the cell disruption and leakage, but also interact with gDNA when treated separately and prevents its PCR amplification or replication. Therefore, the HSER-CQDs interacts with the DNA when incubated separately, but when they are treated with the cells they interact with the cell wall and no strong interactions can happen with the total DNA content, as a result of which we saw a prominent band from the PCR amplification using the supernatant after the treatment.

Mechanism of Action by Interaction with Bacterial Cells, DNA, Changes in Secondary Structure, and Membrane Insertion
The previous studies under microscope showed the loss in cell integrity and disruption of the cells. To further confirm the interaction of HSER-CQDs with the bacterial cell wall causing disruption and leakage of the cellular content, the HSER-CQDs were treated with VRSA cells, and the supernatant after 4 h of treatment was used to study the presence of DNA. The treated VRSA cells supernatant on PCR amplification, using the 16S rRNA primers, confirmed the presence of the band of DNA at 106 bp, whereas the control without any treatment showed no band, as shown in Figure  6A. Thus, it can be concluded that the HSER-CQDs treated cell samples were indeed having cell wall damage and leakage of DNA. Further, the interaction of HSER-CQDs in presence of gDNA was performed separately. Then the PCR amplification of gDNA after treatment with HSER-CQDs for 16S rRNA showed no band, but for the control (gDNA without treatment) a prominent band at 106 bp was obtained, as shown in Figure 6B. Thus, it was seen that the HSER-CQDs not only interacted with the cell wall, which caused the cell disruption and leakage, but also interact with gDNA when treated separately and prevents its PCR amplification or replication. Therefore, the HSER-CQDs interacts with the DNA when incubated separately, but when they are treated with the cells they interact with the cell wall and no strong interactions can happen with the total DNA content, as a result of which we saw a prominent band from the PCR amplification using the supernatant after the treatment. Based on net charge, cell-penetrating peptides can be either hydrophobic, amphipathic, or cationic [43]. The secondary structure of many cell-penetrating peptides and antimicrobial peptides has been studied in connection to their interaction with membranes. Some peptides, like penetratin, adopt either α-helix or β-sheet structure when interacting with the membrane, while polycationic peptides (like Tat and short poly-arginines) remain in unstructured random coils [44]. These peptides have high affinity to membranes with high concentrations of anionic lipids, and require formation of salt-bridges to obtain favorable energetic values for insertion in the membrane [45]. The pH (Low) Insertion Peptides (pHLIP peptides (low pH-dependent)) were shown to transform between three Based on net charge, cell-penetrating peptides can be either hydrophobic, amphipathic, or cationic [43]. The secondary structure of many cell-penetrating peptides and antimicrobial peptides has been studied in connection to their interaction with membranes. Some peptides, like penetratin, adopt either α-helix or β-sheet structure when interacting with the membrane, while polycationic peptides (like Tat and short poly-arginines) remain in unstructured random coils [44]. These peptides have high affinity to membranes with high concentrations of anionic lipids, and require formation of salt-bridges to obtain favorable energetic values for insertion in the membrane [45]. The pH (Low) Insertion Peptides (pHLIP peptides (low pH-dependent)) were shown to transform between three states, from unstructured highly-soluble to α-helix transmembrane state [46]. The naturally-occurring SMAP-29 peptide was shown to exhibit different antibacterial activities associated with its orientation when it was immobilized on magnetic beads [47]. Novicidin is an example of peptides that form a stabilized helix inside the lipid membrane [48]. A study on Novicidin showed that the binding process is ruled by membrane partitioning (how much membrane surface area per peptide), not only by helicity or charge [49].
We have showed in this work that the HSER peptide adapted a loop-shaped structure in MD simulation, and displayed random secondary structure signature by FTIR analysis (that can be interpreted as α-helix in other cases). Following the immobilization of HSER on CQDs, β-sheet structure signature was detected, which also correlates with short-range inter-chain interactions. The data clearly shows that the immobilized peptide backbone structure is different from the free one. Based on previous insights, this change in secondary structure can explain the change in peptide antimicrobial activity. The β-sheet structure realigns the peptide sequence in an alternating and amphipathic way that can break some of the electrostatic strains in the side-chains; allowing them to directly interact with the membrane. Lastly, the ROS generation against the bacterial cells was seen using DCFDA probe and spectrophotometer. The results showed in Figure S3 that the cells treated with HSER-CQDs showed negligible ROS generations in comparison to the positive control treated with H 2 O 2 . The HSER and CQDs treated cells showed higher ROS generations in comparison to HSER-CQDs-treated cells.
Finally, the mechanism of interaction for HSER-CQDs is based on the change of structure of the HSER after coating on CQDs surface, which causes cell wall interaction leading to cell wall disruption, with loss of cell integrity causing the leakage of intercellular component with no ROS generations, as shown in Figure 7; but when incubated separately with gDNA it prevents it from amplification. states, from unstructured highly-soluble to α-helix transmembrane state [46]. The naturally-occurring SMAP-29 peptide was shown to exhibit different antibacterial activities associated with its orientation when it was immobilized on magnetic beads [47]. Novicidin is an example of peptides that form a stabilized helix inside the lipid membrane [48]. A study on Novicidin showed that the binding process is ruled by membrane partitioning (how much membrane surface area per peptide), not only by helicity or charge [49]. We have showed in this work that the HSER peptide adapted a loop-shaped structure in MD simulation, and displayed random secondary structure signature by FTIR analysis (that can be interpreted as α-helix in other cases). Following the immobilization of HSER on CQDs, β-sheet structure signature was detected, which also correlates with short-range inter-chain interactions. The data clearly shows that the immobilized peptide backbone structure is different from the free one. Based on previous insights, this change in secondary structure can explain the change in peptide antimicrobial activity. The β-sheet structure realigns the peptide sequence in an alternating and amphipathic way that can break some of the electrostatic strains in the side-chains; allowing them to directly interact with the membrane. Lastly, the ROS generation against the bacterial cells was seen using DCFDA probe and spectrophotometer. The results showed in Figure S3 that the cells treated with HSER-CQDs showed negligible ROS generations in comparison to the positive control treated with H2O2. The HSER and CQDs treated cells showed higher ROS generations in comparison to HSER-CQDs-treated cells.
Finally, the mechanism of interaction for HSER-CQDs is based on the change of structure of the HSER after coating on CQDs surface, which causes cell wall interaction leading to cell wall disruption, with loss of cell integrity causing the leakage of intercellular component with no ROS generations, as shown in Figure 7; but when incubated separately with gDNA it prevents it from amplification. Figure 7. The mechanism of action of HSER-CQD, due to change in the structure of the peptide after interacting with CQDs, causing cell wall interaction, leads to leakage of cytoplasmic content and interaction with DNA, causing its inactivation. Figure 7. The mechanism of action of HSER-CQD, due to change in the structure of the peptide after interacting with CQDs, causing cell wall interaction, leads to leakage of cytoplasmic content and interaction with DNA, causing its inactivation.

Influence and Cytotoxicity Test of HSER-CQD on Eukaryotic Cells
The cytotoxicity of HSER-CQDs was estimated using the MTT assay PNT1A (prostate epithelial cells), Du-145(prostate adenocarcinoma cells), MDA MB 468 (mammary gland adenocarcinoma cells), and HBL-100 (mammary gland epithelial cells). MTT assay is based on reduction of MTT by succinate dehydrogenase reflecting metabolic healthy conditions of cells [50,51]. The viability was studied using different concentrations of HSER-CQDs, as shown in Figure S5A-S5D. In case of normal prostrate epithelial cells (PNT1A), the CQDs did not show any sign of toxicity at any concentration tested. However, HSER-CQDs incubated with normal mammary gland epithelial cells (HBL-100) at concentration of 125 µg/mL showed reduction in viability of cells by 26%, but lower concentration showed no toxicity at all. Furthermore, the mammary adenocarcinoma cells showed prominent reduction in viability until 31.25 µg/mL, whereas prostate adenocarcinoma cells showed prominent reduction in viability in different concentrations of the HSER-CQDs conjugate. Thus, cytotoxicity was seen against adenocarcinoma cells, but no prominent cytotoxicity was seen against the normal epithelial cell lines in the presence of HSER-CQDs.
Further, the hemolytic effects of the HSER-CQDs at different concentrations was studied using blood with positive control as 0.1% Triton-X (100% hemolysis) and negative control as PBS (no hemolysis). The 75 and 50 µg/mL concentrations of the HSER-CQDs conjugate showed negligible hemolysis, almost below 1.5% of hemolysis, as shown in Figure S5E. Thus, the concentration at which the conjugate works showed no toxicity towards red blood cells.
Finally, the cytotoxicity was confirmed by microscopic observation under an inverted microscopy. The results after treatment of HBL-100, PNT1A, and MDA MB 468 using HSER-CQDs showed no morphological changes in comparison to the control cells. Whereas in case of Du-145, the treated cells, when compared to control cells, showed considerable change in the morphology of few cells with loss of cell integrity and cellular leakage (shown in Figure 8). Thus, the results showing the high viability of normal cells with some toxic activity towards Du-145 adenocarcinoma cells in presence of HSER-CQDs proved that they are biocompatible and non-toxic towards normal human cells in vivo.

Conclusions
In the present study, HSER was designed from retinoic acid receptor responder protein 2 of Homo sapiens due to its defensive response against gram positive and gram negative bacteria. The structural analysis of HSER was computationally done using MD simulation and synthesis was performed. But the peptide showed no prominent antibacterial activity against VRSA and E. coli. Thereafter, the synthesis of the CQDs was done based on our previous work and conjugated with HSER to increase its activity. The characterization was done using spectrophotometer, HPLC coupled with ESI-qTOF mass spectrometer, UV-transilluminator, zeta potential and size, and FTIR. The prominent antibacterial activity was seen against VRSA and carbapenem-resistant E. coli using

Conclusions
In the present study, HSER was designed from retinoic acid receptor responder protein 2 of Homo sapiens due to its defensive response against gram positive and gram negative bacteria. The structural analysis of HSER was computationally done using MD simulation and synthesis was performed. But the peptide showed no prominent antibacterial activity against VRSA and E. coli. Thereafter, the synthesis of the CQDs was done based on our previous work and conjugated with HSER to increase its activity. The characterization was done using spectrophotometer, HPLC coupled with ESI-qTOF mass spectrometer, UV-transilluminator, zeta potential and size, and FTIR. The prominent antibacterial activity was seen against VRSA and carbapenem-resistant E. coli using growth curve analysis and viability percentage, along with the determination of MIC against respective bacteria. The antibacterial activity was further confirmed by microscopic analysis, showing loss of cell integrity, leakage of cellular content due to cell disruption, and with cellular debris. The live/dead cells assay showed the presence of a high amount of dead bacterial cells. The mechanism of action is based on change in structure of HSER after interacting with the CQDs, which was supported by FTIR results showing that the cell wall disruption occurred along with some interaction in presence of gDNA, which stop its amplification. Finally, the HSER-CQDs were found to be compatible with RBCs and nontoxic against normal human epithelial cells, with the help of MTT and hemolytic assay and further confirmed by microscopic analysis. Thus, HSER-CQDs can be used as a substitute to antibiotics.