Evaluation of PLA-PEG Micellar Nanocarriers with Trastuzumab for Targeted Delivery of Doxorubicin to HER2+ Breast Cancer Cells: A Molecular Dynamics Simulation

Evaluation of PLA-PEG Micellar Nanocarriers with Trastuzumab for Targeted Delivery of Doxorubicin to HER2+ Breast Cancer Cells: A Molecular Dynamics Simulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of PLA-PEG Micellar Nanocarriers with Trastuzumab for Targeted Delivery of Doxorubicin to HER2+ Breast Cancer Cells: A Molecular Dynamics Simulation Zohreh Arefi Khorrami, Mohammad Khedri, Mostafa Keshavarz, Bahram Nasernejad This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7359044/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Context Breast cancer that is HER2-positive is still recognized as one of the more aggressive forms of breast cancer, which strongly suggests the need for more effective targeted forms of therapy. To develop better therapeutic approaches, understanding how drug delivery systems and monoclonal antibodies interact will be important. This work explores a new targeted delivery system for doxorubicin that employs PLA-PEG micellar copolymers with trastuzumab for better therapeutic effect against HER2-positive breast cancer. Molecular docking analysis suggests that there is a hydrogen bonding interaction between the COOH terminal group of the PLA-PEG micelle and the amine groups of trastuzumab, indicating favorable interactions with a COOH linker that was established on the PEG terminus. Among four different PLA-PEG molecular weight combinations evaluated using molecular dynamics simulation, PLA5K-PEG5K demonstrated optimal stability and absorption properties, as determined by structural and energetic analyses. The PLA5K-PEG5K-doxorubicin system with trastuzumab preserved structural integrity in aqueous solution and also indicates a favorable absorption and stability over time. Also, the behavior of this system near a POPE membrane was investigated, which obtained high interaction energy values, indicating great potential to deliver drugs into cells. These computational findings allow for the theoretical groundwork of a better-targeted delivery system, which could lead to improved outcomes for patients suffering from HER2-positive breast cancer. Methods Molecular docking studies were performed to assess protein-polymer binding between trastuzumab and PLA-PEG copolymers. Molecular dynamics (MD) simulations were completed to assess the stability and adsorption of the different PLA-PEG molecular weight combinations in an aqueous environment. System stability was examined through solvent-accessible surface area analysis, energy analysis, and radius of gyration measurements. Evaluation of membrane interactions was performed with a POPE model to assess their potential as a delivery vehicle, cellular delivery potential, and the energetics of the interactions. All MD simulations were run under periodic boundary conditions, and each system was simulated for 50 ns using Gromacs. Drug Delivery Targeted Drug Delivery Monoclonal Antibody Nanoparticle Molecular Dynamics Molecular Docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Cancer arises from uncontrolled growth and proliferation of cells, leading to tumors formation and an unacceptable systemic spread [ 1 , 2 ]. If they are not identified and treated in time, unfortunately, they can lead to death due to collapse and spreading in the body [ 3 ]. Oncologists utilize different treatment options, including surgery, radiotherapy, and stem cell therapy, depending on the type and stage of cancer [ 4 ]. However, the method that is used for a wide range of cancers is chemotherapy, in which anti-cancer drugs are administered into the patient's body in various ways, including oral, parenteral, transdermal, buccal, nasal, etc. [ 5 , 6 ]. Although chemotherapy is widely used, conventional chemotherapy has limited efficacy due to the non-selective nature of typical cytotoxicity, which often leads to the destruction of cancer cells and healthy cells [ 7 ]. Drug delivery is a further development in chemotherapy involving the oncologist, specialist, and biomedical engineer [ 8 ]. The aim of drug delivery is to formulate an optimal drug delivery system that releases the intended concentration of the drug to hit the target cells at the right time [ 9 ]. The drug faces many obstacles on its way through the body, most of which are related to the epithelial surfaces of the body [ 9 ]. The ideal drug delivery system can successfully overcome all of these obstacles [ 9 ]. The parameter that determines the ideality of a drug delivery system is bioavailability, which refers to the speed and extent of drug absorption and its availability at the site of action [ 9 ]. According to the study by Anya et al. [ 9 ], among the key parameters that lead to increased drug bioavailability and, subsequently, increased therapeutic efficacy are the type of drug carrier and monoclonal antibodies. There are a variety of carriers for drug delivery, from lipid and polymer carriers to carbon nanotubes and magnetic nanoparticles [ 10 ]. Ideal carriers should have certain properties, such as appropriate size distribution, biodegradability, biocompatibility, RES evasion, and long circulation half-life [ 11 ]. These features not only fulfill an ideal carrier profile but also improve drug bioavailability and therapeutic efficacy [ 12 ]. Monoclonal antibodies work by highly specific molecular recognition, similar to a lock-and-key system [ 13 ]. Cell surface receptors act as specialized molecular locks to which only matching monoclonal antibodies would bind [ 13 ]. The overexpression of these receptors on cancer cells offers an opportunity for targeted therapy; thus, monoclonal antibodies specific for these receptors can be incorporated into a drug delivery complex, allowing us to convert an existing conventional drug delivery to a targeted drug delivery system [ 14 ]. Currently, drug delivery research involves the characterization of delivery systems based on design principles and in-vitro synthesis, which is often confirmed by Fourier Transform Infrared Spectroscopy (FTIR) analysis, while drug release process in tumor microenvironments are assessed in an in-vivo step [ 15 ]. These experimental methods require a lot of energy, time, and cost. In-silico drug delivery is a relatively new field that at least partially overcomes these hurdles because it allows for computations that explore key variables, such as ligand-protein binding types, complex stability in physiological conditions, and drug release features in the proximity of tumors of viable drug delivery systems (ViDDS), the main goals of this investigation [ 16 ]. This study targets HER2-positive breast cancer and employs doxorubicin, PLA-PEG micellar copolymer, and trastuzumab as therapeutic agent, carrier, and targeting moiety, respectively. Breast cancer is one of the most aggressive and metastatic cancers in women worldwide, especially its HER2-positive subtype [ 17 ]. Therefore, the drawbacks of traditional chemotherapy, such as toxicity and low efficacy, have led to the creation of targeted delivery strategies [ 18 ]. In this regard, monoclonal antibodies conjugated to micellar copolymers show significant therapeutic efficacy. For example, Ahn et al. (2015) showed that a polymeric micelle containing a platinum drug linked to the Fab' fragment of an anti-TF antibody via a maleimide group led to a 15-fold increase in cell attachment within 1 hour and suppressed the growth of gastric tumors for more than 40 days [ 19 ]. In another study, Xu et al. (2019) showed that PEG-PCL nanoparticles containing docetaxel linked to the PD-L1 monoclonal antibody, while showing acceptable uptake, induced apoptosis in pancreatic cancer cells [ 20 ]. In another study, Yu et al. (2024) used mixed micelles containing the polymeric prodrug doxorubicin and chlorin e6 conjugated to a monoclonal antibody for combined chemo-photodynamic antitumor therapy, which resulted in increased drug accumulation at the tumor site, tumor growth suppression, reduced cardiotoxicity, and combined therapy (chemotherapy and photodynamic therapy) [ 21 ]. Also, Zhang et al. (2023) demonstrated that the use of DAR PDL1 conjugated PLG − PEG polymer and SN38 drug improved tumor accumulation 2.8-fold in colorectal cancer models compared to SN38 alone with an enhanced therapeutic outcome [ 22 ]. All these studies show that the use of monoclonal antibodies in drug delivery systems increases treatment efficacy. Thus, this study utilizes trastuzumab (Herceptin®) monoclonal antibody for targeting HER2-positive breast cancer [ 23 ]. Based on the design principles of Anya et al [ 9 ], the PLA-PEG micellar copolymer was chosen as the carrier system to take advantage of the hydrophobic PLA core for cost-effective doxorubicin encapsulation, and the hydrophilic PEG corona for improved stability and circulation time [ 24 ]. Finally, molecular docking and dynamics simulations were performed to evaluate the possible binding between antibody and carrier, the temporal evolution, and the stability of the designed complex in physiological conditions. Molecular docking is one of the computational approaches used to predict the preferred orientation of a ligand to a target protein, and thus it is a key technique in structure-based drug design and discovery [ 25 ]. There are several molecular docking tools available for predicting protein-ligand interactions. Among the most used platforms are Molegro Virtual Docker (MVD), which has an easy-to-use interface with strong cavity detection algorithms [ 26 ]; AutoDock, a widely known open-source suite that uses a Lamarckian Genetic Algorithm for binding predictions [ 27 ]; and its successor AutoDock Vina, which aims for high performance and accuracy with better scoring functions and optimization algorithms [ 28 ]. Molecular dynamics simulations are grounded on Newton's second law of motion and are a powerful tool to examine how system properties evolve over time at the atomic level in accordance with known physical principles, thus providing new insight into the behavior of drug delivery systems in physiological environments [ 29 ]. GROMACS was used for stability and absorption analyses [ 30 , 31 ], while NAMD was used for the release process in the cellular microenvironment in this study [ 32 ]. The current study is the first in-silico investigation of the PLA-PEG-TRASTUZUMAB complex as a doxorubicin delivery system for HER2-positive breast cancer therapy. Although the current study is only limited to molecular docking and dynamics studies, future studies will include potential in vitro and in vivo validation of predicted results. 2. Materials and Methods Materials The structure of the drug complex PLA-PEG-Trastuzumab-DOX was prepared using the following components: The doxorubicin structure was derived from the DrugBank database. The crystallographic structure of Trastuzumab was obtained from the RCSB Protein Data Bank (PDB ID: 1N8Z). Molecular weights of PLA-PEGs were designed via Avogadro software and the CHARMM-GUI server before optimizing their geometry in Gaussian software. Topology files were prepared by the PolyParGen server. The Visual Molecular Dynamics (VMD) software was used to construct a POPE membrane. All the molecular dynamics simulations were carried out on the Ubuntu operating system using GROMACS 5.1.2, except for the release study, which was performed using NAMD due to the large system size. 2.1. Molecular Docking Simulation To evaluate the potential types of interactions between PLA-PEG and the antibody, molecular docking simulations were employed in this study. For conjugation of PLA-PEG to the antibody using the EDC/NHS technique, a carboxylic acid (COOH) linker was attached to the terminal end of the PEG segment [ 33 ]. In order to perform molecular docking, ligand-receptor structures are first optimized in the form of a preprocessing step that includes removing water molecules and ions from the molecular structure [ 34 ]. The space around the protein is then searched to identify potential ligand binding sites [ 34 ]. After that, the possible interactions between the ligand-receptors will be predicted, and the output from these calculations gives the predicted energy value of each position with the type of interactions related to each pose [ 34 ]. Actually, molecular docking predicts several positions for ligand-receptor binding, and to find the best pose, many validation methods are used, such as checking the energy of each pose, RMSD, etc. [ 34 ]. In this study, we employed additional validation techniques for our work using the Molegro Virtual Docker and AutoDock software tools. Using this two software, we investigated the interactions between Trastuzumab and PLA-PEG. This work started by preparing the PDB structure of trastuzumab from the protein database RCSB with a 1N8Z identifier. The obtained structure was modified by removing unwanted components in AutoDock software. We first used the modified structure of trastuzumab and PLA-PEG to predict possible interactions in Molegro Virtual Docker software. After preparing the protein structure, we set the number of poses and runs to five and ten, respectively. The MoleDock SE algorithm was also selected for scoring. We evaluated the docking results of Molegro Virtual Docker software with a MolDock score, where a more negative score indicates more stability of the protein-ligand complex. Next, in order to perform molecular docking in AutoDock software, first the geometery files of Trastuzumab and PLA-PEG were prepared. A simulation box was created afterwards, wholly covering the ligand-protein region. After the main run, the results of molecular docking simulation in this software were checked by validation methods such as energy and RMSD. In other words, the above software tools are capable of predicting and analyzing any possible interactions between trastuzumab and PLA-PEG. 2.2. Molecular Dynamics Simulation In this work, we utilized GROMACS version 5.1.2 for all of our molecular dynamic’s simulation, while our molecular graphics interface was provided by the VMD software. Simulations were run under the OPLS force field, with Newton's equation of motion integrated with the leapfrog algorithm. We implemented the LINCS algorithm to allow for using a time step of as large as 2 femtoseconds throughout our simulations. Our simulation protocol comprised four key stages: energy minimization (EM), temperature equilibration (NVT), equilibrium of pressure (NPT), and the main simulation run [ 35 ]. For energy minimization, we applied the steepest descent algorithm. Also, we set 1000 kJ/mol/nm as the maximum force between consecutive time steps. In the equilibration steps, we first stabilized the system temperature at 300 K with a V-rescale thermostat and time constant of 1 in our simulation setting and then run the NVT step related to it. Then we set pressure at 1 bar with Parrinello-Rahman barostat and time constants of 2 picoseconds. The other setting for our work includes the cutoff radius that we set to 1 nm in all simulations for both electrostatic and van der Waals interactions, coupled with a period of 10 picoseconds Verlet scheme. We have dramatically reduced the edge effects for an infinite system by applying periodic boundary conditions in all three spatial dimensions. Structures of the carrier, drug, and monoclonal antibody were prepared for the initial step of simulation. Initially, we went ahead to prepare some kinds of molecular weights of PLA-PEG including PLA1.5K-PEG2K, PLA2K-PEG2K, PLA5K-PEG5K, PLA8K-PEG2K [ 36 – 39 ]. The number of monomers for each weight was obtaned using the molecular mass of a single monomer. PLA, with the general formula C3H4O2, has a molecular mass of 72.062, while PEG with its repeating units (CH₂CH₂O)n, has a molecular mass of 44.052. With these calculations, the following monomer counts were obtained: PLA5K-PEG5K (69 PLA, 114 PEG), PLA2K-PEG2K (28 PLA, 45 PEG), PLA1.5K-PEG2K (21 PLA, 45 PEG), PLA8K-PEG2K (111 PLA, 45 PEG). Considering the high number of monomers some compounds have, structures were drawn using the CHARMM-GUI server and Avogadro software. Optimization of the geometries of each structure was done with DFT theory in the Gaussian software with using the basis set 6–31 + + G(d,p). We then attached a COOH functional group as a linker to the PEG terminus of these structures—a standard approach in protein-polymer conjugation techniques (as employed with trastuzumab)—to create an active carboxyl group (typically activated using EDC/NHS chemistry) that enables direct chemical conjugation to the protein [ 33 ]. The structure of the anticancer drug doxorubicin was downloaded from the DrugBank database. Following the preparation of the initial structures, topology files were generated. The topology file contains all the critical information about simulations like atom types, presence of molecules, forces fields and other parameters [ 40 ]. The method for generating the topology file varies depending on the type of initial structure. For protein structures, such as the monoclonal antibody trastuzumab, GROMACS can generate the topology file directly from the geometry files [ 41 ]. For other non-protein structures, like polymers, parameterization, and topology file generation is done through the online servers LigParGen, PolyParGen, and ATB [ 42 ]. These output files frequently require some manual tuning. For our case, we used GROMACS and PolyParGen server to create the topology file of the trastuzumab and PLA-PEG copolymer, respectively. These carefully prepared initial structures and topology files therefore constitute a foundation upon which our molecular dynamics simulations can rely to determine interactions and behavior of our drug delivery system components with high precision and accuracy. Finally, we built a simulation box of dimensions 10×10×10 cubic nanometers and then solvated it with water molecules; thereafter four separate steps were conducted: energy minimization, temperature equilibration, pressure equilibration, and main run. All of the following sections have been simulated in these stages: simulation of PLA-PEG micellar copolymer, simulation of PLA-PEG micellar copolymer and drug, Simulation of trastuzumab-PLA-PEG-drug complex, and Simulation of polymer-drug-trastuzumab complex in the membrane along with HER2 + receptor. 2.2.1. Simulation of PLA-PEG Micellar Copolymer in the Water In the first stage of this molecular dynamic’s simulation, we examined the behavior of four different molecular weights of PLA-PEG copolymers in an aqueous environment. We simulated 10 molecules of each weight: PLA1.5K-PEG2K, PLA2K-PEG2K, PLA5K-PEG5K, and PLA8K-PEG2K. Our simulations clearly explained the micellization process of these copolymers in water. 2.2.2. Simulation of PLA-PEG Micellar Copolymer with Drug In the second phase of our molecular dynamic’s simulation, we studied the behavior of 10 polymers of PLA-PEG in an aqueous medium with 10 drug molecules, to investigate the stability and drug encapsulation efficacy in the four different weights of PLA-PEG. 2.2.3. Simulation of Trastuzumab-PLA-PEG-Drug Complex The third simulation stage was dedicated to the behavior of the optimal composition identified from previous stages: one PLA5K-PEG5K polymer, one drug molecule, and one monoclonal antibody in an aqueous environment. System stability and absorption over time were investigated by some thermodynamic analysis. 2.2.4. Simulation of polymer-drug-trastuzumab complex in the membrane along with HER2 + receptor The final simulation step was conducted with the NAMD software (Due to the large size of the system) at 50 ns to investigate the behavior of the drug delivery system in a more biologically relevant environment. First of all, a POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) membrane was constructed using VMD software. This membrane is used as a simplified model of the cell membrane. Afterward, the PLA-PEG-Trastuzumab complex previously optimized was added to this system. Additionally, from the NCBI database, the structure of the HER2 + receptor was imported and added to the simulation for performing a test modeling of the interaction of the drug delivery system with its target cell surface receptor. 3. Results 3.1. Molecular Docking Simulation of PLA-PEG and Trastuzumab Interactions The reliability of the results obtained in AutoDock is usually checked by obtaining the RMSD (Root Mean Square Deviation) values. An RMSD less than 1 Å is excellent, while 2–3 Å is good, 3–5 Å is weak, and above 5 Å is unacceptable [ 43 ]. As shown in Fig. 2 -Table 1, according to our AutoDock results, mode 1 was the most favorable, with a binding affinity of -4.4 Kcal/mol and an RMSD of 2.4. Further analysis of AutoDock results revealed specific interactions between PLA-PEG and trastuzumab. The copolymer exhibited four hydrogen bonds to amino acid residues TYR87 (Tyrosine 87), LYS43 (Lysine 43), GLN39 (Glutamine 39) and GLY41 (Glycine 41) of trastuzumab as noted in the Fig. 2 . This implies a successful binding between the COOH linker from PLA-PEG and amine group of antibody. The reliability of the results obtained in AutoDock is usually checked by obtaining the RMSD (Root Mean Square Deviation) values. An RMSD less than 1 Å is excellent, while 2-3 Å is good, 3-5 Å is weak, and above 5 Å is unacceptable [43]. As shown in Figure 2-Table 1, according to our AutoDock results, mode 1 was the most favorable, with a binding affinity of -4.4 Kcal/mol and an RMSD of 2.4. Further analysis of AutoDock results revealed specific interactions between PLA-PEG and trastuzumab. The copolymer exhibited four hydrogen bonds to amino acid residues TYR87 (Tyrosine 87), LYS43 (Lysine 43), GLN39 (Glutamine 39) and GLY41 (Glycine 41) of trastuzumab as noted in the Figure 2. This implies a successful binding between the COOH linker from PLA-PEG and amine group of antibody. Comparing results from AutoDock and Molegro Virtual Docker gave two similar hydrogen bonds and two different ones. This may have been because of computational differences between the software and differences in how each program identifies and classifies interactions. AutoDock results were interpreted in Discovery Studio and LigPlot software to emphasize the interactions more accurately, as shown in Fig. 3 . Molecular docking studies with the mentioned software tools, while proving the possibility of binding, provide possible interactions that PLA-PEG may have with trastuzumab. The use of different software and the similarity of their outputs strengthens our confidence in the predicted connection model. These results are a computer validation of the structural basis of the proposed drug delivery complex, which provides a suitable field for experimental validations in the future. 3.2. Simulation of PLA-PEG Micellar Copolymer in the Water After simulating 10 molecules of each molecular weight of PLA-PEG copolymers: PLA1.5K-PEG2K, PLA2K-PEG2K, PLA5K-PEG5K, and PLA8K-PEG2K in an aqueous environment, we investigated some thermodynamic parameters to analyze the stability and adsorption of the drug complex over time. The analysis was started by investigating the interaction energies such as the van der Waals and electrostatic potentials using the g_mmpbsa analysis [ 44 ]. Our results indicated that among the four compositions, PLA8K-PEG2K and PLA1.5K-PEG2K had the greatest magnitude of electrostatic potential and van der Waals energy, and hence, with average total energies of -765.52 and − 345.33 kJ/mol, respectively, they are the most energetically stable compounds, as shown in Fig. 4. We also investigated the radius of gyration, which provides information about mass distribution around the center of mass in a molecule [ 45 ]. A smaller radius of gyration will indicate a more compact structure and correlate to higher stability in an aqueous environment [ 45 ]. The Radial Distribution Function (RDF) analysis shows the atomic adsorption and structural compactness, as a higher RDF peak indicating a more structural adsorption and stability [ 46 ]. While Solvent Accessible Surface Area (SASA) analysis strongly shows the stability of a structure in an aqueous environment over time, it decreases with more compact structures less exposed to solvent, typically correlating with greater stability [ 47 ]. Furthermore, RMSD indicates the average distance from a reference structure in a structure, capturing overall structural shifts and stability [ 48 ], In contrast, RMSF evaluates the amount each atom fluctuates from its average position [ 49 ]. These analyses revealed the following results. Figure 5 -a showed that PLA1.5K-PEG2K had a favorable radius of gyration (An average of 4.8 nm). Figure 5 -b showed that PLA5K-PEG5K had the highest RDF value (approximately 3.8) for atomic adsorption and, hence, the most compact structural arrangement overall. Figure 5 -c confirms PLA5K-PEG5K's compactness with its lowest SASA values (193.59 nm 2 ). Figures S2 and S3 also show PLA8K-PEG2K's stability with the smallest RMSD and RMSF value with an average of 6.19 Å and 3.13 Å, respectively. Overall, visualizing these results suggests PLA5K-PEG5K and PLA8K-PEG2K formed the most stable structures, with the greatest degree of compaction and molecular adsorption over time. 3.3. Simulation of PLA-PEG Micellar Copolymer with Drug After simulating 10 PLA-PEG polymers in an aqueous medium with 10 drug molecules, the energy analysis using the g_mmpbsa command revealed that although PLA1.5K-PEG2K exhibited the highest electrostatic potential energy and van der Waals forces, the more stable and decreasing energy pattern observed for PLA5K-PEG5K and demonstrates that this structure possesses the most stable energy profile with a total average of -1029.34 KJ/mol (Fig. 6). This stability pattern indicates that PLA5K-PEG5K represents the most stable configuration among all investigated polymers. Based on other analyses, PLA5K-PEG5K consistently demonstrates superior stability and drug interaction properties. With the smallest radius of gyration (An average of 4.5 nm) as shown in Fig. 7 -a, this combination achieves the most compact and stable conformation in aqueous environments while carrying the drug. It also shows the highest RDF peak (6.9) and lowest SASA value (213.867 nm 2 ) and exhibits an excellent drug encapsulation efficiency. While PLA8K-PEG2K forms maximum hydrogen bonds (Fig. 8 d) and has the lowest RMSD fluctuations (An average of 5.93 Å) with the quickest convergence to stability (Figure S5), PLA5K-PEG5K with an average of 3.27 Å has the lowest atomic fluctuations in the RMSF analysis (Figure S6). On the whole, combined data from supplementary analysis (Figures S5-S6) and Figs. 6– 7 indicate PLA5K-PEG5K is the best polymer composition for drug delivery - highlighting the need for rational design to improve drug bioavailability. 3.4. Simulation of Trastuzumab-PLA-PEG-Drug Complex The third simulation stage was dedicated to the behavior of the optimal composition identified from previous stages: one PLA5K-PEG5K polymer, one drug molecule, and one monoclonal antibody in an aqueous environment. System stability increased, as g_mmpbsa energy analysis plot in Fig. 8 -a shows. In other words, based on Fig. 8 -b, the total energy of the system dropped from − 85.838 to − 631.715 kJ/mol, which is the average of − 409.3 kJ/mol, thus denoting the corresponding complex maintains its structure in the aqueous environment over time and does not collapse. Also, the decreasing trend of SASA shows that the relevant complex undergoes aggregation over time and offers acceptable adsorption. All of this indicates the stability and significant adsorption of the relevant complex in the aqueous environment and over time, as obtained from Fig. 8 -c. Therefore, the reduction in SASA shows improved encapsulation of the drug and strong interactions between the constituents. Hydrogen bond analysis showed that there was an increase in the number of hydrogen bonds with time. The increase in hydrogen bonding, as evident in Fig. 8 -d, signifies a stable and cohesive structure and hence adds to the stability of the drug delivery system. These results, taken together, indicate that the PLA5K-PEG5K copolymer forms a stable complex with the drug and trastuzumab, in such a way that provides favorable energy profiles, reduces solvent exposure, and increases intermolecular interactions. This stability is an essential prerequisite for the potential efficacy of the drug delivery system targeted for cancer therapy. Also shown in Fig. 9 is the third simulation stage output. 3.5. Simulation of polymer-drug-trastuzumab complex in the membrane along with HER2 + receptor In the final simulation step that we conducted using NAMD software, due to the large size of the system, we investigated the behavior of the drug complex: PLA5K-PEG5K-DOX-Trustuzumab along with a POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) membrane. Based on Fig. 10 , the energy profile shows that the energy value reaches − 1100 kJ/mol over time. This significant amount shows the strength of interactions between the drug complex and the membrane and, more generally, the stability of the system in the presence of a lipid membrane. 4. Discussion Compared to the previous coarse-grained simulations, all-atom molecular dynamics simulations afford a higher degree of resolution, particularly in regard to the understanding at the atomic-level. Kamrani and Hadizadeh (2019) used coarse-grained MD simulations to model PLA-PEG and PCL-PEG aggregation [ 50 ], while Chansuna et al. (2014) used a DPD type simulation in PLA-PEG-PLA triblock copolymers [ 51 ]. Regardless of their computational benefits, our all-atom simulation allowed for greater resolution regarding the drug-polymer interactions and led to a more accurate assessment on the stability of the structure along with the binding energies. Our findings indicated PLA5K-PEG5K had the most ideal stability (total energy of -1029.34 kJ/mol) and exceptional drug encapsulation attributes. This finding aligns with Kamrani and Hadizadeh's observation that heavier polymers demonstrate better drug acceptance and structural stability [ 50 ]. Their study showed that increasing PLA segment size enhanced system stability through reduced perturbation in radius of gyration measurements [ 50 ]. The energy profile analysis conducted in our research indicates stronger interactions than the Flory-Huggins interaction parameters provided by Kamrani and Hadizadeh in their prior work, which became more negative with improved polymer-drug compatibility [ 50 ]. In the radius of gyration analysis, our PLA5K-PEG5K structure had the most compact structure (4.5 nm average) when loaded with doxorubicin. Compactness is vital for drug delivery efficacy based on the work by Chansuna et al. showing how micelles adopt improved structural organization due to decreases in critical micelle concentration (cmc) as LA/EG ratios increased [ 51 ]. The RDF analysis in our study (peak value of 6.9 for PLA5K-PEG5K) indicates excellent structural compactness, which correlates with Kamrani and Hadizadeh's findings where sharp RDF peaks indicated dense micelle structures [ 50 ]. Their study demonstrated that PLA acts as the core while PEG acts as the shell, consistent with our observations [ 50 ]. Our molecular docking results indicated strong trastuzumab-polymer interactions (RMSD: 2.392, docking: -89.4666) having formation of four hydrogen bonds. The strong interactions are important for the purpose of a targeted delivery. Laudadio et al. (2021) examined drug-polymer interactions using full atomistic MD simulations and identified that drug binding energies were sensitive to drug hydrophilicity [ 52 ]. In their investigation using a hydrophobic drug similar to doxorubicin, they showed that hydrophobic drugs prefer to interact with the cores of the polymer, which is consistent with our results [ 52 ]. The SASA analysis we performed in our study (213.867 nm² of PLA5K-PEG5K with drug) indicated a reduction in solvent exposure and enhanced encapsulation. This is also consistent with the findings of Laudadio et al, where lower SASA values relate to better drug encapsulation and stability [ 52 ]. Our theoretical investigation into the drug-polymer and polymer-polymer interactions yields more information than just computational covalent interactions. The findings of the research presented here when compared to synthesized studies yields some interesting correlations. For example, in their study, Peng et al (2019) successfully synthesized micelles from Herceptin conjugated PCL-PEG copolymers for treatment on HER2-positive breast cancer [ 53 ]. Although the authors used PCL instead of PLA, the authors demonstrated an experimental method of conjugating antibody using aldehyde-amine reactions, now confirming our computational predictions of a stable trastuzumab-polymer interaction [ 53 ]. The authors were able to successfully target tumors while also showing improved therapeutic efficacy, thus supporting our computational prediction of stable complex formation between trastuzumab, polymer and drug molecules [ 53 ]. Our final stage simulation that investigated the polymer-drug-trastuzumab complex with POPE membrane showed strong interactions (the energy reached − 1100 kJ/mol), suggesting excellent compatibility with the membrane. The findings of our computational predictions are in agreement with the experimental findings of Peng et al. where their Herceptin-conjugated nanoparticles entered HER2-positive tumor cells through caveolin-mediated pathways [ 53 ]. While our simulations provide precise insights into antibody-polymer-drug interactions and the formation of stable PLA5K-PEG5K-trastuzumab complexes is consistent with the experimental results of Peng et al., our work has limitations that need to be addressed in future studies. Among these, this work utilized POPE membrane, whereas designing MDA-MB-231 membrane is essential for more realistic simulation of cancer cell membrane. In addition, we should consider the pH effects in future simulations potentially because cancer cell surfaces are more acidic. Greater simulation times than 400 nanoseconds and greater simulation box volumes could also provide insight into the proposed system. We also recommend exploring other molecular weights of carriers and, most importantly, using umbrella sampling simulation to more accurately investigate the drug release. 5. Conclusion In this molecular dynamics simulation study, we were able to effectively show that the material PLA5K-PEG5K copolymer composition, when conjugated with trastuzumab, is the most favorable for targeting doxorubicin to HER2 + breast cancer cells. Through the comprehensive thermodynamic analysis laid out in this work we determined that PLA5K-PEG5K demonstrated a high level of stability with the most compact structure (4.5 nm radius of gyration), drug encapsulation efficiency (RDF peak of 6.9), and energy profile (-1029.34 kJ/mol). Additionally, we discovered and confirmed by a molecular docking simulation (RMSD 2.392, MolDock score − 89.4666) that a stable PLA-PEG-trastuzumab complex could be formed with several hydrogen bonding interactions. The final membrane simulation revealed that there were strong interactions between the complex and the membrane (-1100 kJ/mol) validating the potential of this system and its ability to target cancer cells. This work is important in providing insight into rationally designing polymer-based drug delivery systems and we envision our study to provide experimental support for a targeted nanocarrier strategy in the future to treat HER2 + breast cancer. Abbreviations PLA: Poly(lactic acid) or Polylactic acid; PEG: Poly(ethylene glycol); NCBI: National Center for Biotechnology Information; RCSB PDB: Research Collaboratory for Structural Bioinformatics Protein Data Bank; CHARMM-GUI: Chemistry at Harvard Macromolecular Mechanics - Graphical User Interface; MD: Molecular Dynamics; NAMD: Nanoscale Molecular Dynamics; GROMACS: Groningen Machine for Chemical Simulations; VMD: Visual Molecular Dynamics; 1N8Z: Crystal structure of extracellular domain of human HER2 complexed with Herceptin Fab. Declarations Clinical trial number: not applicable. Ethical Approval Not applicable. This study involves computational molecular dynamics (MD) simulations using data exclusively obtained from publicly available databases including Protein Data Bank (PDB) and RCSB. No experiments were conducted on humans or animals, and no patient data or biological samples were collected or used. As this research is purely computational in nature and utilizes only publicly available data, it does not require specific institutional ethical approval. Consent to Participate Not applicable. Consent to Publish Not applicable. Data Availability Statement The initial structural data used in this study are publicly available from the Protein Data Bank (PDB) and RCSB database. The molecular dynamics simulation files, trajectory data, and analysis results generated during this study are available from the corresponding author upon reasonable request. Author Contributions Zohreh Arefi: Conceptualization, review proposal, Literature search, data extraction, Software, original draft writing, and visualization. Mohammad Khedri: Conceptualization, review proposal, review and editing Dr. Mostafa Keshavarz Morraveji: supervision, project administration, review and editing. Dr. Bahram Nasernejad: supervision, project administration, review and editing. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Competing Interests The authors declare that they have no competing interests. References Sriharikrishnaa S, Suresh PS, Prasada K S (2023). An introduction to fundamentals of cancer biology. In Optical Polarimetric Modalities for Biomedical Research pp. 307-330, Cham: Springer International Publishing.doi: https://doi.org/'https://doi.org/10.1007/978-3-031-31852-8_11. RA Weinberg (1983). A Molecular Basis of Cancer. Scientific American. 249 , 126-143. https://doi.org/https://doi.org/10.1038/scientificamerican1183-126. Sati P, Sharma E, Dhyani P, Attri DC, Rana R, Kiyekbayeva L, Büsselberg D, Samuel SM, Sharifi-Rad J (2024). Paclitaxel and its semi-synthetic derivatives: comprehensive insights into chemical structure, mechanisms of action, and anticancer properties. 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Biomaterials. 222:119420 . https://doi.org/https://doi.org/10.1016/j.biomaterials.2019.119420. Additional Declarations No competing interests reported. Supplementary Files articlesupplementary.docx Graphicalabstract.jpg Graphical abstract Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7359044","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":510111214,"identity":"f502a8c9-1e95-48f4-bfd7-82529087d9fa","order_by":0,"name":"Zohreh Arefi 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20:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7359044/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7359044/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90911539,"identity":"6ac1a010-6f60-4896-85a8-cbcb04e86608","added_by":"auto","created_at":"2025-09-09 13:43:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":62620,"visible":true,"origin":"","legend":"\u003cp\u003ea) MolDock Score value of five poses simulated in MolegroVirtual Docker software, and b) The type of pose 1 interactions between trastuzumab and PLA-PEG in the Molegro Virtual Docker software.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7359044/v1/f7f8198be7ebfb0a18d2de8e.jpg"},{"id":90911540,"identity":"f890bc18-55f1-4538-a18e-3f1ed611b9ff","added_by":"auto","created_at":"2025-09-09 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13:43:34","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":159729,"visible":true,"origin":"","legend":"\u003cp\u003eComparative energy analysis of PLA-PEG copolymers during the simulation of PLA-PEG micellar copolymer in the water: Electrostatic, van der Waals, and total energy profiles.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7359044/v1/ff86cf662ab930fff2a1afcc.jpg"},{"id":90912558,"identity":"e892de4e-d4a2-4942-8420-8e4079fe963a","added_by":"auto","created_at":"2025-09-09 13:51:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":131932,"visible":true,"origin":"","legend":"\u003cp\u003eComprehensive analysis of PLA-PEG copolymers in the first stage of molecular dynamics simulation: (a) Average of Radius of gyration, (b) Radial Distribution Function (RDF), and (c) Solvent Accessible Surface Area (SASA).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7359044/v1/3954ba7221dcd1130071ff5f.jpg"},{"id":90911550,"identity":"728f8a8a-3ee9-46d6-a7e2-0ccdaa2e0f56","added_by":"auto","created_at":"2025-09-09 13:43:34","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":165737,"visible":true,"origin":"","legend":"\u003cp\u003eenergy analysis of PLA-PEG copolymers with drug during the second step of molecular dynamics simulation: Electrostatic, van der Waals, and total energy profiles.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7359044/v1/130fee6bc800b38afbe97539.jpg"},{"id":90911545,"identity":"20810ce1-13f3-4788-8a3f-327bf07dc848","added_by":"auto","created_at":"2025-09-09 13:43:34","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":145250,"visible":true,"origin":"","legend":"\u003cp\u003eComprehensive analysis of Simulation of PLA-PEG micellar copolymer with drug: (a) Average of Radius of gyration, (b) Radial Distribution Function (RDF), (c) Solvent Accessible Surface Area (SASA), and (d) Hydrogen bond.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7359044/v1/fecf0ba7c02edfb30884a323.jpg"},{"id":90914117,"identity":"e570e3c2-d9f0-46e5-9b34-1262e26de858","added_by":"auto","created_at":"2025-09-09 13:59:34","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":119234,"visible":true,"origin":"","legend":"\u003cp\u003eKey analyses of the PLA5K-PEG5K–drug-trastuzumab complex for the 50 ns molecular dynamics simulation: a) Average van der Waals and electrostatic along with the total energy of the system; b) total energy profile showing stabilization of a system with time; c) SASA trend showing compactness; d) hydrogen bond count showing enhanced intermolecular interactions.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7359044/v1/1dc08ac81abbad28d220f75d.jpg"},{"id":90914888,"identity":"68d5a995-e547-4f40-ada8-c23b25c087d2","added_by":"auto","created_at":"2025-09-09 14:07:34","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":124158,"visible":true,"origin":"","legend":"\u003cp\u003eThe output of the Simulation of Trastuzumab-PLA-PEG-Drug Complex.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7359044/v1/80008734f3f9f8af8296cbe5.jpg"},{"id":90916190,"identity":"2716af7e-9878-41da-bebf-c81220ecca4c","added_by":"auto","created_at":"2025-09-09 14:15:34","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":170734,"visible":true,"origin":"","legend":"\u003cp\u003eNAMD’s MD simulation output: a) An overview of doxorubicin-PLA-PEG-Trastuzumab MD simulation along with a POPE membrane using NAMD software, and b) Energy average of this complex.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7359044/v1/74505339e49c1d0995a31ea8.jpg"},{"id":91148506,"identity":"6ec8a908-9550-464a-9273-68c8f7cc15dd","added_by":"auto","created_at":"2025-09-12 06:44:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2234366,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7359044/v1/696b6322-59c7-4e8a-b465-bbb8cf9f4c0c.pdf"},{"id":90911562,"identity":"d7c37955-2c0d-4b64-b723-c5664e341e14","added_by":"auto","created_at":"2025-09-09 13:43:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1016024,"visible":true,"origin":"","legend":"","description":"","filename":"articlesupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7359044/v1/19bc944202d8d08160ba8580.docx"},{"id":90914116,"identity":"4025c2f8-b239-481d-b97f-862557e6da3e","added_by":"auto","created_at":"2025-09-09 13:59:34","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":179488,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7359044/v1/90f0b9975cdb62b50d12901a.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of PLA-PEG Micellar Nanocarriers with Trastuzumab for Targeted Delivery of Doxorubicin to HER2+ Breast Cancer Cells: A Molecular Dynamics Simulation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer arises from uncontrolled growth and proliferation of cells, leading to tumors formation and an unacceptable systemic spread [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. If they are not identified and treated in time, unfortunately, they can lead to death due to collapse and spreading in the body [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Oncologists utilize different treatment options, including surgery, radiotherapy, and stem cell therapy, depending on the type and stage of cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the method that is used for a wide range of cancers is chemotherapy, in which anti-cancer drugs are administered into the patient's body in various ways, including oral, parenteral, transdermal, buccal, nasal, etc. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Although chemotherapy is widely used, conventional chemotherapy has limited efficacy due to the non-selective nature of typical cytotoxicity, which often leads to the destruction of cancer cells and healthy cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Drug delivery is a further development in chemotherapy\u0026ensp;involving the oncologist, specialist, and biomedical engineer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The aim of drug delivery is to formulate an optimal drug delivery system that releases the intended concentration of the drug to hit the target cells at the\u0026ensp;right time [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The drug faces many obstacles on its way through the body, most of which are related to the epithelial surfaces of the body [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The ideal drug delivery system can successfully overcome all of these obstacles [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The parameter that determines the ideality of a drug delivery system is bioavailability, which refers to the speed and extent of drug absorption and its availability at the site of action [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. According to the study by Anya et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], among the key parameters that lead to increased drug bioavailability and, subsequently, increased therapeutic efficacy are the type of drug carrier and monoclonal antibodies. There are a variety of carriers for drug delivery, from lipid and polymer carriers to carbon nanotubes and magnetic nanoparticles [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Ideal carriers should have certain properties, such as appropriate\u0026ensp;size distribution, biodegradability, biocompatibility, RES evasion, and long circulation half-life [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These features not only fulfill an ideal carrier profile\u0026ensp;but also improve drug bioavailability and therapeutic efficacy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Monoclonal antibodies work\u0026ensp;by highly specific molecular recognition, similar to a lock-and-key system [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Cell surface receptors act as specialized molecular locks to which only\u0026ensp;matching monoclonal antibodies would bind [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The overexpression of these receptors on cancer cells offers an opportunity for targeted therapy; thus, monoclonal antibodies specific for these receptors can be incorporated into a drug delivery complex, allowing us to convert an existing conventional\u0026ensp;drug delivery to a targeted drug delivery system [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Currently, drug delivery research involves the characterization of delivery systems based on design principles and in-vitro synthesis, which is often confirmed by Fourier Transform\u0026ensp;Infrared Spectroscopy (FTIR) analysis, while drug release process in tumor microenvironments are assessed in an in-vivo step [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These experimental methods require a lot of energy, time, and cost. In-silico drug delivery is a relatively new field that at least partially overcomes these hurdles because it allows for computations that explore key variables, such as\u0026ensp;ligand-protein binding types, complex stability in physiological conditions, and drug release features in the proximity of tumors of viable drug delivery systems (ViDDS), the main goals of this investigation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This\u0026ensp;study targets HER2-positive breast cancer and employs doxorubicin, PLA-PEG micellar copolymer, and trastuzumab as therapeutic agent, carrier, and targeting moiety, respectively. Breast cancer is one of the most\u0026ensp;aggressive and metastatic cancers in women worldwide, especially its HER2-positive subtype [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, the drawbacks of traditional chemotherapy, such\u0026ensp;as toxicity and low efficacy, have led to the creation of targeted delivery strategies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this regard, monoclonal antibodies conjugated to micellar copolymers show significant therapeutic efficacy. For example, Ahn et al. (2015) showed that a polymeric micelle containing a platinum drug linked to the Fab' fragment of an anti-TF antibody via a maleimide group led to a 15-fold increase in cell attachment within 1 hour and suppressed the growth of gastric tumors for more than 40 days [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In another study, Xu et al. (2019) showed that PEG-PCL nanoparticles containing docetaxel linked to the PD-L1 monoclonal antibody, while showing acceptable uptake, induced apoptosis in pancreatic cancer cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In another study, Yu et al. (2024) used mixed micelles containing the polymeric prodrug doxorubicin and chlorin e6 conjugated to a monoclonal antibody for combined chemo-photodynamic antitumor therapy, which resulted in increased drug accumulation at the tumor site, tumor growth suppression, reduced cardiotoxicity, and combined therapy (chemotherapy and photodynamic therapy) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Also, Zhang et al. (2023) demonstrated that the use of DAR PDL1 conjugated PLG\u0026thinsp;\u0026minus;\u0026thinsp;PEG polymer and SN38 drug improved tumor accumulation 2.8-fold in colorectal cancer models compared to SN38\u0026ensp;alone with an enhanced therapeutic outcome [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. All these studies show that the use of monoclonal antibodies in drug delivery systems increases treatment efficacy. Thus,\u0026ensp;this study utilizes trastuzumab (Herceptin\u0026reg;) monoclonal antibody for targeting HER2-positive breast cancer [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Based on the design principles of Anya et al [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], the PLA-PEG micellar copolymer was chosen\u0026ensp;as the carrier system to take advantage of the hydrophobic PLA core for cost-effective doxorubicin encapsulation, and the hydrophilic PEG corona for improved stability and circulation time [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Finally, molecular docking and dynamics simulations were performed to evaluate the possible binding between antibody and carrier, the temporal\u0026ensp;evolution, and the stability of the designed complex in physiological conditions. Molecular docking\u0026ensp;is one of the computational approaches used to predict the preferred orientation of a ligand to a target protein, and thus it is a key technique in structure-based drug design and discovery [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. There are several molecular docking\u0026ensp;tools available for predicting protein-ligand interactions. Among the most used platforms are Molegro Virtual Docker (MVD), which has an easy-to-use interface with strong cavity detection algorithms [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]; AutoDock,\u0026ensp;a widely known open-source suite that uses a Lamarckian Genetic Algorithm for binding predictions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]; and its successor AutoDock Vina, which aims for high performance and accuracy with better scoring functions and optimization algorithms [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Molecular dynamics simulations are grounded on Newton's second law of motion and are a powerful tool to examine how system properties evolve over time at the atomic level in accordance with known physical principles, thus providing new insight into the behavior of drug delivery systems\u0026ensp;in physiological environments [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. GROMACS was used for stability and absorption\u0026ensp;analyses [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], while NAMD was used for the release process in the cellular microenvironment in this study [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The current study is the first in-silico investigation of the PLA-PEG-TRASTUZUMAB complex as\u0026ensp;a doxorubicin delivery system for HER2-positive breast cancer therapy. Although the\u0026ensp;current study is only limited to molecular docking and dynamics studies, future studies will include potential in vitro and in vivo validation of predicted results.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cb\u003eMaterials\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe structure of the drug complex PLA-PEG-Trastuzumab-DOX was prepared using the following components: The doxorubicin structure was derived from the DrugBank database. The crystallographic structure\u0026ensp;of Trastuzumab was obtained from the RCSB Protein Data Bank (PDB ID: 1N8Z). Molecular weights\u0026ensp;of PLA-PEGs were designed via Avogadro software and the CHARMM-GUI server before optimizing their geometry in Gaussian software. Topology\u0026ensp;files were prepared by the PolyParGen server. The Visual Molecular Dynamics (VMD) software was used to construct\u0026ensp;a POPE membrane. All the molecular dynamics simulations were carried out on the Ubuntu operating system using GROMACS 5.1.2, except for the release study, which was performed using NAMD due to the large\u0026ensp;system size.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Molecular Docking Simulation\u003c/h2\u003e\u003cp\u003eTo evaluate the potential types of interactions between PLA-PEG and the antibody, molecular docking simulations were employed in this study. For conjugation of PLA-PEG to the antibody using the EDC/NHS technique, a carboxylic acid (COOH) linker was attached to the terminal end of the PEG segment [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In order to perform molecular docking, ligand-receptor structures are first optimized in the form of a preprocessing step that includes removing water molecules and ions from the molecular structure [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The space around the protein is then searched to identify potential ligand binding sites [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. After that, the possible interactions between the ligand-receptors will be predicted, and the output from these calculations gives the predicted energy value of each position with the type of interactions related to each pose [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Actually, molecular docking predicts several positions for ligand-receptor binding, and to find the best pose, many validation methods are used, such as checking the energy of each pose, RMSD, etc. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this study, we employed additional validation techniques for our work using the Molegro Virtual Docker and AutoDock software tools. Using this two software, we investigated the interactions between Trastuzumab and PLA-PEG. This work started by preparing the PDB structure of trastuzumab from the protein database RCSB with a 1N8Z identifier. The obtained structure was modified by removing unwanted components in AutoDock software. We first used the modified structure of trastuzumab and PLA-PEG to predict possible interactions in Molegro Virtual Docker software. After preparing the protein structure, we set the number of poses and runs to five and ten, respectively. The MoleDock SE algorithm was also selected for scoring. We evaluated the docking results of Molegro Virtual Docker software with a MolDock score, where a more negative score indicates more stability of the protein-ligand complex. Next, in order to perform molecular docking in AutoDock software, first the geometery files of Trastuzumab and PLA-PEG were prepared. A simulation box was created afterwards, wholly covering the ligand-protein region. After the main run, the results of molecular docking simulation in this software were checked by validation methods such as energy and RMSD. In other words, the above software tools are capable of predicting and analyzing any possible interactions between trastuzumab and PLA-PEG.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Molecular Dynamics Simulation\u003c/h2\u003e\u003cp\u003eIn this work, we utilized GROMACS version 5.1.2 for all of our molecular dynamic\u0026rsquo;s simulation, while our molecular graphics interface was provided by the VMD software. Simulations were run under the OPLS force field, with Newton's equation of motion integrated with the leapfrog algorithm. We implemented the LINCS algorithm to allow for using a time step of as large as 2 femtoseconds throughout our simulations. Our simulation protocol comprised four key stages: energy minimization (EM), temperature equilibration (NVT), equilibrium of pressure (NPT), and the main simulation run [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. For energy minimization, we applied the steepest descent algorithm. Also, we set 1000 kJ/mol/nm as the maximum force between consecutive time steps. In the equilibration steps, we first stabilized the system temperature at 300 K with a V-rescale thermostat and time constant of 1 in our simulation setting and then run the NVT step related to it. Then we set pressure at 1 bar with Parrinello-Rahman barostat and time constants of 2 picoseconds. The other setting for our work includes the cutoff radius that we set to 1 nm in all simulations for both electrostatic and van der Waals interactions, coupled with a period of 10 picoseconds Verlet scheme. We have dramatically reduced the edge effects for an infinite system by applying periodic boundary conditions in all three spatial dimensions. Structures of the carrier, drug, and monoclonal antibody were prepared for the initial step of simulation. Initially, we went ahead to prepare some kinds of molecular weights of PLA-PEG including PLA1.5K-PEG2K, PLA2K-PEG2K, PLA5K-PEG5K, PLA8K-PEG2K [\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The number of monomers for each weight was obtaned using the molecular mass of a single monomer. PLA, with the general formula C3H4O2, has a molecular mass of 72.062, while PEG with its repeating units (CH₂CH₂O)n, has a molecular mass of 44.052. With these calculations, the following monomer counts were obtained: PLA5K-PEG5K (69 PLA, 114 PEG), PLA2K-PEG2K (28 PLA, 45 PEG), PLA1.5K-PEG2K (21 PLA, 45 PEG), PLA8K-PEG2K (111 PLA, 45 PEG). Considering the high number of monomers some compounds have, structures were drawn using the CHARMM-GUI server and Avogadro software. Optimization of the geometries of each structure was done with DFT theory in the Gaussian software with using the basis set 6\u0026ndash;31\u0026thinsp;+\u0026thinsp;+\u0026thinsp;G(d,p). We then attached a COOH functional group as a linker to the PEG terminus of these structures\u0026mdash;a standard approach in protein-polymer conjugation techniques (as employed with trastuzumab)\u0026mdash;to create an active carboxyl group (typically activated using EDC/NHS chemistry) that enables direct chemical conjugation to the protein [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The structure of the anticancer drug doxorubicin was downloaded from the DrugBank database. Following the preparation of the initial structures, topology files were generated. The topology file contains all the critical information about simulations like atom types, presence of molecules, forces fields and other parameters [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The method for generating the topology file varies depending on the type of initial structure. For protein structures, such as the monoclonal antibody trastuzumab, GROMACS can generate the topology file directly from the geometry files [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. For other non-protein structures, like polymers, parameterization, and topology file generation is done through the online servers LigParGen, PolyParGen, and ATB [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. These output files frequently require some manual tuning. For our case, we used GROMACS and PolyParGen server to create the topology file of the trastuzumab and PLA-PEG copolymer, respectively. These carefully prepared initial structures and topology files therefore constitute a foundation upon which our molecular dynamics simulations can rely to determine interactions and behavior of our drug delivery system components with high precision and accuracy. Finally, we built a simulation box of dimensions 10\u0026times;10\u0026times;10 cubic nanometers and then solvated it with water molecules; thereafter four separate steps were conducted: energy minimization, temperature equilibration, pressure equilibration, and main run. All of the following sections have been simulated in these stages: simulation of PLA-PEG micellar copolymer, simulation of PLA-PEG micellar copolymer and drug, Simulation of trastuzumab-PLA-PEG-drug complex, and Simulation of polymer-drug-trastuzumab complex in the membrane along with HER2\u0026thinsp;+\u0026thinsp;receptor.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e2.2.1.\u003c/b\u003e \u003cb\u003eSimulation of PLA-PEG Micellar Copolymer in the Water\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eIn the first stage of this molecular dynamic\u0026rsquo;s simulation, we examined the behavior of four different molecular weights of PLA-PEG copolymers in an aqueous environment. We simulated 10 molecules of each weight: PLA1.5K-PEG2K, PLA2K-PEG2K, PLA5K-PEG5K, and PLA8K-PEG2K. Our simulations clearly explained the micellization process of these copolymers in water.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e2.2.2.\u003c/b\u003e \u003cb\u003eSimulation of PLA-PEG Micellar Copolymer with Drug\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eIn the second phase of our molecular dynamic\u0026rsquo;s simulation, we studied the behavior of 10 polymers of PLA-PEG in an aqueous medium with 10 drug molecules, to investigate the stability and drug encapsulation efficacy in the four different weights of PLA-PEG.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3. \u003cem\u003eSimulation of Trastuzumab-PLA-PEG-Drug Complex\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe third simulation stage was dedicated to the behavior of the optimal composition identified from previous stages: one PLA5K-PEG5K polymer, one drug molecule, and one monoclonal antibody in an aqueous environment. System stability and absorption over time were investigated by some thermodynamic analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e2.2.4.\u003c/b\u003e \u003cb\u003eSimulation of polymer-drug-trastuzumab complex in the membrane along with HER2\u0026thinsp;+\u0026thinsp;receptor\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe final simulation step was conducted with the NAMD software (Due to the large size of the system) at 50 ns to investigate the behavior of the drug delivery system in a more biologically relevant environment. First of all, a POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) membrane was constructed using VMD software. This membrane is used as a simplified model of the cell membrane. Afterward, the PLA-PEG-Trastuzumab complex previously optimized was added to this system. Additionally, from the NCBI database, the structure of the HER2\u0026thinsp;+\u0026thinsp;receptor was imported and added to the simulation for performing a test modeling of the interaction of the drug delivery system with its target cell surface receptor.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Molecular Docking Simulation of PLA-PEG and Trastuzumab Interactions\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe reliability of the results obtained in AutoDock is usually checked by obtaining the RMSD (Root Mean Square Deviation) values. An RMSD less than 1 \u0026Aring; is excellent, while 2\u0026ndash;3 \u0026Aring; is good, 3\u0026ndash;5 \u0026Aring; is weak, and above 5 \u0026Aring; is unacceptable [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-Table\u0026nbsp;1, according to our AutoDock results, mode 1 was the most favorable, with a binding affinity of -4.4 Kcal/mol and an RMSD of 2.4. Further analysis of AutoDock results revealed specific interactions between PLA-PEG and trastuzumab. The copolymer exhibited four hydrogen bonds to amino acid residues TYR87 (Tyrosine 87), LYS43 (Lysine 43), GLN39 (Glutamine 39) and GLY41 (Glycine 41) of trastuzumab as noted in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. This implies a successful binding between the COOH linker from PLA-PEG and amine group of antibody.\u003c/p\u003e\u003cp\u003eThe reliability of the results obtained in AutoDock is usually checked by obtaining the RMSD (Root Mean Square Deviation) values. An RMSD less than 1 \u0026Aring; is excellent, while 2-3 \u0026Aring; is good, 3-5 \u0026Aring; is weak, and above 5 \u0026Aring; is unacceptable [43]. As shown in Figure 2-Table 1, according to our AutoDock results, mode 1 was the most favorable, with a binding affinity of -4.4 Kcal/mol and an RMSD of 2.4. Further analysis of AutoDock results revealed specific interactions between PLA-PEG and trastuzumab. The copolymer exhibited four hydrogen bonds to amino acid residues TYR87 (Tyrosine 87), LYS43 (Lysine 43), GLN39 (Glutamine 39) and GLY41 (Glycine 41) of trastuzumab as noted in the Figure 2. This implies a successful binding between the COOH linker from PLA-PEG and amine group of antibody.\u003c/p\u003e\u003cp\u003eComparing results from AutoDock and Molegro Virtual Docker gave two similar hydrogen bonds and two different ones. This may have been because of computational differences between the software and differences in how each program identifies and classifies interactions. AutoDock results were interpreted in Discovery Studio and LigPlot software to emphasize the interactions more accurately, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMolecular docking studies with the mentioned software tools, while proving the possibility of binding, provide possible interactions that PLA-PEG may have with trastuzumab. The use of different software and the similarity of their outputs strengthens our confidence in the predicted connection model. These results are a computer validation of the structural basis of the proposed drug delivery complex, which provides a suitable field for experimental validations in the future.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Simulation of PLA-PEG Micellar Copolymer in the Water\u003c/h2\u003e\u003cp\u003eAfter simulating 10 molecules of each molecular weight of PLA-PEG copolymers: PLA1.5K-PEG2K, PLA2K-PEG2K, PLA5K-PEG5K, and PLA8K-PEG2K in an aqueous environment, we investigated some thermodynamic parameters to analyze the stability and adsorption of the drug complex over time. The analysis was started by investigating the interaction energies such as the van der Waals and electrostatic potentials using the g_mmpbsa analysis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Our results indicated that among the four compositions, PLA8K-PEG2K and PLA1.5K-PEG2K had the greatest magnitude of electrostatic potential and van der Waals energy, and hence, with average total energies of -765.52 and \u0026minus;\u0026thinsp;345.33 kJ/mol, respectively, they are the most energetically stable compounds, as shown in Fig.\u0026nbsp;4.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe also investigated the radius of gyration, which provides information about mass distribution around the center of mass in a molecule [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. A smaller radius of gyration will indicate a more compact structure and correlate to higher stability in an aqueous environment [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The Radial Distribution Function (RDF) analysis shows the atomic adsorption and structural compactness, as a higher RDF peak indicating a more structural adsorption and stability [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. While Solvent Accessible Surface Area (SASA) analysis strongly shows the stability of a structure in an aqueous environment over time, it decreases with more compact structures less exposed to solvent, typically correlating with greater stability [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Furthermore, RMSD indicates the average distance from a reference structure in a structure, capturing overall structural shifts and stability [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], In contrast, RMSF evaluates the amount each atom fluctuates from its average position [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. These analyses revealed the following results. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e-a showed that PLA1.5K-PEG2K had a favorable radius of gyration (An average of 4.8 nm). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e-b showed that PLA5K-PEG5K had the highest RDF value (approximately 3.8) for atomic adsorption and, hence, the most compact structural arrangement overall. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e-c confirms PLA5K-PEG5K's compactness with its lowest SASA values (193.59 nm\u003csup\u003e2\u003c/sup\u003e). Figures S2 and S3 also show PLA8K-PEG2K's stability with the smallest RMSD and RMSF value with an average of 6.19 \u0026Aring; and 3.13 \u0026Aring;, respectively. Overall, visualizing these results suggests PLA5K-PEG5K and PLA8K-PEG2K formed the most stable structures, with the greatest degree of compaction and molecular adsorption over time.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Simulation of PLA-PEG Micellar Copolymer with Drug\u003c/h2\u003e\u003cp\u003eAfter simulating 10 PLA-PEG polymers in an aqueous medium with 10 drug molecules, the energy analysis using the g_mmpbsa command revealed that although PLA1.5K-PEG2K exhibited the highest electrostatic potential energy and van der Waals forces, the more stable and decreasing energy pattern observed for PLA5K-PEG5K and demonstrates that this structure possesses the most stable energy profile with a total average of -1029.34 KJ/mol (Fig.\u0026nbsp;6). This stability pattern indicates that PLA5K-PEG5K represents the most stable configuration among all investigated polymers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBased on other analyses, PLA5K-PEG5K consistently demonstrates superior stability and drug interaction properties. With the smallest radius of gyration (An average of 4.5 nm) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003e-a, this combination achieves the most compact and stable conformation in aqueous environments while carrying the drug. It also shows the highest RDF peak (6.9) and lowest SASA value (213.867 nm\u003csup\u003e2\u003c/sup\u003e) and exhibits an excellent drug encapsulation efficiency. While PLA8K-PEG2K forms maximum hydrogen bonds (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003ed) and has the lowest RMSD fluctuations (An average of 5.93 \u0026Aring;) with the quickest convergence to stability (Figure S5), PLA5K-PEG5K with an average of 3.27 \u0026Aring; has the lowest atomic fluctuations in the RMSF analysis (Figure S6). On the whole, combined data from supplementary analysis (Figures S5-S6) and Figs.\u0026nbsp;6\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003e indicate PLA5K-PEG5K is the best polymer composition for drug delivery - highlighting the need for rational design to improve drug bioavailability.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Simulation of Trastuzumab-PLA-PEG-Drug Complex\u003c/h2\u003e\u003cp\u003eThe third simulation stage was dedicated to the behavior of the optimal composition identified from previous stages: one PLA5K-PEG5K polymer, one drug molecule, and one monoclonal antibody in an aqueous environment. System stability increased, as g_mmpbsa energy analysis plot in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003e-a shows. In other words, based on Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003e-b, the total energy of the system dropped from \u0026minus;\u0026thinsp;85.838 to \u0026minus;\u0026thinsp;631.715 kJ/mol, which is the average of \u0026minus;\u0026thinsp;409.3 kJ/mol, thus denoting the corresponding complex maintains its structure in the aqueous environment over time and does not collapse. Also, the decreasing trend of SASA shows that the relevant complex undergoes aggregation over time and offers acceptable adsorption. All of this indicates the stability and significant adsorption of the relevant complex in the aqueous environment and over time, as obtained from Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003e-c. Therefore, the reduction in SASA shows improved encapsulation of the drug and strong interactions between the constituents. Hydrogen bond analysis showed that there was an increase in the number of hydrogen bonds with time. The increase in hydrogen bonding, as evident in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003e-d, signifies a stable and cohesive structure and hence adds to the stability of the drug delivery system.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese results, taken together, indicate that the PLA5K-PEG5K copolymer forms a stable complex with the drug and trastuzumab, in such a way that provides favorable energy profiles, reduces solvent exposure, and increases intermolecular interactions. This stability is an essential prerequisite for the potential efficacy of the drug delivery system targeted for cancer therapy. Also shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e is the third simulation stage output.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Simulation of polymer-drug-trastuzumab complex in the membrane along with HER2\u0026thinsp;+\u0026thinsp;receptor\u003c/h2\u003e\u003cp\u003eIn the final simulation step that we conducted using NAMD software, due to the large size of the system, we investigated the behavior of the drug complex: PLA5K-PEG5K-DOX-Trustuzumab along with a POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) membrane. Based on Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e, the energy profile shows that the energy value reaches \u0026minus;\u0026thinsp;1100 kJ/mol over time. This significant amount shows the strength of interactions between the drug complex and the membrane and, more generally, the stability of the system in the presence of a lipid membrane.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCompared to the previous coarse-grained simulations, all-atom molecular dynamics simulations afford a higher degree of resolution, particularly in regard to the understanding at the atomic-level. Kamrani and Hadizadeh (2019) used coarse-grained MD simulations to model PLA-PEG and PCL-PEG aggregation [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], while Chansuna et al. (2014) used a DPD type simulation in PLA-PEG-PLA triblock copolymers [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Regardless of their computational benefits, our all-atom simulation allowed for greater resolution regarding the drug-polymer interactions and led to a more accurate assessment on the stability of the structure along with the binding energies. Our findings indicated PLA5K-PEG5K had the most ideal stability (total energy of -1029.34 kJ/mol) and exceptional drug encapsulation attributes. This finding aligns with Kamrani and Hadizadeh's observation that heavier polymers demonstrate better drug acceptance and structural stability [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Their study showed that increasing PLA segment size enhanced system stability through reduced perturbation in radius of gyration measurements [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The energy profile analysis conducted in our research indicates stronger interactions than the Flory-Huggins interaction parameters provided by Kamrani and Hadizadeh in their prior work, which became more negative with improved polymer-drug compatibility [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In the radius of gyration analysis, our PLA5K-PEG5K structure had the most compact structure (4.5 nm average) when loaded with doxorubicin. Compactness is vital for drug delivery efficacy based on the work by Chansuna et al. showing how micelles adopt improved structural organization due to decreases in critical micelle concentration (cmc) as LA/EG ratios increased [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The RDF analysis in our study (peak value of 6.9 for PLA5K-PEG5K) indicates excellent structural compactness, which correlates with Kamrani and Hadizadeh's findings where sharp RDF peaks indicated dense micelle structures [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Their study demonstrated that PLA acts as the core while PEG acts as the shell, consistent with our observations [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Our molecular docking results indicated strong trastuzumab-polymer interactions (RMSD: 2.392, docking: -89.4666) having formation of four hydrogen bonds. The strong interactions are important for the purpose of a targeted delivery. Laudadio et al. (2021) examined drug-polymer interactions using full atomistic MD simulations and identified that drug binding energies were sensitive to drug hydrophilicity [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In their investigation using a hydrophobic drug similar to doxorubicin, they showed that hydrophobic drugs prefer to interact with the cores of the polymer, which is consistent with our results [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The SASA analysis we performed in our study (213.867 nm\u0026sup2; of PLA5K-PEG5K with drug) indicated a reduction in solvent exposure and enhanced encapsulation. This is also consistent with the findings of Laudadio et al, where lower SASA values relate to better drug encapsulation and stability [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Our theoretical investigation into the drug-polymer and polymer-polymer interactions yields more information than just computational covalent interactions. The findings of the research presented here when compared to synthesized studies yields some interesting correlations. For example, in their study, Peng et al (2019) successfully synthesized micelles from Herceptin conjugated PCL-PEG copolymers for treatment on HER2-positive breast cancer [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Although the authors used PCL instead of PLA, the authors demonstrated an experimental method of conjugating antibody using aldehyde-amine reactions, now confirming our computational predictions of a stable trastuzumab-polymer interaction [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The authors were able to successfully target tumors while also showing improved therapeutic efficacy, thus supporting our computational prediction of stable complex formation between trastuzumab, polymer and drug molecules [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Our final stage simulation that investigated the polymer-drug-trastuzumab complex with POPE membrane showed strong interactions (the energy reached \u0026minus;\u0026thinsp;1100 kJ/mol), suggesting excellent compatibility with the membrane. The findings of our computational predictions are in agreement with the experimental findings of Peng et al. where their Herceptin-conjugated nanoparticles entered HER2-positive tumor cells through caveolin-mediated pathways [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. While our simulations provide precise insights into antibody-polymer-drug interactions and the formation of stable PLA5K-PEG5K-trastuzumab complexes is consistent with the experimental results of Peng et al., our work has limitations that need to be addressed in future studies. Among these, this work utilized POPE membrane, whereas designing MDA-MB-231 membrane is essential for more realistic simulation of cancer cell membrane. In addition, we should consider the pH effects in future simulations potentially because cancer cell surfaces are more acidic. Greater simulation times than 400 nanoseconds and greater simulation box volumes could also provide insight into the proposed system. We also recommend exploring other molecular weights of carriers and, most importantly, using umbrella sampling simulation to more accurately investigate the drug release.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this molecular dynamics simulation study, we were able to effectively show that the material PLA5K-PEG5K copolymer composition, when conjugated with trastuzumab, is the most favorable for targeting doxorubicin to HER2\u0026thinsp;+\u0026thinsp;breast cancer cells. Through the comprehensive thermodynamic analysis laid out in this work we determined that PLA5K-PEG5K demonstrated a high level of stability with the most compact structure (4.5 nm radius of gyration), drug encapsulation efficiency (RDF peak of 6.9), and energy profile (-1029.34 kJ/mol). Additionally, we discovered and confirmed by a molecular docking simulation (RMSD 2.392, MolDock score \u0026minus;\u0026thinsp;89.4666) that a stable PLA-PEG-trastuzumab complex could be formed with several hydrogen bonding interactions. The final membrane simulation revealed that there were strong interactions between the complex and the membrane (-1100 kJ/mol) validating the potential of this system and its ability to target cancer cells. This work is important in providing insight into rationally designing polymer-based drug delivery systems and we envision our study to provide experimental support for a targeted nanocarrier strategy in the future to treat HER2\u0026thinsp;+\u0026thinsp;breast cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePLA: Poly(lactic acid) or Polylactic acid; PEG: Poly(ethylene glycol); NCBI: National Center for Biotechnology Information; RCSB PDB: Research Collaboratory for Structural Bioinformatics Protein Data Bank; CHARMM-GUI: Chemistry at Harvard Macromolecular Mechanics - Graphical User Interface; MD: Molecular Dynamics; NAMD: Nanoscale Molecular Dynamics; GROMACS: Groningen Machine for Chemical Simulations; VMD: Visual Molecular Dynamics; 1N8Z: Crystal structure of extracellular domain of human HER2 complexed with Herceptin Fab.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. This study involves computational molecular dynamics (MD) simulations using data exclusively obtained from publicly available databases including Protein Data Bank (PDB) and RCSB. No experiments were conducted on humans or animals, and no patient data or biological samples were collected or used. As this research is purely computational in nature and utilizes only publicly available data, it does not require specific institutional ethical approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe initial structural data used in this study are publicly available from the Protein Data Bank (PDB) and RCSB database. The molecular dynamics simulation files, trajectory data, and analysis results generated during this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZohreh Arefi: Conceptualization, review proposal, Literature search, data extraction, Software, original draft writing, and visualization.\u003c/p\u003e\n\u003cp\u003eMohammad Khedri: Conceptualization, review proposal, review and editing\u003c/p\u003e\n\u003cp\u003eDr. Mostafa Keshavarz Morraveji: supervision, project administration, review and editing.\u003c/p\u003e\n\u003cp\u003eDr. Bahram Nasernejad: supervision, project administration, review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSriharikrishnaa S, Suresh PS, Prasada K S (2023). 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Biomaterials. \u003cstrong\u003e222:119420\u003c/strong\u003e. https://doi.org/https://doi.org/10.1016/j.biomaterials.2019.119420.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Drug Delivery, Targeted Drug Delivery, Monoclonal Antibody, Nanoparticle, Molecular Dynamics, Molecular Docking","lastPublishedDoi":"10.21203/rs.3.rs-7359044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7359044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eContext\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBreast cancer that is HER2-positive is still recognized as one of the more aggressive forms of breast cancer, which strongly suggests the need for more effective targeted forms of therapy. To develop better therapeutic approaches, understanding how drug delivery systems and monoclonal antibodies interact will be important. This work explores a new targeted delivery system for doxorubicin that employs PLA-PEG micellar copolymers with trastuzumab for better therapeutic effect against HER2-positive breast cancer. Molecular docking analysis suggests that there is a hydrogen bonding interaction between the COOH terminal group of the PLA-PEG micelle and the amine groups of trastuzumab, indicating favorable interactions with a COOH linker that was established on the PEG terminus. Among four different PLA-PEG molecular weight combinations evaluated using molecular dynamics simulation, PLA5K-PEG5K demonstrated optimal stability and absorption properties, as determined by structural and energetic analyses. The PLA5K-PEG5K-doxorubicin system with trastuzumab preserved structural integrity in aqueous solution and also indicates a favorable absorption and stability over time. Also, the behavior of this system near a POPE membrane was investigated, which obtained high interaction energy values, indicating great potential to deliver drugs into cells. These computational findings allow for the theoretical groundwork of a better-targeted delivery system, which could lead to improved outcomes for patients suffering from HER2-positive breast cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular docking studies were performed to assess protein-polymer binding between trastuzumab and PLA-PEG copolymers. Molecular dynamics (MD) simulations were completed to assess the stability and adsorption of the different PLA-PEG molecular weight combinations in an aqueous environment. System stability was examined through solvent-accessible surface area analysis, energy analysis, and radius of gyration measurements. Evaluation of membrane interactions was performed with a POPE model to assess their potential as a delivery vehicle, cellular delivery potential, and the energetics of the interactions. All MD simulations were run under periodic boundary conditions, and each system was simulated for 50 ns using Gromacs.\u003c/p\u003e","manuscriptTitle":"Evaluation of PLA-PEG Micellar Nanocarriers with Trastuzumab for Targeted Delivery of Doxorubicin to HER2+ Breast Cancer Cells: A Molecular Dynamics Simulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 13:43:29","doi":"10.21203/rs.3.rs-7359044/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52817cba-ebb8-48cd-be50-277006e69420","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-07T21:08:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-09 13:43:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7359044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7359044","identity":"rs-7359044","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}