Preparation of pH-sensitive carboxymethyl cellulose/bovine serum protein complex particles and investigation of their drug carrying capacity

Preparation of pH-sensitive carboxymethyl cellulose/bovine serum protein complex particles and investigation of their drug carrying capacity | 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 Preparation of pH-sensitive carboxymethyl cellulose/bovine serum protein complex particles and investigation of their drug carrying capacity Kaiqiang Zheng, Ziang Quan, Xiaohui Wang, ShiHao Zhou, Kuo Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4755038/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Mar, 2025 Read the published version in Cellulose → Version 1 posted 9 You are reading this latest preprint version Abstract In this work, structurally stable and high-performance drug deliver composite particles were prepared successfully through a portable and simple electrostatic self-assembly method with carboxymethyl cellulose (CMC) and bovine serum protein (BSA). When regulating the pH value of the system lower than the isoelectric point of BSA, it exhibited positivity and was assembled with CMC through electrostatic attraction. The prepared composite particles were characterized, and different factors impacting on the composite materials were investigated. Amoxicillin and theophylline were selected as the experimental drugs to test the drug sustained-release performance of the composite particles. Results indicated that the composite particles possessed uniform shape, with an average particle size of 255 nm before heating and a PDI of 0.16 before heating. After heating, the particle size increased to 296 nm with PDI of 0.219. The encapsulation rate of amoxicillin and theophylline were found to be 44.1% and 58.9%, and the sustained-release curve demonstrated excellent drug loading efficiency and sustained release ability. This study demonstrates the potential application of CMC, a biocompatible natural high molecular weight material, in the delivery of small molecule drugs. It also demonstrates the development potential of composite systems composed of proteins such as BSA and polysaccharides. composite particles carboxymethyl cellulose (CMC) Bovine serum protein (BSA) electrostatic interaction drug load Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction The investigation of drug-carrying micro/nano particles is a prominent area of research in the field of drug delivery systems(McClements 2015 ).Composite particles, due to the high specific surface area caused by their small size, have the potential to enhance drug stability and utilization while reducing side effects(Yang et al. 2016 ; Liu et al. 2020 ; Lukova et al. 2023 ).Furthermore, they can be tailored for different functionalization purposes(Brown et al. 2006 ).As a result, composite particles play a crucial role in various applications within the field of drug delivery(Dai and Si 2019 ; Joshy et al. 2017 ). The selections of materials and preparation methods for constructing composite particles significantly impact the efficiency of their drug loading and sustained release(Moschakis and Biliaderis 2017 ; Damiri et al. 2023 ).Various materials can serve as drug carriers, including metallic inorganic materials, inorganic non-metallic materials, synthetic polymer and so on. While these carriers demonstrate good performance, they also present certain issues such as poor biocompatibility, toxicity or complex preparation processes involving organic compounds that may compromise safety(Gyarmati and Pukánszky 2017 ).In contrast, natural polymer materials are renewable,biodegradable and possess excellent biocompatibility properties(Javanbakht and Shaabani 2019 ; Yang et al. 2023 ; Papageorgiou 2018 ). Composite particles composed of natural biodegradable polymer materials are synthesized in a straightforward, environmentally friendly manner to serve as carriers for drug delivery(Timilsena et al. 2019 ; Patil and Patel 2020 ; Jamroży et al. 2024 ).These complex have the capability to encapsulate drugs, improve drug utilization, control drug release, and reduce certain side effects(Fazal et al. 2023 ; Paliya et al. 2023 ; Yan et al. 2023 ).Polysaccharides and proteins are abundant natural biopolymers that serve as fundamental units in life. The assembly between them forms the basis for the existence and continuation of all organisms(Devi et al. 2017 ; Xu et al. 2020 ).Therefore, it is of great significance to investigate the assembly of both components in order to construct a delivery system with high biocompatibility and biosafety(Ezhilarasi et al. 2013 ; Jones and McClements 2010 ; Xiao et al. 2023 ).Xiong et al. conducted a study on the formation of nanoparticles embedded with resveratrol by combining ovalbumin and carboxymethyl cellulose sodium. The nanocomplexes were formed by the electrostatic assembly between OVA and CMC and presented a spherical morphology, compared to native resveratrol, the bio-accessibility of resveratrol embedded in nanocomplexes and nanoparticles was increased to 60% and 80%(Xiong et al. 2018 ).Liang et al. prepared biodegradable nanoparticles (NPs). Assembled with sodium carboxymethyl cellulose (CMC) and zein to produce zein–CMC NPs. Paclitaxel (PTX) was 95.5% encapsulated at a zein–CMC weight ratio of 1 : 3 and the NPs were spherical with an average particle size of approximately 159.4 nm, The NPs demonstrated good stability over a broad range of pH ranging from 3.7 to 11.0(Liang et al. 2015 ).Fuge et al. prepared ultra long and stable ovalbumin and carboxymethyl cellulose nanoparticles, investigated the effects of different influencing factors on their stability, and successfully loaded curcumin efficiently into these particles, these particles with the loose structure of wool ball could effectively load curcumin. Curcumin-loaded of OVA/CMC nanoparticles show good DPPH· scavenging activity, Ferric-reducing ability and ABTS scavenging activity compared with curcumin/water(Niu et al. 2021 ). The main preparation methods for these composite include physical mixing, enzyme conjugation and chemical cross-linking(Zeeb et al. 2017 ).Chemical cross-linking may involve the use of other crosslinking agents, which can have a certain impact on the environment and health during the preparation process. The technical requirements for enzyme conjugation are intricate, making the process challenging. In contrast, physical mixing, take self-assembly for example, have milder and more controllable conditions, and have no impact on organisms and the environment(Niu et al. 2021 ; Yang et al. 2014 ).When the environmental conditions are favorable, polyelectrolytes with opposite charges will spontaneously bind(Mariani et al. 2016 ; Maciel et al. 2017 ).However, changes in the external equilibrium environment can lead to the self-assembly forming a single-phase solution or precipitate(Mariani et al. 2016 ; Liu et al. 2009 ; Li et al. 2012 ).Therefore, various factors such as pH value, concentration, mass ratio, and ion concentration can impact the formation of nanoparticles(Mariani et al. 2016 ; Li et al. 2012 ; Eghbal and Choudhary 2018 ).Davi conducted a study on the role of pH value in the formation of CA/CMC microparticles. The research demonstrated that different pH values and substance ratios during the composite coagulation process would result in different CA and CMC microparticles being produced(Souza et al. 2024 ). BSA, as the animal serum protein with the best similarity to human serum albumin (HSA), is widely used in biochemical laboratories and is referred to as the fifth component. It is consisted of 583 amino acid residues with a molecular weight of 66.4 kDa and an isoelectric point of 4.7(Kaibara et al. 2000 ). CMC contains numerous carboxyl groups (-COO) in its molecular structure, making it soluble in water and capable of forming ionic bonds with amino group molecules in aqueous solutions. Its charge distribution depends on the number of carboxyl groups present in each glucose unit, and it remains stable within a pH range of 2–10.(Geng et al. 2014 ; Song and Chen 2015 ).Additionally, CMC serves as a non-toxic, biocompatible, and biodegradable natural polymer. It plays a significant role in various types of occasions.(Kong et al. 2022 ; Sedlář et al. 2023 ).For example, it can enhance drug efficiency and delivery(Li and Wang 2015 ; Song et al. 2012 ; Zhu et al. 2013 ). In this study, we utilized a combination of electrostatic self-assembly and thermal induction to prepare CMC/BSA micro/nano particles as carriers for small molecule substances, aiming to improve material stability and achieve controlled sustained release. The impacts of various factors on CMC/BSA complex particles were investigated, resulting in the successful preparation of excellent CMC/BSA complex system. And as model drugs, amoxicillin and theophylline were selected to demonstrated the encapsulation and sustained-release performance of the composite system for drug delivery. Furthermore, our study aims to expand the construction of functionalization for different polysaccharide-protein complexes by exploring their feasibility through result analysis. 2. Materials and Testing instruments 2.1.Materials CMC (degree of substitution 0.95, viscosity at 1%, average molecular weight 90 KDa) was synthesized in the laboratory. BSA (96%) was purchased from Maclean Biochemical Technology Co. LTD(Shanghai,China). Amoxicillin (CP) was purchased from Maclean Biochemical Technology Co. LTD(Shanghai,China). Theophylline (medical grade) was purchased from Changchun Tairen Technology Co. LTD (Chuangchun, China). Glacial acetic acid (AR) was obtained from Tianjin Opuseng Chemical Co. LTD (Tianjin, China). NaCl (AR) was purchased from Tianjin Yongda Chemical Reagent Co, LTD (Tianjin, China). 2.2 Characterization of CMC/BSA composite particles The mean particle size and poly dispersion index of the particles were determined by dynamic light scattering using Malvern Nano-ZS90 (Malvern,UK) at 25°C with an angle detection at 173°C. The data selected were the mean values of three independent repeats. Zeta potential is an important index for characterizing the stability of colloidal dispersions, with higher absolute values indicating greater stability. The sample was balanced within the instrument for 120 seconds and then measured three times to collect data from at least 10 consecutive readings each time, ensuring accuracy. Fourier infrared analysis was performed using the Bruker TENSOR Fourier Infrared spectroscopy instrument (Bruker, Germany) measuring in the range of 500-4000cm, and analyzing functional groups and recombination. The JSM-7610F Plus scanning electron microscope (JEOL, Japan) was used to observe and study the morphology of the composite material CMC/BSA (1:3), both unheated and heated. The sample diluted ten times was dropped on conductive adhesive, dried, sprayed with gold, and then observed for its morphology. 2.3 Preparation of CMC/BSA composite particles Since proteins are amphoteric electrolytes, the pH value of BSA was adjusted to an appropriate level before preparation to enhance the influence of electrostatic forces. BSA and CMC were dissolved in ultra-pure water (1mg/ml) and stirred at room temperature (25℃) for 2 hours with a gentle magnetic force. The BSA solution should be refrigerated at 4℃ overnight after preparation to ensure complete dissolution. Subsequently, the pH-adjusted BSA solution was added dropwise into the CMC solution at different ratios, followed by gentle stirring for 30 minutes. The resulting compound was then heated in a water bath at 80°C for 30 minutes to obtain the desired product. The composite mechanism of BSA and CMC is shown in Scheme. 1. 2.4 Drug encapsulation Amoxicillin and theophylline (3000µg; 1000µg/ml) were dissolved in ultra-pure water. A certain amount of drug solution was then added to heated CMC-BSA complex solution and slowly stirred for 1 hour, resulting in a final drug content of (3000µg/1500µg) in a 15ml sample. The loaded drug sample was poured into an ultrafiltration centrifuge tube (10000MW) and centrifuged at 4000rpm for 45 minutes. The absorbance of the filtrate after centrifugation was measured using a UV-VIS spectrophotometer within the corresponding range, and the packaging rate and load rate were calculated based on the standard curve. 2.5 In vitro drug release Under physiological conditions, in vitro drug release was studied using the dialysis method. Specifically, a 15 ml solution of the compound containing the drug and an equivalent concentration of free drug were placed into a dialysis bag (Total amount of amoxicillin and theophylline drugs is 12000ug/3000ug),and subjected to dialysis with 200 ml PBS (pH 7.4) at a constant temperature of 37°C in a water bath, with slow magnetic stirring for 24 hours. At appropriate intervals, 5ml of PBS was collected and replenished with an equal amount of fresh PBS each time. The amount of drug released was measured using a UV-VIS spectrophotometer. 3. Resaults and discussion 3.1 Exploration of the influence factors of CMC and BSA composite system 3.1.1 The effect of pH on the turbidity of CMC and BSA composite system Due to BSA being a zwitterionic protein with an isoelectric point of 4.7, it exhibits different surface electrical properties when the pH value of the system is different. However, CMC exhibits negative charge in aqueous solution systems due to the ionization of sodium ions. Therefore, when the pH value of the system is different, the composite state of the CMC and BSA composite system is also different. If we want to utilize the electrostatic attraction between CMC and BSA as the driving force for their self-assembl, we must explore the influence of the pH value of the composite system. The zeta potential values at different pH levels were measured (Fig. 1 ). From Fig. 1 , it can be seen that when the pH value of the system is higher than 4.7, the BSA surface exhibits negative charge, while when the pH value of the system is lower than 4.7, its surface exhibits positive charge. Therefore, the protein's charge is pH-dependent. In order to obtain the pH range suitable for self-assembly of CMC and BSA, the turbidity of composite systems with different ratios were tested as a function of pH value, and the results The are shown in Fig. 2 . From Fig. 2 , it can be seen that throughout the entire pH range, there is an initial increase followed by a decrease in turbidity. Overall, it is evident that pH values have a substantial influence on the combination of these two substances. Taking CMC/BSA(1:3) as an example, when the pH value exceeds pHa, the complex turbidity of CMC/BSA remains relatively constant. Generally speaking, when the pH value surpasses the protein's isoelectric point, the protein exhibits overall anionic properties. However, weak electrostatic interactions may still occur between local positive charge patches of the protein and anionic groups of polysaccharides. When the pH value ranged from pHa to pHb, there is a slight increase in turbidity. During this time frame, due to a lower pH than that of the protein's isoelectric point, oppositely charged substances properly combine to form a soluble complex which maintains homogeneity and forms a single-phase system.If the pH value falls below pHb, there will be a sharp rise in solution turbidity where electrostatic forces play a dominant role. At this stage, a large number of CMC and BSA self-assemble to form composite particles. With a further decrease in pH value, electric neutralization is achieved at pHd. This results in a change in the color of the solution and the formation of a large number of insoluble composite condensates with the highest stability and content, leading to phase separation. When the pH value drops below pHd and more H + ions are added to the solution, the polysaccharide molecules become gradually protonated, reducing the net negative charge. As a result, the interaction strength between CMC and BSA decreases, leading to reduced electrostatic attraction. Consequently, the condensate begins to dissociate, causing the mixed solution to become transparent while maintaining stable turbidity. Furthermore, adjusting the ratio of polysaccharide to protein will alter the pH of the turbidity curve formed by their complexation. Increasing BSA concentration shifts this curve towards higher pH due to an increase in protein content and consequently an increase in CMC chains available for interaction with proteins. Conversely, increasing CMC content raises negative charge levels in the composite system necessitating more positive charge for neutralization. Thus shifting the turbidity curve towards lower pH values. This phenomenon is evident when comparing CMC/BSA(3:1) at a pH value of 4.5 where its clarified solution indicates minimal recombination between them suggesting that electrostatic forces do not play a dominant role. Therefore different ratios at same pH also have an effect on their recombination process. 3.1.2 The effect of pH on the Zeta potential of CMC and BSA composite system The Zeta potential is closely related to the interaction between CMC and BSA. Therefore, we selected three typical raw material ratios and tested the Zeta potential of the composite system with pH changes, as shown in Fig. 3 . When the zeta potential of the composite solution approaches 0, stronger electrostatic interactions will result in the formation of insoluble condensates and phase separation. This is consistent with the observation of condensates in turbidity as pH decreases. Furthermore, a higher absolute value of zeta potential indicates an increased surface charge, leading to repulsion between particles and thus contributing to the overall stability of the system. Conversely, if the absolute value of the zeta potential is very low, particles are more likely to attract each other, resulting in instability within the system. In conclusion, considering that electrostatic self-assembly plays a crucial role in particle recombination for CMC/BSA, it is essential to carefully select an appropriate pH range and consider different ratios for optimal results. 3.1.3 The effect of pH on the fluorescence spectrum of CMC and BSA composite system The fluorescence spectrum of CMC-BSA is depicted in Fig. 3 . In pure BSA and BSA with a high pH, the fluorescence intensity decreases steadily with the continuous addition of CMC. However, the maximum emission peak does not shift, while the peak of the maximum absorption is related to pH. This is due to the fact that altering the pH of a protein impacts its structural and functional characteristics. At a low pH value, the BSA absorption peak was at 334.6nm. As the concentration of CMC increases, not only does the fluorescence intensity weaken, but it also shifts from 334.6nm to 327.6nm, exhibiting a noticeable blue shift. These findings suggest that CMC has the ability to quench the fluorescence of BSA, leading to a change in the microenvironment of the protein upon addition of CMC, resulting in a more hydrophobic environment. The blue shift in fluorescence is typically associated with the fluorescence characteristics and charge distribution within the fluorescent molecule. Any changes in charge distribution within the fluorescer molecule can cause a shift in emission peak towards shorter wavelengths. This phenomenon can be achieved through introduction or chemical modification of heteroatoms within the molecular structure. Fluorescence studies same have demonstrated that variations in pH levels impact the interaction betweethe two components. From the results of the influence of pH values on the turbidity, Zeta potential, and fluorescence intensity of the composite system, it can be seen that When the pH exceeds the protein's isoelectric point, although it exhibits anionic properties overall, weak electrostatic interactions may occur between local positive charge patches on the protein and polysaccharide anionic groups, resulting in changes in particle size. Conversely, when adjusting below the isoelectric point, decreasing pH leads to more deposition of BSA on CMC chains, forming soluble complexes through electrostatic interactions that alter solution appearance. So we come to the conclusion that when a lower pH value which is below the isoelectric point of BSA is selected, BSA and CMC can achieve a relatively stable composite through electrostatic self-assembly. Therefore, this pH range 4.0-4.5 is selected when preparing CMC/BSA composite particles. 3.1.4 The effect of pH and ratio on the CMC/BSA composite particles In order to explore and obtain accurate pH values accurately obtain CMC/BSA composite particles with excellent structure, morphology, and properties within the appropriate pH range that can form stable CMC/BSA composite systems, particle size and PDI tests were conducted on composite particles with different pH and composition ratios. The results are shown in Fig. 4 . From the results in Fig. 4 , it can be concluded that pH value and the ratio of CMC and BSA have an impact on the particle size and PDI of composite particles. When CMC and BSA were combined into particles in different ratios, in each system with a constant pH, as the amount of CMC increased, the particle size and PDI of CMC/BSA composite particles showed an overall upward trend. This is because CMC and BSA self-assemble together driven by electrostatic attraction. When there was less CMC, BSA was the main component in the system. Except for the BSA composited with CMC, excess BSA accumulated on the surface of the composite particles, resulting in a slightly larger average particle size and poor dispersion of the system. When BSA and CMC can be perfectly matched, the system was mostly composed of structurally intact CMC/BSA composite particles, with the minimum average particle size and the best particle dispersion. When CMC continued to increase, there was an excess of CMC in the system. In addition to CMC/BSA composite particles, there was also a three-dimensional structure formed by the entanglement of CMC long chain structures, which continuously increased the average particle size of the system, the mechanism is shown in Scheme 2 . By comparing the changes in particle size and PDI of composite particles in different pH systems horizontally, it can be found that at a pH of 4.3, the particle size and PDI values of composite particles showed the minimum values, indicating that in this pH system, CMC and BSA reached the best composite state, and the dispersion of composite particles was also the best. Based on the analysis of the above particle size and PDI results, when BSA and CMC were combined in a 3:1 ratio at a pH of 4.3 in the system, the resulting composite particle structure and dispersion were the most excellent. 3.1.5 The effect of temperature on the CMC/BSA composite particles Due to the sensitivity of protein structure to temperature, during heating, the protein will change its structure and expose embedded non-polar peptides, thereby enhancing hydrophobic interactions between adjacent non-polar fragments of peptides. Therefore, under heating conditions, the process of BSA and CMC composite will become more complex. In order to explore the effect of heating temperature on composite particles, particles composed of BSA and CMC in a 3:1 ratio were heated at 40°C, 60°C, and 80°C in a pH 4.3 system, and their particle size and PDI were characterized. The results are shown in Fig. 5 . From Fig. 5 , it can be seen that after heating, the particle size and PDI slightly increase, indicating that heating has a certain impact on the structure and distribution of composite particles, with PDI being the best after heating at 80 ℃. 3.1.6 The impact of salt solution on CMC/BSA composite particles The addition of salt solution (NaCl) can shield electrostatic interactions. The addition of sodium chloride will cause Na + to bind to negatively charged polysaccharides, while Cl − will bind to positively charged proteins, producing an electrostatic shielding effect, reducing the possibility of electrostatic attraction between proteins and polysaccharides, thus preventing electrolytes that originally carried two opposite charges from generating electrostatic recombination. Therefore, the tolerance of composite particles to salt solution is an important indicator for measuring their use as a carrier system. In order to evaluate the tolerance of CMC/BSA composite particles to salt solution, different concentrations of NaCl (10/4/1wt%) were added to the system to observe their changes. The results are shown in Fig. 6 . From Fig. 6 , it can be seen that with the increase of NaCl in the system, the turbidity of the solution decreased and gradually clarified. When 1wt% NaCl was added to the system, the effect on the appearance of the solution was minimal. At a concentration of 4wt% NaCl, the solution was slightly clear. Adding 10% NaCl solution clearly changed from turbid to clear. This can be attributed to the electrostatic shielding effect of salt, which reduced electrostatic repulsion between particles and leaded to the binding of biomolecules in the solution with surface particles. The above results indicate that CMC/BSA exhibits certain salt resistance in low salinity solutions. When heated to 80℃, even with the addition of 4wt% NaCl solution, the turbidity of the solution system remained almost unchanged. This indicated that after heating, CMC and BSA may self assemble through mechanisms such as hydrogen bonding, hydrophobic bonding, and disulfide bonding, in addition to electrostatic attraction. Due to the presence of these additional forces, the structure and properties of the heated composite particles exhibit greater stability. 3.1.7 Structure and morphology of CMC/BSA composite particles prepared under optimized conditions Based on the above analysis, when the ratio of BSA to CMC is 3:1 and the system pH is 4.3, the CMC/BSA composite particles obtained have the best dispersibility and the most stable structure. In order to obtain the structure and microstructure of the composite particles prepared under the above optimized conditions, FT-IR and SEM tests were conducted on the composite particles prepared under these conditions, and the results are shown in Fig. 7 . As illustrated in Fig. 7 (a), the BSA FT-IR spectrum displays a peak at 1656 cm − 1 , corresponding to the stretching vibration of conjugated peptide bonds. Additionally, the peak at 1542 cm − 1 represents the vibration of secondary hydrogen bonds, while the peak at 1400 cm − 1 is attributed to C-N vibration of protein residues. In contrast, the CMC FT-IR spectrum shows a vibration absorption peak at 3340 cm − 1 for the hydroxyl group and a peak at 1607 cm − 1 for symmetric and asymmetric vibration absorption of C = O in the COO-Na group. Additionally, the peak value at 1426 cm − 1 is associated with symmetric tensile vibration of carboxyl group, and peaks at 1061 cm − 1 represent symmetric and asymmetric vibration absorption peaks of -C-O-C-. Notably, in the complex sample spectrum, there was a shift from 1416 cm − 1 to 1408 cm − 1 for the symmetric stretching vibration peak of the carboxyl group in CMC. The quadratic N-H bending peak at 1542 cm − 1 in BSA experienced a significant shift to 1583 cm − 1 due to electrostatic interaction between the carboxyl group of CMC and the amino group of Ly. Furthermore, it is important to note that the peak value at 1583 cm − 1 in complex is a superposition of asymmetric tensile vibrations previously observed at 1607 cm − 1 for CMC. Figure 7 (b) presents the morphology of CMC/BSA composite particles. From Fig. 7 (b), it can be seen that the composite particles exhibit a regular particle shape and good dispersion. 3.2 Drug loading Heating CMC/BSA composite particles to form composite particles with a certain three-dimensional spatial structure, which exhibit negative charge due to the outer layer being CMC, thus providing a specific drug loading space for positively charged drugs. Due to the electrostatic interaction between the amino groups of amoxicillin and theophylline and the electrostatic attraction of carboxyl groups on CMC, we selected two typical aqueous solution systems of positively charged small molecule drugs, amoxicillin and theophylline, as model drugs to test the drug loading performance of the composite particles. The results are shown in Table 1 . From the results, it can be seen that when the concentration of amoxicillin is 300ug/ml, the encapsulation efficiency of the composite particles is 44.1%, and the loading rate is 4.41%. When the concentration of theophylline is 100ug/ml, the encapsulation efficiency of the composite particles is 58.9%, and the loading rate is 5.89%. The results indicate that the composite particles have good loading capacity for small molecule drugs, and their structure can effectively adsorb small molecule drugs. Table 1 Drug encapsulations and loaded efficiencies Drug concentration EE(%) LC(%) Amoxicillin (300µg/ml) 44.1 ± 0.2 4.41 ± 0.2 Theophylline (100µg/ml) 58.92 ± 4.16 5.89 ± 0.42 3.3 vitro release In a phosphate buffer solution at 37℃ and pH = 7.4, continuous tests were conducted on the sustained-release performance of two drugs using CMC/BSA composite particles. The results are shown in Fig. 8 . From the sustained-release curve of theophylline, it can be seen that the release rate continuously increased with time during the initial released stage (within 4 hours). At 4 hours, the drug release rate was 70%, and then the growth rate slowed down. After 12 hours, the cumulative release rate was 89.9%, ultimately reaching the equilibrium of drug release. The sustained-release curve of amoxicillin showed a similar trend. In the initial release stage, the drug release amount significantly increased, and after reaching a certain release amount, the drug release slowed down, ultimately reaching a balance of drug release. From the release curve, it can be seen that the CMC/BSA composite particles have good sustained release performance, and the cumulative drug release rate can reach about 90%. From the release curve, it can be seen that CMC/BSA composite particles have good sustained-release performance and are expected to be applied in sustained-release and controlled release systems, greatly improving drug efficacy and reducing toxic side effects. 4. Conclusion In this work, CMC/BSA composite particles loaded with amoxicillin and theophylline were prepared using an environmentally friendly, convenient, and sustainable method. The composite materials were characterized, and the influencing factors of the composite were investigated. The optimal particle size and PDI are 255nm and 0.16 when the ratio of CMC to BSA is 1:3 at pH 4.3, and 296nm and 0.219 when heated at 80℃. The loading rates of amoxicillin and theophylline were found to be 44.1% and 58.9%, respectively, with a slow release of drugs achieved in sustained release experiments in vitro. Furthermore, due to the low toxicity of the selected material, it has potential for use as a carrier for various other drugs in future applications. Declarations Acknowledgments : The authors wish to acknowledge Department of Education Science and Technology Research Project of Jilin Provincial (JJKH20220239KJ); The authors wish to acknowledge Science and Technology innovation development planning project of Jilin City Science and Technology Bureau (20240103010); The authors acknowledge the assistance of JLICT Center of Characterization and Analysis Author contributions All authors contributed to the study conception and design. The first draft of he manuscript was written by Zheng, and all authors have provided comments on previous versions of the manuscript, and have read and approved the final version. Funding This study was supported by Department of Education of Jilin Provincial (JJKH20220239KJ); This study was supported by Jilin City Science and Technology Bureau (20240103010) Data availability The authors confirm that all relevant data are included in the paper, and the raw data are available upon request from the corresponding author. Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Brown MB, Martin GP, Jones SA, Akomeah FK (2006) Dermal and Transdermal Drug Delivery Systems: Current and Future Prospects. Drug Delivery 13:175–187. https://doi.org/10.1080/10717540500455975 Dai L, Si C (2019) Recent Advances on Cellulose-Based Nano-Drug Delivery Systems: Design of Prodrugs and Nanoparticles. 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J Mater Chem 22:2512–2519. https://doi.org/10.1039/C1JM13735B Souza DSDSD, Tartare VAP, Bega BDS, et al (2024) The pH role in casein-carboxymethylcellulose nano/microparticles formation. Colloids and Surfaces A: Physicochemical and Engineering Aspects 682:132953. https://doi.org/10.1016/j.colsurfa.2023.132953 Timilsena YP, Akanbi TO, Khalid N, et al (2019) Complex coacervation: Principles, mechanisms and applications in microencapsulation. International Journal of Biological Macromolecules 121:1276–1286. https://doi.org/10.1016/j.ijbiomac.2018.10.144 Xiao L, Xu Z, Fan X, Li Y (2023) Encapsulation of mixed-valence copper oxide nanoparticles in aluminum carboxymethylcellulose composite microspheres: an efficient synergistic catalyst for boosting aldehyde–alkyne–amine coupling reactions. Cellulose 30:8691–8708. https://doi.org/10.1007/s10570-023-05370-x Xiong W, Ren C, Li J, Li B (2018) Enhancing the photostability and bioaccessibility of resveratrol using ovalbumin–carboxymethylcellulose nanocomplexes and nanoparticles. Food Funct 9:3788–3797. https://doi.org/10.1039/C8FO00300A Xu Y, Ma X-Y, Gong W, et al (2020) Nanoparticles based on carboxymethylcellulose-modified rice protein for efficient delivery of lutein. Food Funct 11:2380–2394. https://doi.org/10.1039/C9FO02439E Yan S, Regenstein JM, Qi B, Li Y (2023) Construction of protein-, polysaccharide- and polyphenol-based conjugates as delivery systems. Critical Reviews in Food Science and Nutrition 1–19. https://doi.org/10.1080/10408398.2023.2293253 Yang J, Duan J, Zhang L, et al (2016) Spherical nanocomposite particles prepared from mixed cellulose–chitosan solutions. Cellulose 23:3105–3115. https://doi.org/10.1007/s10570-016-1029-4 Yang P, Li Z, Fang B, Liu L (2023) Self-healing hydrogels based on biological macromolecules in wound healing: A review. International Journal of Biological Macromolecules 253:127612. https://doi.org/10.1016/j.ijbiomac.2023.127612 Yang Y, Wang S, Wang Y, et al (2014) Advances in self-assembled chitosan nanomaterials for drug delivery. Biotechnology Advances 32:1301–1316. https://doi.org/10.1016/j.biotechadv.2014.07.007 Zeeb B, McClements DJ, Weiss J (2017) Enzyme-Based Strategies for Structuring Foods for Improved Functionality. Annu Rev Food Sci Technol 8:21–34. https://doi.org/10.1146/annurev-food-030216-025753 Zhu K, Ye T, Liu J, et al (2013) Nanogels fabricated by lysozyme and sodium carboxymethyl cellulose for 5-fluorouracil controlled release. International Journal of Pharmaceutics 441:721–727. https://doi.org/10.1016/j.ijpharm.2012.10.022 Schemes Schemes 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme1.png Scheme 1. The mechanism of CMC/BSA composite particles Scheme2.png Scheme 2. The mechanism of CMC/BSA composite particles in different circumstances Cite Share Download PDF Status: Published Journal Publication published 24 Mar, 2025 Read the published version in Cellulose → Version 1 posted Editorial decision: Revision requested 12 Aug, 2024 Reviews received at journal 01 Aug, 2024 Reviewers agreed at journal 24 Jul, 2024 Reviewers agreed at journal 22 Jul, 2024 Reviewers agreed at journal 22 Jul, 2024 Reviewers invited by journal 22 Jul, 2024 Editor assigned by journal 17 Jul, 2024 Submission checks completed at journal 17 Jul, 2024 First submitted to journal 17 Jul, 2024 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. 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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-4755038","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":334989231,"identity":"9af3095d-d04e-44d3-a4fe-4d6198a0383d","order_by":0,"name":"Kaiqiang Zheng","email":"","orcid":"","institution":"Jilin Institute of Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"Kaiqiang","middleName":"","lastName":"Zheng","suffix":""},{"id":334989232,"identity":"f74a78a5-593c-470f-8962-eda60f5ce3e7","order_by":1,"name":"Ziang Quan","email":"","orcid":"","institution":"Jilin Institute of Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"Ziang","middleName":"","lastName":"Quan","suffix":""},{"id":334989233,"identity":"e4fa513f-8b7e-45b1-b296-2ff995a1a32d","order_by":2,"name":"Xiaohui Wang","email":"","orcid":"","institution":"Jilin Institute of Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiaohui","middleName":"","lastName":"Wang","suffix":""},{"id":334989234,"identity":"ea719729-800c-4ede-94bd-92339358c1ad","order_by":3,"name":"ShiHao Zhou","email":"","orcid":"","institution":"Jilin Institute of Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"ShiHao","middleName":"","lastName":"Zhou","suffix":""},{"id":334989235,"identity":"7bcb688f-ccc4-4027-a184-abcbfca99dc7","order_by":4,"name":"Kuo Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kuo","middleName":"","lastName":"Wang","suffix":""},{"id":334989236,"identity":"b62b67f4-39f9-4b98-974f-bcd26dcef0fb","order_by":5,"name":"Meng Cui","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYFAC5oMPEv9IyPEzMx9+QKQWtmSDjw02xpLtbGkGRGrhMROc2ZCWuOE8j4IEURr4px1LY+bdcZhx82EeBgOGGptoglokbicfe8x75jCz2WHeAw8YjqXlNhDSYiCdlm7Mw3aYzewwX4IBY8NhYrTkmEkDtfAYN/MYSBCtRXJmW5qEATOxWiRupyUbfDhjYyBxGBjICcT4hX928sEHCRUS9f39hw8/+FBjQ1gLKkggTfkoGAWjYBSMAlwAAO5fPyFjiFWBAAAAAElFTkSuQmCC","orcid":"","institution":"Jilin Institute of Chemical Technology","correspondingAuthor":true,"prefix":"","firstName":"Meng","middleName":"","lastName":"Cui","suffix":""}],"badges":[],"createdAt":"2024-07-17 09:21:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4755038/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4755038/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10570-025-06479-x","type":"published","date":"2025-03-24T15:57:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62146640,"identity":"b9144b3a-bc47-4342-aadf-c5d848e301d1","added_by":"auto","created_at":"2024-08-09 18:43:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7364,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in surface potential of BSA in different pH value systems\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4755038/v1/74a9068427666014971c2e51.png"},{"id":62146984,"identity":"3bc99db4-e8e3-4b49-9be3-56b28e4e610f","added_by":"auto","created_at":"2024-08-09 18:51:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12748,"visible":true,"origin":"","legend":"\u003cp\u003eTurbidity variation curve of CMC and BSA composite system with pH\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4755038/v1/764baa93d08223d1f179d245.png"},{"id":62146986,"identity":"df167d3d-8b01-41b6-9a46-1655456c443a","added_by":"auto","created_at":"2024-08-09 18:51:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10376,"visible":true,"origin":"","legend":"\u003cp\u003eZeta potential of different ratios of BSA and CMC\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4755038/v1/935470b5ff5ac68f5807d8e0.png"},{"id":62146645,"identity":"72f615e3-1340-48c9-a324-70e04b4fb1fa","added_by":"auto","created_at":"2024-08-09 18:43:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":30653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 3. \u003c/strong\u003e(a)The fluorescence spectrum of BSA (high pH); (b)The fluorescence spectrum of BSA (pure); (c)The fluorescence spectrum of BSA (low pH)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4755038/v1/9dc695af3b7649865eff8dd2.png"},{"id":62147272,"identity":"b706f920-3ec8-4bec-965e-00db3ae6bf1f","added_by":"auto","created_at":"2024-08-09 18:59:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":27928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.4.\u003c/strong\u003eParticles size and PDI of the composite particles. (a)particles size and PDI of BSA:CMC(4:1); (b)particles size and PDI of BSA:CMC(3:1); (c)particles size and PDI of BSA:CMC(2:1); (d)particles size and PDI of BSA:CMC(1:1); (e)particles size and PDI of BSA:CMC(1:2);(f)particles size and PDI of BSA:CMC(1:3)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4755038/v1/344b986da1383a8f75d80f5f.png"},{"id":62146641,"identity":"2868c84a-ee8b-44a8-9bf1-397419c398f3","added_by":"auto","created_at":"2024-08-09 18:43:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":12367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.5.\u003c/strong\u003eparticles size and PDI of BSA:CMC(3:1)after 40/60/80℃ heated\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4755038/v1/f4c0313f60632dd9cb5cc6d8.png"},{"id":62146987,"identity":"82f8ea56-a6ba-4fc9-aa4f-7fdce27076f2","added_by":"auto","created_at":"2024-08-09 18:51:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":433095,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.6.\u003c/strong\u003eThe turbidity of the system at different NaCl addition amounts and temperatures (a: 1wt% NaCl, b: 4wt% NaCl, c: 10wt% NaCl , d: Turbidity of CMC/BSA composite particle system (BSA: CMC=3:1) at different heating temperatures)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4755038/v1/c1b768f89c5d3a8866e9e1cb.png"},{"id":62146646,"identity":"9395c312-9cce-48ab-8cfd-706355223014","added_by":"auto","created_at":"2024-08-09 18:43:32","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":77874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.7\u003c/strong\u003e(a)the FT-IR spectrum of the CMC/BSA composite particles and (b) the morphology of CMC/BSA composite particles\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4755038/v1/53f030b821f7df3919a3140c.png"},{"id":62146648,"identity":"5e0d3321-25cc-4fdd-8849-e763b8f14255","added_by":"auto","created_at":"2024-08-09 18:43:32","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":15593,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.8.\u003c/strong\u003e(a)the release of Theophylline; (b)the release of Amoxicillin\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4755038/v1/43533b3377b973abc220d9ec.png"},{"id":79604958,"identity":"19567960-7729-445c-8c95-e09ba44d1d4c","added_by":"auto","created_at":"2025-03-31 16:09:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1478529,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4755038/v1/8f57251e-b520-449b-95e9-deb8538c56c8.pdf"},{"id":62146643,"identity":"fd3bf7c4-6e79-4052-a0e2-61892bfc364b","added_by":"auto","created_at":"2024-08-09 18:43:32","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":102868,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. The mechanism of CMC/BSA composite particles\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-4755038/v1/ef5a3dbf5a45d8ad8c679501.png"},{"id":62146650,"identity":"c2043d8f-9f85-4777-883a-89b570d75406","added_by":"auto","created_at":"2024-08-09 18:43:32","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":150876,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 2. The mechanism of CMC/BSA composite particles in different circumstances\u003c/p\u003e","description":"","filename":"Scheme2.png","url":"https://assets-eu.researchsquare.com/files/rs-4755038/v1/361042e529718570ea10e886.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preparation of pH-sensitive carboxymethyl cellulose/bovine serum protein complex particles and investigation of their drug carrying capacity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe investigation of drug-carrying micro/nano particles is a prominent area of research in the field of drug delivery systems(McClements \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).Composite particles, due to the high specific surface area caused by their small size, have the potential to enhance drug stability and utilization while reducing side effects(Yang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lukova et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).Furthermore, they can be tailored for different functionalization purposes(Brown et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).As a result, composite particles play a crucial role in various applications within the field of drug delivery(Dai and Si \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Joshy et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe selections of materials and preparation methods for constructing composite particles significantly impact the efficiency of their drug loading and sustained release(Moschakis and Biliaderis \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Damiri et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).Various materials can serve as drug carriers, including metallic inorganic materials, inorganic non-metallic materials, synthetic polymer and so on. While these carriers demonstrate good performance, they also present certain issues such as poor biocompatibility, toxicity or complex preparation processes involving organic compounds that may compromise safety(Gyarmati and Puk\u0026aacute;nszky \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).In contrast, natural polymer materials are renewable,biodegradable and possess excellent biocompatibility properties(Javanbakht and Shaabani \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Papageorgiou \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eComposite particles composed of natural biodegradable polymer materials are synthesized in a straightforward, environmentally friendly manner to serve as carriers for drug delivery(Timilsena et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Patil and Patel \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jamroży et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).These complex have the capability to encapsulate drugs, improve drug utilization, control drug release, and reduce certain side effects(Fazal et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Paliya et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).Polysaccharides and proteins are abundant natural biopolymers that serve as fundamental units in life. The assembly between them forms the basis for the existence and continuation of all organisms(Devi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).Therefore, it is of great significance to investigate the assembly of both components in order to construct a delivery system with high biocompatibility and biosafety(Ezhilarasi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Jones and McClements \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Xiao et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).Xiong et al. conducted a study on the formation of nanoparticles embedded with resveratrol by combining ovalbumin and carboxymethyl cellulose sodium. The nanocomplexes were formed by the electrostatic assembly between OVA and CMC and presented a spherical morphology, compared to native resveratrol, the bio-accessibility of resveratrol embedded in nanocomplexes and nanoparticles was increased to 60% and 80%(Xiong et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).Liang et al. prepared biodegradable nanoparticles (NPs). Assembled with sodium carboxymethyl cellulose (CMC) and zein to produce zein\u0026ndash;CMC NPs. Paclitaxel (PTX) was 95.5% encapsulated at a zein\u0026ndash;CMC weight ratio of 1 : 3 and the NPs were spherical with an average particle size of approximately 159.4 nm, The NPs demonstrated good stability over a broad range of pH ranging from 3.7 to 11.0(Liang et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).Fuge et al. prepared ultra long and stable ovalbumin and carboxymethyl cellulose nanoparticles, investigated the effects of different influencing factors on their stability, and successfully loaded curcumin efficiently into these particles, these particles with the loose structure of wool ball could effectively load curcumin. Curcumin-loaded of OVA/CMC nanoparticles show good DPPH\u0026middot; scavenging activity, Ferric-reducing ability and ABTS scavenging activity compared with curcumin/water(Niu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe main preparation methods for these composite include physical mixing, enzyme conjugation and chemical cross-linking(Zeeb et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).Chemical cross-linking may involve the use of other crosslinking agents, which can have a certain impact on the environment and health during the preparation process. The technical requirements for enzyme conjugation are intricate, making the process challenging. In contrast, physical mixing, take self-assembly for example, have milder and more controllable conditions, and have no impact on organisms and the environment(Niu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).When the environmental conditions are favorable, polyelectrolytes with opposite charges will spontaneously bind(Mariani et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Maciel et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).However, changes in the external equilibrium environment can lead to the self-assembly forming a single-phase solution or precipitate(Mariani et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).Therefore, various factors such as pH value, concentration, mass ratio, and ion concentration can impact the formation of nanoparticles(Mariani et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Eghbal and Choudhary \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).Davi conducted a study on the role of pH value in the formation of CA/CMC microparticles. The research demonstrated that different pH values and substance ratios during the composite coagulation process would result in different CA and CMC microparticles being produced(Souza et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBSA, as the animal serum protein with the best similarity to human serum albumin (HSA), is widely used in biochemical laboratories and is referred to as the fifth component. It is consisted of 583 amino acid residues with a molecular weight of 66.4 kDa and an isoelectric point of 4.7(Kaibara et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCMC contains numerous carboxyl groups (-COO) in its molecular structure, making it soluble in water and capable of forming ionic bonds with amino group molecules in aqueous solutions. Its charge distribution depends on the number of carboxyl groups present in each glucose unit, and it remains stable within a pH range of 2\u0026ndash;10.(Geng et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Song and Chen \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).Additionally, CMC serves as a non-toxic, biocompatible, and biodegradable natural polymer. It plays a significant role in various types of occasions.(Kong et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sedl\u0026aacute;ř et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).For example, it can enhance drug efficiency and delivery(Li and Wang \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we utilized a combination of electrostatic self-assembly and thermal induction to prepare CMC/BSA micro/nano particles as carriers for small molecule substances, aiming to improve material stability and achieve controlled sustained release. The impacts of various factors on CMC/BSA complex particles were investigated, resulting in the successful preparation of excellent CMC/BSA complex system. And as model drugs, amoxicillin and theophylline were selected to demonstrated the encapsulation and sustained-release performance of the composite system for drug delivery. Furthermore, our study aims to expand the construction of functionalization for different polysaccharide-protein complexes by exploring their feasibility through result analysis.\u003c/p\u003e"},{"header":"2. Materials and Testing instruments","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1.Materials\u003c/h2\u003e \u003cp\u003eCMC (degree of substitution 0.95, viscosity at 1%, average molecular weight 90 KDa) was synthesized in the laboratory. BSA (96%) was purchased from Maclean Biochemical Technology Co. LTD(Shanghai,China). Amoxicillin (CP) was purchased from Maclean Biochemical Technology Co. LTD(Shanghai,China). Theophylline (medical grade) was purchased from Changchun Tairen Technology Co. LTD (Chuangchun, China). Glacial acetic acid (AR) was obtained from Tianjin Opuseng Chemical Co. LTD (Tianjin, China). NaCl (AR) was purchased from Tianjin Yongda Chemical Reagent Co, LTD (Tianjin, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Characterization of CMC/BSA composite particles\u003c/h2\u003e \u003cp\u003eThe mean particle size and poly dispersion index of the particles were determined by dynamic light scattering using Malvern Nano-ZS90 (Malvern,UK) at 25\u0026deg;C with an angle detection at 173\u0026deg;C. The data selected were the mean values of three independent repeats. Zeta potential is an important index for characterizing the stability of colloidal dispersions, with higher absolute values indicating greater stability. The sample was balanced within the instrument for 120 seconds and then measured three times to collect data from at least 10 consecutive readings each time, ensuring accuracy.\u003c/p\u003e \u003cp\u003eFourier infrared analysis was performed using the Bruker TENSOR Fourier Infrared spectroscopy instrument (Bruker, Germany) measuring in the range of 500-4000cm, and analyzing functional groups and recombination.\u003c/p\u003e \u003cp\u003eThe JSM-7610F Plus scanning electron microscope (JEOL, Japan) was used to observe and study the morphology of the composite material CMC/BSA (1:3), both unheated and heated. The sample diluted ten times was dropped on conductive adhesive, dried, sprayed with gold, and then observed for its morphology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of CMC/BSA composite particles\u003c/h2\u003e \u003cp\u003eSince proteins are amphoteric electrolytes, the pH value of BSA was adjusted to an appropriate level before preparation to enhance the influence of electrostatic forces. BSA and CMC were dissolved in ultra-pure water (1mg/ml) and stirred at room temperature (25℃) for 2 hours with a gentle magnetic force. The BSA solution should be refrigerated at 4℃ overnight after preparation to ensure complete dissolution. Subsequently, the pH-adjusted BSA solution was added dropwise into the CMC solution at different ratios, followed by gentle stirring for 30 minutes. The resulting compound was then heated in a water bath at 80\u0026deg;C for 30 minutes to obtain the desired product. The composite mechanism of BSA and CMC is shown in Scheme. 1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Drug encapsulation\u003c/h2\u003e \u003cp\u003eAmoxicillin and theophylline (3000\u0026micro;g; 1000\u0026micro;g/ml) were dissolved in ultra-pure water. A certain amount of drug solution was then added to heated CMC-BSA complex solution and slowly stirred for 1 hour, resulting in a final drug content of (3000\u0026micro;g/1500\u0026micro;g) in a 15ml sample. The loaded drug sample was poured into an ultrafiltration centrifuge tube (10000MW) and centrifuged at 4000rpm for 45 minutes. The absorbance of the filtrate after centrifugation was measured using a UV-VIS spectrophotometer within the corresponding range, and the packaging rate and load rate were calculated based on the standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 In vitro drug release\u003c/h2\u003e \u003cp\u003eUnder physiological conditions, in vitro drug release was studied using the dialysis method. Specifically, a 15 ml solution of the compound containing the drug and an equivalent concentration of free drug were placed into a dialysis bag (Total amount of amoxicillin and theophylline drugs is 12000ug/3000ug),and subjected to dialysis with 200 ml PBS (pH 7.4) at a constant temperature of 37\u0026deg;C in a water bath, with slow magnetic stirring for 24 hours. At appropriate intervals, 5ml of PBS was collected and replenished with an equal amount of fresh PBS each time. The amount of drug released was measured using a UV-VIS spectrophotometer.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Resaults and discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Exploration of the influence factors of CMC and BSA composite system\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 The effect of pH on the turbidity of CMC and BSA composite system\u003c/h2\u003e \u003cp\u003eDue to BSA being a zwitterionic protein with an isoelectric point of 4.7, it exhibits different surface electrical properties when the pH value of the system is different. However, CMC exhibits negative charge in aqueous solution systems due to the ionization of sodium ions. Therefore, when the pH value of the system is different, the composite state of the CMC and BSA composite system is also different. If we want to utilize the electrostatic attraction between CMC and BSA as the driving force for their self-assembl, we must explore the influence of the pH value of the composite system. The zeta potential values at different pH levels were measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). From Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, it can be seen that when the pH value of the system is higher than 4.7, the BSA surface exhibits negative charge, while when the pH value of the system is lower than 4.7, its surface exhibits positive charge. Therefore, the protein's charge is pH-dependent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to obtain the pH range suitable for self-assembly of CMC and BSA, the turbidity of composite systems with different ratios were tested as a function of pH value, and the results The are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. From Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, it can be seen that throughout the entire pH range, there is an initial increase followed by a decrease in turbidity. Overall, it is evident that pH values have a substantial influence on the combination of these two substances.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaking CMC/BSA(1:3) as an example, when the pH value exceeds pHa, the complex turbidity of CMC/BSA remains relatively constant. Generally speaking, when the pH value surpasses the protein's isoelectric point, the protein exhibits overall anionic properties. However, weak electrostatic interactions may still occur between local positive charge patches of the protein and anionic groups of polysaccharides. When the pH value ranged from pHa to pHb, there is a slight increase in turbidity. During this time frame, due to a lower pH than that of the protein's isoelectric point, oppositely charged substances properly combine to form a soluble complex which maintains homogeneity and forms a single-phase system.If the pH value falls below pHb, there will be a sharp rise in solution turbidity where electrostatic forces play a dominant role. At this stage, a large number of CMC and BSA self-assemble to form composite particles. With a further decrease in pH value, electric neutralization is achieved at pHd. This results in a change in the color of the solution and the formation of a large number of insoluble composite condensates with the highest stability and content, leading to phase separation. When the pH value drops below pHd and more H\u0026thinsp;+\u0026thinsp;ions are added to the solution, the polysaccharide molecules become gradually protonated, reducing the net negative charge. As a result, the interaction strength between CMC and BSA decreases, leading to reduced electrostatic attraction. Consequently, the condensate begins to dissociate, causing the mixed solution to become transparent while maintaining stable turbidity. Furthermore, adjusting the ratio of polysaccharide to protein will alter the pH of the turbidity curve formed by their complexation. Increasing BSA concentration shifts this curve towards higher pH due to an increase in protein content and consequently an increase in CMC chains available for interaction with proteins. Conversely, increasing CMC content raises negative charge levels in the composite system necessitating more positive charge for neutralization. Thus shifting the turbidity curve towards lower pH values. This phenomenon is evident when comparing CMC/BSA(3:1) at a pH value of 4.5 where its clarified solution indicates minimal recombination between them suggesting that electrostatic forces do not play a dominant role. Therefore different ratios at same pH also have an effect on their recombination process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 The effect of pH on the Zeta potential of CMC and BSA composite system\u003c/h2\u003e \u003cp\u003eThe Zeta potential is closely related to the interaction between CMC and BSA. Therefore, we selected three typical raw material ratios and tested the Zeta potential of the composite system with pH changes, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e. When the zeta potential of the composite solution approaches 0, stronger electrostatic interactions will result in the formation of insoluble condensates and phase separation. This is consistent with the observation of condensates in turbidity as pH decreases. Furthermore, a higher absolute value of zeta potential indicates an increased surface charge, leading to repulsion between particles and thus contributing to the overall stability of the system. Conversely, if the absolute value of the zeta potential is very low, particles are more likely to attract each other, resulting in instability within the system. In conclusion, considering that electrostatic self-assembly plays a crucial role in particle recombination for CMC/BSA, it is essential to carefully select an appropriate pH range and consider different ratios for optimal results.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3 The effect of pH on the fluorescence spectrum of CMC and BSA composite system\u003c/h2\u003e \u003cp\u003eThe fluorescence spectrum of CMC-BSA is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e. In pure BSA and BSA with a high pH, the fluorescence intensity decreases steadily with the continuous addition of CMC. However, the maximum emission peak does not shift, while the peak of the maximum absorption is related to pH. This is due to the fact that altering the pH of a protein impacts its structural and functional characteristics. At a low pH value, the BSA absorption peak was at 334.6nm. As the concentration of CMC increases, not only does the fluorescence intensity weaken, but it also shifts from 334.6nm to 327.6nm, exhibiting a noticeable blue shift. These findings suggest that CMC has the ability to quench the fluorescence of BSA, leading to a change in the microenvironment of the protein upon addition of CMC, resulting in a more hydrophobic environment. The blue shift in fluorescence is typically associated with the fluorescence characteristics and charge distribution within the fluorescent molecule. Any changes in charge distribution within the fluorescer molecule can cause a shift in emission peak towards shorter wavelengths. This phenomenon can be achieved through introduction or chemical modification of heteroatoms within the molecular structure. Fluorescence studies same have demonstrated that variations in pH levels impact the interaction betweethe two components.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom the results of the influence of pH values on the turbidity, Zeta potential, and fluorescence intensity of the composite system, it can be seen that When the pH exceeds the protein's isoelectric point, although it exhibits anionic properties overall, weak electrostatic interactions may occur between local positive charge patches on the protein and polysaccharide anionic groups, resulting in changes in particle size. Conversely, when adjusting below the isoelectric point, decreasing pH leads to more deposition of BSA on CMC chains, forming soluble complexes through electrostatic interactions that alter solution appearance. So we come to the conclusion that when a lower pH value which is below the isoelectric point of BSA is selected, BSA and CMC can achieve a relatively stable composite through electrostatic self-assembly. Therefore, this pH range 4.0-4.5 is selected when preparing CMC/BSA composite particles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4 The effect of pH and ratio on the CMC/BSA composite particles\u003c/h2\u003e \u003cp\u003eIn order to explore and obtain accurate pH values accurately obtain CMC/BSA composite particles with excellent structure, morphology, and properties within the appropriate pH range that can form stable CMC/BSA composite systems, particle size and PDI tests were conducted on composite particles with different pH and composition ratios. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom the results in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e, it can be concluded that pH value and the ratio of CMC and BSA have an impact on the particle size and PDI of composite particles. When CMC and BSA were combined into particles in different ratios, in each system with a constant pH, as the amount of CMC increased, the particle size and PDI of CMC/BSA composite particles showed an overall upward trend. This is because CMC and BSA self-assemble together driven by electrostatic attraction. When there was less CMC, BSA was the main component in the system. Except for the BSA composited with CMC, excess BSA accumulated on the surface of the composite particles, resulting in a slightly larger average particle size and poor dispersion of the system. When BSA and CMC can be perfectly matched, the system was mostly composed of structurally intact CMC/BSA composite particles, with the minimum average particle size and the best particle dispersion. When CMC continued to increase, there was an excess of CMC in the system. In addition to CMC/BSA composite particles, there was also a three-dimensional structure formed by the entanglement of CMC long chain structures, which continuously increased the average particle size of the system, the mechanism is shown in Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBy comparing the changes in particle size and PDI of composite particles in different pH systems horizontally, it can be found that at a pH of 4.3, the particle size and PDI values of composite particles showed the minimum values, indicating that in this pH system, CMC and BSA reached the best composite state, and the dispersion of composite particles was also the best.\u003c/p\u003e \u003cp\u003eBased on the analysis of the above particle size and PDI results, when BSA and CMC were combined in a 3:1 ratio at a pH of 4.3 in the system, the resulting composite particle structure and dispersion were the most excellent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.1.5 The effect of temperature on the CMC/BSA composite particles\u003c/h2\u003e \u003cp\u003eDue to the sensitivity of protein structure to temperature, during heating, the protein will change its structure and expose embedded non-polar peptides, thereby enhancing hydrophobic interactions between adjacent non-polar fragments of peptides. Therefore, under heating conditions, the process of BSA and CMC composite will become more complex. In order to explore the effect of heating temperature on composite particles, particles composed of BSA and CMC in a 3:1 ratio were heated at 40\u0026deg;C, 60\u0026deg;C, and 80\u0026deg;C in a pH 4.3 system, and their particle size and PDI were characterized. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e. From Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e, it can be seen that after heating, the particle size and PDI slightly increase, indicating that heating has a certain impact on the structure and distribution of composite particles, with PDI being the best after heating at 80 ℃.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.1.6 The impact of salt solution on CMC/BSA composite particles\u003c/h2\u003e \u003cp\u003eThe addition of salt solution (NaCl) can shield electrostatic interactions. The addition of sodium chloride will cause Na\u003csup\u003e+\u003c/sup\u003e to bind to negatively charged polysaccharides, while Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e will bind to positively charged proteins, producing an electrostatic shielding effect, reducing the possibility of electrostatic attraction between proteins and polysaccharides, thus preventing electrolytes that originally carried two opposite charges from generating electrostatic recombination. Therefore, the tolerance of composite particles to salt solution is an important indicator for measuring their use as a carrier system. In order to evaluate the tolerance of CMC/BSA composite particles to salt solution, different concentrations of NaCl (10/4/1wt%) were added to the system to observe their changes. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e, it can be seen that with the increase of NaCl in the system, the turbidity of the solution decreased and gradually clarified. When 1wt% NaCl was added to the system, the effect on the appearance of the solution was minimal. At a concentration of 4wt% NaCl, the solution was slightly clear. Adding 10% NaCl solution clearly changed from turbid to clear. This can be attributed to the electrostatic shielding effect of salt, which reduced electrostatic repulsion between particles and leaded to the binding of biomolecules in the solution with surface particles. The above results indicate that CMC/BSA exhibits certain salt resistance in low salinity solutions.\u003c/p\u003e \u003cp\u003eWhen heated to 80℃, even with the addition of 4wt% NaCl solution, the turbidity of the solution system remained almost unchanged. This indicated that after heating, CMC and BSA may self assemble through mechanisms such as hydrogen bonding, hydrophobic bonding, and disulfide bonding, in addition to electrostatic attraction. Due to the presence of these additional forces, the structure and properties of the heated composite particles exhibit greater stability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.1.7 Structure and morphology of CMC/BSA composite particles prepared under optimized conditions\u003c/h2\u003e \u003cp\u003eBased on the above analysis, when the ratio of BSA to CMC is 3:1 and the system pH is 4.3, the CMC/BSA composite particles obtained have the best dispersibility and the most stable structure. In order to obtain the structure and microstructure of the composite particles prepared under the above optimized conditions, FT-IR and SEM tests were conducted on the composite particles prepared under these conditions, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e(a), the BSA FT-IR spectrum displays a peak at 1656 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to the stretching vibration of conjugated peptide bonds. Additionally, the peak at 1542 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represents the vibration of secondary hydrogen bonds, while the peak at 1400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is attributed to C-N vibration of protein residues. In contrast, the CMC FT-IR spectrum shows a vibration absorption peak at 3340 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the hydroxyl group and a peak at 1607 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for symmetric and asymmetric vibration absorption of C\u0026thinsp;=\u0026thinsp;O in the COO-Na group. Additionally, the peak value at 1426 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is associated with symmetric tensile vibration of carboxyl group, and peaks at 1061 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represent symmetric and asymmetric vibration absorption peaks of -C-O-C-. Notably, in the complex sample spectrum, there was a shift from 1416 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1408 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the symmetric stretching vibration peak of the carboxyl group in CMC. The quadratic N-H bending peak at 1542 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in BSA experienced a significant shift to 1583 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e due to electrostatic interaction between the carboxyl group of CMC and the amino group of Ly. Furthermore, it is important to note that the peak value at 1583 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in complex is a superposition of asymmetric tensile vibrations previously observed at 1607 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for CMC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e(b) presents the morphology of CMC/BSA composite particles. From Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e(b), it can be seen that the composite particles exhibit a regular particle shape and good dispersion.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Drug loading\u003c/h2\u003e \u003cp\u003eHeating CMC/BSA composite particles to form composite particles with a certain three-dimensional spatial structure, which exhibit negative charge due to the outer layer being CMC, thus providing a specific drug loading space for positively charged drugs. Due to the electrostatic interaction between the amino groups of amoxicillin and theophylline and the electrostatic attraction of carboxyl groups on CMC, we selected two typical aqueous solution systems of positively charged small molecule drugs, amoxicillin and theophylline, as model drugs to test the drug loading performance of the composite particles. The results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. From the results, it can be seen that when the concentration of amoxicillin is 300ug/ml, the encapsulation efficiency of the composite particles is 44.1%, and the loading rate is 4.41%. When the concentration of theophylline is 100ug/ml, the encapsulation efficiency of the composite particles is 58.9%, and the loading rate is 5.89%. The results indicate that the composite particles have good loading capacity for small molecule drugs, and their structure can effectively adsorb small molecule drugs.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDrug encapsulations and loaded efficiencies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrug concentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEE(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmoxicillin (300\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e44.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTheophylline (100\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e58.92\u0026thinsp;\u0026plusmn;\u0026thinsp;4.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 vitro release\u003c/h2\u003e \u003cp\u003eIn a phosphate buffer solution at 37℃ and pH\u0026thinsp;=\u0026thinsp;7.4, continuous tests were conducted on the sustained-release performance of two drugs using CMC/BSA composite particles. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e. From the sustained-release curve of theophylline, it can be seen that the release rate continuously increased with time during the initial released stage (within 4 hours). At 4 hours, the drug release rate was 70%, and then the growth rate slowed down. After 12 hours, the cumulative release rate was 89.9%, ultimately reaching the equilibrium of drug release.\u003c/p\u003e \u003cp\u003eThe sustained-release curve of amoxicillin showed a similar trend. In the initial release stage, the drug release amount significantly increased, and after reaching a certain release amount, the drug release slowed down, ultimately reaching a balance of drug release. From the release curve, it can be seen that the CMC/BSA composite particles have good sustained release performance, and the cumulative drug release rate can reach about 90%.\u003c/p\u003e \u003cp\u003eFrom the release curve, it can be seen that CMC/BSA composite particles have good sustained-release performance and are expected to be applied in sustained-release and controlled release systems, greatly improving drug efficacy and reducing toxic side effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this work, CMC/BSA composite particles loaded with amoxicillin and theophylline were prepared using an environmentally friendly, convenient, and sustainable method. The composite materials were characterized, and the influencing factors of the composite were investigated. The optimal particle size and PDI are 255nm and 0.16 when the ratio of CMC to BSA is 1:3 at pH 4.3, and 296nm and 0.219 when heated at 80℃. The loading rates of amoxicillin and theophylline were found to be 44.1% and 58.9%, respectively, with a slow release of drugs achieved in sustained release experiments in vitro. Furthermore, due to the low toxicity of the selected material, it has potential for use as a carrier for various other drugs in future applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e: The authors wish to acknowledge Department of Education Science and Technology Research Project of Jilin Provincial (JJKH20220239KJ); The authors wish to acknowledge Science and Technology innovation development planning project of Jilin City Science and Technology Bureau (20240103010); The authors acknowledge the assistance of JLICT Center of Characterization and Analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e All authors contributed to the study conception and design. The first draft of he manuscript was written by Zheng, and all authors have provided comments on previous versions of the manuscript, and have read and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was supported by Department of Education of Jilin Provincial (JJKH20220239KJ); This study was supported by Jilin City Science and Technology Bureau (20240103010)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e The authors confirm that all relevant data are included in the paper, and the raw data are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBrown MB, Martin GP, Jones SA, Akomeah FK (2006) Dermal and Transdermal Drug Delivery Systems: Current and Future Prospects. 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International Journal of Pharmaceutics 441:721\u0026ndash;727. https://doi.org/10.1016/j.ijpharm.2012.10.022\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellulose","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cels","sideBox":"Learn more about [Cellulose](https://www.springer.com/journal/10570)","snPcode":"10570","submissionUrl":"https://submission.nature.com/new-submission/10570/3","title":"Cellulose","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"composite particles, carboxymethyl cellulose (CMC), Bovine serum protein (BSA), electrostatic interaction, drug load","lastPublishedDoi":"10.21203/rs.3.rs-4755038/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4755038/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this work, structurally stable and high-performance drug deliver composite particles were prepared successfully through a portable and simple electrostatic self-assembly method with carboxymethyl cellulose (CMC) and bovine serum protein (BSA). When regulating the pH value of the system lower than the isoelectric point of BSA, it exhibited positivity and was assembled with CMC through electrostatic attraction. The prepared composite particles were characterized, and different factors impacting on the composite materials were investigated. Amoxicillin and theophylline were selected as the experimental drugs to test the drug sustained-release performance of the composite particles. Results indicated that the composite particles possessed uniform shape, with an average particle size of 255 nm before heating and a PDI of 0.16 before heating. After heating, the particle size increased to 296 nm with PDI of 0.219. The encapsulation rate of amoxicillin and theophylline were found to be 44.1% and 58.9%, and the sustained-release curve demonstrated excellent drug loading efficiency and sustained release ability. This study demonstrates the potential application of CMC, a biocompatible natural high molecular weight material, in the delivery of small molecule drugs. It also demonstrates the development potential of composite systems composed of proteins such as BSA and polysaccharides.\u003c/p\u003e","manuscriptTitle":"Preparation of pH-sensitive carboxymethyl cellulose/bovine serum protein complex particles and investigation of their drug carrying capacity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 18:43:27","doi":"10.21203/rs.3.rs-4755038/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-12T16:08:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-02T02:33:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3467464249326546582890610126385808580","date":"2024-07-24T08:09:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143932292542768560765997297850430444004","date":"2024-07-22T11:08:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"106288943474154148327002315401042679275","date":"2024-07-22T08:51:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-22T07:57:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-17T10:29:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-17T10:27:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellulose","date":"2024-07-17T09:20:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellulose","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cels","sideBox":"Learn more about [Cellulose](https://www.springer.com/journal/10570)","snPcode":"10570","submissionUrl":"https://submission.nature.com/new-submission/10570/3","title":"Cellulose","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c81848a3-103c-4c67-bf7b-2e50297c35da","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-31T16:04:34+00:00","versionOfRecord":{"articleIdentity":"rs-4755038","link":"https://doi.org/10.1007/s10570-025-06479-x","journal":{"identity":"cellulose","isVorOnly":false,"title":"Cellulose"},"publishedOn":"2025-03-24 15:57:24","publishedOnDateReadable":"March 24th, 2025"},"versionCreatedAt":"2024-08-09 18:43:27","video":"","vorDoi":"10.1007/s10570-025-06479-x","vorDoiUrl":"https://doi.org/10.1007/s10570-025-06479-x","workflowStages":[]},"version":"v1","identity":"rs-4755038","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4755038","identity":"rs-4755038","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}