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.
3.3 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.