Radiation-induced nanogel engineering based on pectin for pH-responsive rutin delivery for cancer treatment

Crosslinking induced by radiation is a common technique to synthesize nanogels from water-soluble polymers like pectin and poly(acrylic acid). Here are some reasons why this approach works well (Matusiak et al. 2020). High-energy radiation like gamma rays can easily penetrate water easily and produce radical species that are reactive within the polymer chains. This process makes it possible to synthesize nanogels in mild environments, without requiring harsh chemicals or high temperatures (Ashfaq et al. 2021). The crosslinking of polymers induced by radiation is an approach to the synthesis of hydrogels and their nanoscale analogs, nanogels (NGs) (Dispenza et al. 2017). The research conducted by Rosiak et al. provides the optimal intramolecular crosslinking technique for the synthesis of nanogels (Rosiak et al. 2005). The process of radical formation in the chains of polymers initiates a series of intricate reactions that culminate in the formation of a crosslinked nanogel network. This phenomenon arises as a result of the interaction between the polymer chains and radiation, leading to the generation of free radicals along the polymer backbone. These free radicals play a pivotal role in intramolecular recombination reactions, where adjacent polymer chains interact and form new covalent bonds. This process, known as crosslinking, results in the creation of a three-dimensional network framework within polymers matrix. Crosslinking degree, or the extent to which these new bonds are formed, profoundly influences the properties and behavior of the resulting nanogel. Regarding drug delivery systems, crosslinked nanogel structure assumes paramount significance. The nanogel’s ability to encapsulate and subsequently release drug molecules hinges on the characteristics of this network. The degree of crosslinking governs important attributes such as the nanogel’s stability, swelling behavior, porosity, and responsiveness to external stimuli. A higher degree of crosslinking generally imparts greater structural stability and mechanical integrity to the nanogel. This can enhance its capacity to encapsulate therapeutic molecules, ensuring their retention within the nanogel’s matrix until specific triggers prompt their release. Conversely, nanogels with less crosslinking may be more responsive to external factors, potentially leading to a faster drug release profile. The crosslinked nanogel’s porosity and swelling behavior are also significantly influenced by the extent of crosslinking. A well-crosslinked nanogel might possess controlled swelling properties, allowing it to encapsulate a precise amount of drug molecules and regulate their release. Conversely, a less crosslinked nanogel could exhibit greater swelling, potentially accommodating a larger drug payload. In essence, the intricate interplay between radiation-induced radical formation, intramolecular recombination reactions, and the resulting crosslinked nanogel network underpins the foundation of a versatile and customizable drug delivery platform. By modulating the degree of crosslinking, researchers can fine-tune the nanogel’s properties to match specific therapeutic requirements. This ability to tailor the nanogel’s performance provides great potential for precise and controlled drug delivery, opening up avenues for more effective and efficient treatment strategies (Ulański et al. 1998).

In this study, the synthesis of Pec/PAAc nanogel was performed using an intramolecular crosslinking method facilitated by gamma irradiation. To prepare the solution, 0.05 g of pectin was dissolved in 10 ml of distilled water at room temperature (20–25 °C), yielding a 0.5% (w/v) pectin solution. Subsequently, 1 ml of acrylic acid (AAc) was added to this solution, resulting in a final concentration of 10% (w/v) acrylic acid. The mixture was stirred thoroughly to ensure complete dissolution of all components.

Next, subject the Pec/PAAc solution to sonication for 5 min. This process promotes even dispersion of the components throughout the solution, resulting in a uniform mixture.

Adjust the pH of the solution to 1 by gradually adding a 35.5% hydrochloric acid (HCl) solution. Carefully monitor the pH level during this process and make incremental adjustments until the pH stabilizes at 1. An effective polymerization reaction requires precise pH adjustment.

Then, expose the pH-adjusted solution to gamma irradiation using a 60Co gamma ray source. Follow appropriate safety protocols and use designated facilities, such as the NCRRT (Nuclear Chemistry and Radiation Technology) in Cairo, Egypt. Apply a gamma irradiation dose of 5 kGy to initiate the radical polymerization reactions.

The monomers of acrylic acid undergo radical polymerization as a result of gamma irradiation, which causes intramolecular crosslinking between the polymer chains. Pec and PAAc combine to form nanogel particles by this process, which produces a three-dimensional network structure.

The outlined procedure highlights the fundamental steps needed to synthesize Pec/PAAc nanogel using gamma irradiation as the radical polymerization initiator. Ensuring adherence to safety protocols and that the proper facilities and equipment are used, especially during the gamma irradiation phase. The resulting nanogel structures possess the potential for various applications, notably in controlled drug delivery systems and other areas that benefit from customized nanomaterial characteristics.

The suspension that is produced when the nanogel is synthesized using gamma irradiation is anticipated to have an acidic pH of around 1. To adjust the pH to a neutral value of 6, the following steps should be taken:

With the prepared nanogel solution, the next steps involve the incorporation of Rutin:

The outlined procedure encompasses several essential stages, from adjusting the pH of the nanogel suspension to incorporating rutin into the nanogel matrix. These steps collectively contribute to the controlled and systematic preparation of the (Pec/PAAc) nanogel and the incorporation of the therapeutic compound, rutin, for potential applications in drug delivery and related fields. It's imperative to execute these steps meticulously, maintaining precise conditions and adhering to safety protocols, to ensure the desired properties and functionality of the resulting nanogel-Rutin complex.

FTIR spectra of the pure rutin and the Pec/PAAc/rutin nanogel were acquired in order to study the chemical stability of rutin within the nanogel. Each sample was compressed into discs and combined with 500 mg of potassium bromide powder that was IR-grade for this examination. The spectrum was obtained between 4000 and 600 cm⁻¹.

Particle size was measured at 25 °C by dynamic light scattering using a Malvern Zeta-sizer Nano-ZS (Malvern Instruments, UK). Prior to each measurement, the dispersions were diluted as necessary to obtain the ideal light scattering intensity. The same device was used to test the zeta potential by tracking the motion of particles in an electric field following a 1:100 dilution. Results are presented as the mean of three independent experiments, with standard deviation included (Al-Zuhairy et al. 2023).

A transmission electron microscope (TEM, JEM-1230, Joel, Tokyo, Japan) was used to analyze the morphology of the prepared nanogel. After being diluted, the nanogel was applied on a grid covered in carbon. After that, it was stained negatively using a 2% w/v solution of phosphotungstic acid. The material was stained, allowed to dry at room temperature, and then examined under a microscope. Sample was dried at room temperature before being observed under the microscope.

The chosen nanogel (equivalent to 1 mg of rutin) was weighed and dissolved in 20 mL of ethanol. The colloidal mixture was sonicated for 20 min to ensure complete dissolution of the rutin into the ethanol. Following sonication, the dispersions was filtered using 0.45-μm membrane filter, and the resultant filtrate was then diluted with additional ethanol. An aliquot of this dispersion was subjected to scanning at a wavelength of 356 nm using a UV spectrophotometer (Shimadzu UV1650 Spectrophotometer, Kyoto, Japan). The drug content was subsequently calculated in triplicates (Ali et al. 2022). Additionally, the spreadability of the rutin nanogel was assessed using a glass slide apparatus (El-Adl et al. 2024). One milligram of nanogel was enclosed between two glass slides. The amount of time it took for the top slide to separate from the stack of slides was noted. Equation (1) was then used to calculate the gel spreadability.where S is for spreadability, M is for weight attached to the higher slide (g), L is for glass slide length (cm), and T is for the amount of time the slide takes to separate from the stack of slides.

The study evaluated the drug diffusion potential of a prepared rutin nanogel formulation and a control rutin suspension using a vertical Franz diffusion cell apparatus. Hairless rat dorsal skin was frozen, stabilized with pH 7.4 phosphate buffer, and mounted in the Franz cell (Hassan et al. 2022). The receptor compartment was filled with pH 7.4 phosphate buffer, maintained at 32 ± 0.5 °C with constant stirring. The nanogel formulation and control were then applied to the excised skin. Samples were collected from the acceptor compartment and analyzed spectrophotometrically at 356 nm. This data was used to determine the drug flux at steady state (Jss), the permeability coefficient (Kp), and the enhancement ratio. The Jss was calculated by plotting the cumulative drug penetration per unit area (μg/cm²) versus time and finding the slope of the linear portion. The Kp was then calculated by dividing the Jss by the initial drug concentration (C₀) (Abdou et al. 2017).

Maintaining the integrity of formulations over time requires assessing their chemical and physical stability under varied storage conditions. The formulation was kept for 90 days at 4 °C and 25 °C in sealed glass vials to assess the physical stability of rutin nanogel. For analysis, samples were obtained from each vial at 0-, 45-, and 90-day intervals. For analysis, samples were extracted from each vial at 0-, 45-, and 90-day intervals. Particle size, zeta potential, and drug content were assessed as previously mentioned at the end of the storage time. These values were compared to those of the fresh developed nanogel using a Student’s t test, with a significance level set at p ≤ 0.05. Moreover, the formulation was visually examined for any indications of physical changes, drug leakage, or aggregation (Shoman et al. 2023a).

To examine its binding interactions with the enzymes VEGFR-2 and EGFR^T790M, rutin was subjected to docking studies. The Molsoft program, which offers specific capabilities for simulating protein–ligand interactions, was used to carry out the docking simulations (El-Hddad et al. 2024). This software expects the binding of tiny, flexible molecules (such as substrates or potential drugs) to proteins with established 3D structures, by using grid interaction potentials (http://www.molsoft.com/icm_pro.html).

This research employed VEGFR-2 (PDB ID 4ASD) and EGFR^T790M (PDB ID 3W2O) as biological targets (Elmetwally et al. 2019). Their 3D structures were sourced from the Brookhaven Protein Data Bank (https://www.rcsb.org/). Specifically, sorafenib and erlotinib, which are known inhibitors of VEGFR-2 and EGFR^T790M, were used as reference ligands to assess the accuracy of the docking predictions in comparison to experimental data.

The protein was prepared for docking by addition of polar hydrogen to the protein atoms. The protein binding site was defined by placing a grid over the center of co-crystallized ligand. Before a protein was ready for docking simulations, all the necessary grid maps were calculated prior to docking. Our compound was drawn as a 3D structure, and its energy was minimized. The ligand was extracted from the binding site, and the compound discussed herein was docked into the binding site.

Numerous cancer cell lines were procured from the American Type Culture Collection (ATCC) situated in Manassas, USA. These lines included hepatocellular carcinoma (HepG2), colorectal carcinoma (HCT-116), and breast cancer (MCF-7). In particular, the Roswell Park Memorial Institute (RPMI 1640) medium was used to cultivate these cancer cells according to the proper protocol. The culture medium was supplemented with penicillin (100 units/mL), streptomycin (100 mg/mL), and heat-inactivated fetal bovine serum (10%). The cultivation of these cancer cells was carried out in a humidified atmosphere with 5% (v/v) CO₂ at a temperature of 37 °C.

The 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity of different substances or treatments. A commonly used approach for assessing the viability and proliferation of cells, including the cancer cell lines described, is colorimetric analysis.

Actively proliferating cells from different cancer cell lines were first trypsinized and quantified. They were then seeded into 96-well microtiter plates at appropriate densities, ranging from 2000 to 10,000 cells per 0.33 cm² well. These seeded cells were incubated in a humidified environment at 37 °C for 24 h to allow for cell attachment and acclimation.

Following the initial incubation, the cells were subjected to different doses of the chemicals under investigation, ranging from 0.1 to 1000 μM, for a duration of 72 h. The MTT assay was used to evaluate the treated cells’ vitality. Specifically, the culture media were removed, and the cells were incubated with 200 μl of 5% MTT solution per well.

Due to this, the MTT dye was metabolized by the living cells over the course of 2 h, forming colorful, insoluble formazan crystals.

This allowed the viable cells to metabolize the MTT dye into colored, insoluble formazan crystals over a 2-h period. After this incubation, the formazan crystals were dissolved in 200 μl of acidified isopropanol per well for 30 min after the residual MTT solution was withdrawn. This dissolution step was carried out at room temperature with a MaxQ 2000 plate shaker, covered with aluminum foil and continuously shaking.

The absorbance of the dissolved formazan crystals was measured at 570 nm through the use of Stat FaxR 4200 plate reader. Viability of the cells was calculated as a percentage of the untreated control, and Graph Pad Prism version 5 software was used to calculate the concentration at which the compounds triggered 50% of the maximal inhibition of cell growth (IC₅₀).

Analyzing the assay of VEGFR-2 kinase involved utilizing the Alpha Screen system (PerkinElmer, USA) and an anti-phosphotyrosine antibody in accordance with the manufacturer’s protocol. A solution containing 50 mM Tris–HCl (pH 7.5), 5 mM MnCl2, 5 mM MgCl2, 0.01% Tween-20, and 2 mM DTT was used to conduct the enzyme reactions. A total of 10 μM ATP, 0.1 μg/mL of biotinylated poly-GluTyr (in a 4:1 ratio), and 0.1 nM of VEGFR-2 (Millipore, UK) were all added in the reaction mixture.

Prior to initiating the catalytic reaction with ATP, the investigated substances were subjected to a 5-min room temperature incubation period with the VEGFR-2 enzyme, with final concentrations varying between 0 and 300 μg/mL.

In order to terminate the reactions, 25 μL of a solution comprising 100 mM EDTA, 10-μg/mL donor and acceptor streptavidin beads were added to 62.5 mM HEPES (pH 7.4), 250 mM NaCl, and 0.1% BSA. After an overnight dark incubation, the plate was read using an ELISA Reader (PerkinElmer, USA). The baseline control was represented by wells with biotinylated poly-GluTyr (4:1) and the enzyme but without ATP, and the reaction control was provided by wells with the substrate and the enzyme but not the tested compounds.

The percentage inhibition was computed by comparing contrasting the compound-treated samples with the control incubations. The concentration-inhibition response curve (based on triplicate measurements) was used to identify the concentration of the test drug that induced 50% inhibition (IC₅₀), and the results were compared with sorafenib (Sigma-Aldrich, USA), which acted as the standard VEGFR-2 inhibitor.

The study utilized the homogeneous time resolved fluorescence (HTRF) assay to evaluate the kinase activity of EGFR^T790M mutant. The EGFR^T790M enzyme and ATP were obtained from Sigma. Initially, the tested chemical was first incubated for 5 min with the EGFR^T790M enzyme and its substrates in an enzymatic buffer. This pre-incubation step allowed the compound to interact with the enzyme before the catalytic reaction was initiated. Afterwards, 1.65 μM of ATP was added to the reaction mixture to initiate the enzymatic process. The assay was carried out at room temperature for 30 min. The samples were mixed with detection reagents containing EDTA to stop the enzymatic process. The detection phase was allowed to proceed for 1 h. By utilizing GraphPad Prism 5.0 software, the inhibitory potency of the tested compounds was determined by calculating the half-maximal inhibitory concentration (IC₅₀) values. Three independent measurements were done to determine the concentration of each compound.

The pkCSM online tool was utilized to forecast the pharmacokinetic and toxicity characteristics of the examined substances (https://biosig.lab.uq.edu.au/pkcsm/prediction). The compounds were first prepared by hand-drawing and energy minimization according to the standard small molecule protocol. Then, using the pkCSM prediction algorithm, the ADMET (absorption, distribution, metabolism, excretion, and toxicity) descriptors were determined. This comprehensive in silico assessment provided insights into the pharmacokinetic and toxicological characteristics of the tested substrates.

The use of gamma irradiation is a versatile method for the synthesis of nanogels, which are intricate networks of crosslinked polymer chains. With this method, gamma radiation is applied to the polymer chains, causing the production of free radicals within them. As a result, hydrogel nanoparticles with precisely controlled and customizable properties are often produced using this approach.

As illustrated in Fig. 3a, the process by which gamma irradiation forms nanogels includes carefully choosing monomers and polymers that can undergo radiation-induced crosslinking reactions. The required monomers are added to a polymer solution or dispersion, where the polymers maintain desirable properties including hydrophilicity and biocompatibility that are useful for drug loading in biomedical applications.

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Formation of nanogels in this specific case involves the utilization of poly(acrylic acid) (PAA) and pectin as the polymeric components. Hydrogen bonds are created between the two kinds of polymers as a result of interactions between the carboxylate groups (-COO-) on the PAA chains and the pectin molecules. The hydroxyl groups (-OH) on both the PAA and pectin further engage in hydrogen bonding interactions in acidic (low pH) environments. This interplay between the functional groups serves to bridge the PAA and pectin chains, leading to the creation of a loosely crosslinked network. This initial step is referred to as self-assembly, where the attractive forces between the functional groups of polymer chains cause the chains to start interacting and entwining.

The second phase begins with the application of gamma irradiation as a crosslinking agent. In this process, water subjected to radiolysis, which results in the production of reactive species such as hydroxyl radicals (•OH) and hydrated electrons (e-). These radicals interact with the polymer chains, causing hydrogen abstraction. The resulting interactions promote the creation of crosslinks, which are covalent bonds that link neighboring polymer chains. As a result, a densely interconnected three-dimensional network structure is created (Shoman et al. 2023b).

As nanogel formation process progresses, the crosslinks continue to form, resulting in increasingly tighter interconnections among the polymer chains. This progression culminates in the creation of a comprehensive three-dimensional network structure. This network effectively entraps the solvent within, providing nanogels their distinctive gel-like consistency. Over time, the creation of the nanogel achieves an equilibrium state in which the rate of network disintegration or rearrangement equals the rate of crosslinking. These crosslinks maintain the integrity of the nanogel structure by stabilizing it.

In summary, gamma irradiation–induced nanogel production is a multi-step process. It begins with the initial self-assembly of the polymers through hydrogen bonding, followed by the introduction of crosslinking agents that generate reactive radicals. These reactive species then promote the formation of covalent crosslinks, leading to the creation of a tightly interconnected three-dimensional network. This intricate process ultimately results in the establishment of a stabilized nanogel structure. The ability to tailor the properties of nanogels through this gamma irradiation-based synthesis method makes them versatile candidates for diverse applications, particularly in the realm of controlled drug delivery and biomedical interventions.

Fourier transform infrared spectroscopy (FTIR) is a versatile analytical method employed to recognize and investigate the functional groups within a system. It provides information about the molecular structure and chemical composition of a compound by measuring the interactions between different types of bonds and infrared light. In the case of rutin, which is a flavonoid compound found in various plants, FTIR can reveal the vibrational modes and characteristic peaks associated with its functional groups. Figure 3b provided FTIR for rutin corresponding to specific vibrational modes of functional groups within the molecule. The peak at 3385 cm⁻¹ is likely associated with the O–H stretching vibration of the multiple hydroxyl (-OH) groups present in Rutin, particularly in the sugar moieties and phenolic rings. The peaks at 2987 cm⁻¹ and 2897 cm⁻¹ correspond to the asymmetric and symmetric C-H stretching vibrations in the aliphatic groups, possibly from the carbohydrate portion of the rutin structure.

The characteristic peak at 1773 cm⁻¹ is associated with the carbonyl (C = O) stretching vibration, often attributed to the lactone ring in rutin. The peak at 1660 cm⁻¹ suggests the presence of C = C stretching vibrations, commonly found in aromatic rings. The peak at 1390 cm⁻¹ corresponds to the bending vibrations of C-H bonds in aliphatic groups, while the peak at 1315 cm⁻¹ might indicate the presence of C-O stretching vibrations, possibly arising from ether or ester functional groups. The peak at 1150 cm⁻¹ could relate to C-O stretching vibrations, which might be attributed to the glycosidic linkages in rutin’s sugar moieties. The peak at 963 cm⁻¹ may indicate the presence of aromatic C-H bending vibrations, likely associated with the phenolic rings in rutin. The peak at 835 cm⁻¹ is consistent with the presence of aromatic C-H bending vibrations, further reinforcing the presence of phenolic rings. The positions and intensities of these FTIR peaks provide valuable information about the interaction between (Pec/PAAc) and rutin molecules, as well as the composition and functional groups present in their compound.

The FTIR spectrum of the (Pec/PAAc)/rutin nanogel, as shown in Fig. 3c, reveals several characteristic peaks that provide insights into the interactions between the nanogel components and the Rutin molecule. The peaks at 3400 cm⁻¹ and 3325 cm⁻¹ correspond to the O–H stretching vibrations, indicating the presence of hydroxyl groups contributed by both the (pectin/PAAc) nanogel and the rutin compound, respectively. The peak at 2980 cm⁻¹ suggests the presence of asymmetric C-H stretching vibrations, likely originating from the aliphatic components of the pectin and poly(acrylic acid) in the nanogel. Similarly, the peak at 2890 cm⁻¹ is associated with the symmetric C-H stretching vibrations in aliphatic groups. The broad peak at 1667 cm⁻¹ indicates the presence of C = O stretching vibrations, which may be attributed to the various carbonyl-containing functional groups present in the (Pec/PAAc)/rutin nanogel. The peak at 1390 cm⁻¹ might represent the C-H bending vibrations in the aliphatic structures of the nanogel components. The peaks at 1315 cm⁻¹ and 1143 cm⁻¹ relate to the C-O stretching vibrations, likely associated with the glycosidic linkages or other oxygen-containing groups in the nanogel and rutin. The peak at 963 cm⁻¹ indicates the presence of aromatic C-H bending vibrations, suggesting the incorporation of the phenolic rings from the rutin compound into the nanogel structure. Finally, the peak at 827 cm⁻¹ may correspond to the C-H bond bending vibrations in aliphatic groups.

By comparing the observed peaks in Fig. 3c with the reference FTIR spectrum of rutin in Fig. 3b, and the known functional group vibrations, the changes in peak positions, intensities, and shapes provide strong evidence of the interactions between the (Pec/PAAc) nanogel and the rutin molecule.

The pH-responsive behavior observed in Fig. 4 highlights a compelling avenue for advancing targeted drug delivery systems. This intriguing characteristic of the nanogel/rutin complex indicates its potential to precisely control rutin release based on environmental pH variations. This pH-sensitive release mechanism considers great promise for therapeutic applications. It offers a pathway to significantly improve the accuracy of drug delivery systems. By harnessing this pH-responsive property, the nanogel/rutin complex could usher in a new era of drug delivery precision. The ability to modulate drug release based on pH levels opens doors to targeted delivery strategies tailored to specific sites or tissues within the body. This is particularly advantageous in instances where localized pH variations occur, such as in the acidic environment of the stomach or in disease microenvironments. The complex’s aptitude for releasing its therapeutic payload selectively at the targeted site could significantly boost the effectiveness of treatment. A paramount advantage arising from this pH-sensitive release system is the potential reduction in undesirable side effects. Through the fine-tuned control of drug release, the risk of adverse reactions impacting non-target tissues could be minimized. This facet of targeted drug delivery has the potential to elevate patient comfort and compliance during the course of treatment, further enhancing overall therapeutic outcomes. Optimization of drug dosage is a pivotal aspect of effective treatment, and the pH-responsive behavior of the nanogel/rutin complex offers a path to achieving this optimization. The controlled manner in which the therapeutic agent is released enables a more precise and regulated dosing schedule. As a result, patients may experience improved adherence to treatment plans, potentially leading to enhanced therapeutic success rates. Importantly, the pH-triggered release mechanism could also play a pivotal role in mitigating toxicity concerns associated with certain drugs. By directing drug release primarily to the target site, healthy tissues might be spared exposure to potentially toxic agents. This reduction in toxicity risk could offer a significant advancement in patient safety and overall treatment outcomes. The noteworthy increase in rutin release observed from pH 1 to pH 4 about 65% underscores the dynamic behavior of the nanogel complex. This intriguing phenomenon likely arises from the pH-dependent swelling or degradation of the nanogel matrix, ultimately causing the release of encapsulated rutin. This understanding paves the way for tailored modifications to the nanogel composition, enabling fine-tuning of the pH range for optimal drug release. In conclusion, the pH-responsive behavior demonstrated by the nanogel/rutin complex, as depicted in Fig. 5, holds substantial promise for revolutionizing targeted drug delivery systems. The ability to modulate drug release to pH fluctuations offers a suite of advantages, ranging from targeted delivery and enhanced efficacy to reduced side effects and minimized toxicity. Continued research and development in this realm could yield innovative drug delivery platforms with far-reaching implications for therapeutic interventions.

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In the pH range of 1 to 3, an interesting behavior is observed with the nanogel/rutin complex. During this acidic pH range, the nanogel maintains its structural integrity and exhibits minimal rutin release. This phenomenon can be attributed to the interaction dynamics within the complex. At lower pH levels, specifically from 1 to 3, the carboxyl groups present on poly(acrylic acid) chains become protonated. This protonation alters the charge distribution along the PAAc chains, leading to a non-ionic interaction scenario. Importantly, this change in charge state contributes to the stability of the nanogel structure. An essential factor in this stability is the intermolecular hydrogen bonding interactions that occur between the pectin (Pec) and poly(acrylic acid) (PAAc) molecules. These interactions, facilitated by hydrogen bonds, are essential to maintain the structural cohesion of the nanogel complex. The formation of hydrogen bonds between Pec and PAAc reinforces the complex’s overall architecture, preventing premature or excessive rutin release. In essence, the absence of rutin release at acidic pH levels is a result of the concerted interplay between the protonation of carboxyl groups on PAAc chains, the ensuing non-ionic interactions, and the stabilizing hydrogen bonding between Pec and PAAc molecules. This intricate balance of molecular interactions ensures that the nanogel remains intact and retains its cargo under these specific pH conditions, holding promise for controlled and targeted drug delivery applications.

At the specific pH value of 4, an intriguing event known as “pH-triggered release” occurs within the nanogel/rutin complex. This event is marked by a decomplexation process between the two primary polymer components, namely pectin (Pec) and poly(acrylic acid) (pAAc), which together constitute the matrix of the nanogel.

The decomplexation process is driven by the pH-responsive behavior of the pAAc chains. As the pH rises to 4, the PAAc chains begin to release protons, converting carboxyl groups into negatively charged carboxylate ions. This shift in charge distribution induces repulsive forces between the negatively charged pAAc chains and the corresponding charged segments within the Pec chains.

The electrostatic repulsive forces between the pAAc and Pec chains trigger a sequence of events within the nanogel structure. As repulsive forces gain prominence, the nanogel network undergoes a process of swelling and partial breakdown. This phenomenon causes the nanogel structure to open up, forming pores or gaps within its matrix. The consequences of this structural transformation are significant. The openings or pores generated in the nanogel matrix allow the trapped rutin molecules, which were previously encapsulated within the complex, to diffuse out. This diffusion process is now facilitated by the newly created pathways within the nanogel. Consequently, the increase in rutin release observed at pH 4 can be attributed to this “pH-triggered release” mechanism. The decomplexation-induced nanogel swelling and structural alteration result in the exposure of previously encapsulated rutin molecules to the surrounding environment. This liberation enables rutin to diffuse freely through the opened nanogel structure, leading to an enhanced release of its constituent compounds.

In the alkaline pH range spanning from pH 5 to pH 8, an interesting phenomenon emerges, characterized by a significant reduction of approximately 66% in the release of rutin from the nanogel complex. This behavior can be attributed to the influence of (Na) counter ions, which exhibit a noticeable increase in concentration as the pH shifts towards the higher alkaline values.

At these elevated pH levels, the presence of (Na) counter ions give rise to a phenomenon known as the “charge screening effect”. This effect entails the neutralization or shielding of charged functional groups within the nanogel structure (Ghobashy and Bassioni 2018). The screening of charges can facilitate the reformation of the nanogel network, reducing its porosity and consequently restricting the rutin release. Specifically, the negatively charged carboxylate groups on the pAAc chains, which are crucial components of the nanogel, become subject to shielding due to the interaction with the (Na) counter ions.

The shielding effect initiated by the (Na) counter ions introduces a layer of electrostatic repulsion between the carboxylate groups, which were previously instrumental in the pH-triggered release mechanism, and the surrounding (Na) ions. This repulsion attenuates the interactions that had previously facilitated the controlled release of rutin. As a consequence, the structural dynamics of the nanogel complex are altered. The shielding effect reduces the porosity of the nanogel matrix, hindering the diffusion and release of rutin molecules from within. This restricted diffusion limits the extent to which rutin can permeate through the nanogel structure, thus leading to the observed decrease in rutin release.

A transmission electron microscope (TEM) image of the (pectin, PAAc) nanogel encapsulating rutin offers a detailed visualization of the nanogel’s morphology and structure on a micro- to nanoscale level. TEM, which utilizes electron beams for scanning, delivers high-resolution images that reveal the surface features, textures, and overall structure of the nanogel. The image shows the morphology of the nanogel as a collection of spherical or irregularly shaped nanoparticles formed from the crosslinked pectin and PAAc chains. Detailed observations of the surface texture may reveal features such as bumps, pores, or ridges, providing insights into the polymer interactions and the encapsulation of rutin. The TEM image also allows for the assessment of the size distribution of the nanogel particles, including average particle size and variability. Additionally, regions within the nanogel particles where rutin is encapsulated might be discernible, appearing as darker or lighter spots. The image may also reveal aggregation or clustering of nanogel particles, potentially due to electrostatic interactions, hydrogen bonding, or other forces.

The pH of a solution is a critical parameter that can have a profound impact on the behavior of molecules and complexes within it. In the realm of nanogel/drug complexes, the adjustment of pH from highly acidic (pH 1) to moderately alkaline (pH 8) has been explored in a fascinating study, shedding light on how pH influences the size and stability of these complexes.

One of the most notable findings of this study is the direct relationship between pH and the size of (Pec/PAAc)/Ru nanogel/drug complexes. As the pH of the solution increases, so does the particle size of the complexes. This observation provides valuable insights into the optimization of these complexes for various applications, particularly in drug delivery and nanomedicine.

At pH 1, the complexes exhibit a relatively large particle size, which is expected due to the acidic conditions. However, as the pH gradually increased, a striking trend emerges. The smallest particle size, a mere 16 nm, is achieved at pH 3. This represents a crucial discovery because it indicates that pH 3 is the point at which the nanogel/drug complexes attain their most stable and compact form. At this pH, the nanogel structure appears to be tightly bound to the drug molecule, resulting in a minimal hydrodynamic radius and, consequently, a diminutive particle size.

A particularly intriguing phenomenon is the sharp increase in particle size between pH 4 and 7. Within this range, there is a noticeable transition in the behavior of the complexes. The particle size grows from 185 nm at pH 4 to 296 nm when the pH is adjusted to 7. This abrupt and substantial size change suggests that there exists a critical pH window within this range. During this window, the nanogel structure undergoes significant structural changes, leading to increased swelling. The exact mechanisms behind these changes warrant further investigation, but it is likely related to the deprotonation of specific groups within the nanogel structure, causing the conformation to become less compact. Consequently, the hydrodynamic radius increases, resulting in larger particle sizes.

As the pH surpasses 7 and moves into moderately alkaline conditions, an intriguing phenomenon occurs. The particle sizes start to level off, indicating a pH threshold, presumably above 7, beyond which further structural changes in the nanogel have limited impact on size. This leveling-off effect suggests that at higher pH levels, the nanogel complexes reach a stable configuration where additional structural adjustments yield diminishing returns.

Conversely, when the pH drops below 3, well into the acidic range, the nanogel/drug complexes exhibit larger particle sizes. This can be attributed to the protonation of specific groups within the nanogel structure, leading to a more compact conformation. It is a clear demonstration of how pH, through protonation and deprotonation processes, can dramatically influence the size and stability of these complexes.

The implications of these findings are far-reaching, especially with regard to drug delivery and biomedical applications. The capability for precisely controlling the size and stability of nanogel/drug complexes by adjusting pH offers a valuable tool for tailoring drug release kinetics and optimizing therapeutic efficacy. Future research in this field may delve deeper into the molecular mechanisms underpinning pH-induced changes in nanogel structure and explore how these alterations affect drug release profiles. Additionally, investigating the correlation between pH and the zeta potential of the complexes can provide further insights into their stability and electrostatic interactions.

In conclusion, this study underscores the significance of pH as a critical factor in the behavior of (Pec/PAAc)/Ru nanogel/drug complexes. pH directly influences their size and stability, with pH 3 being the optimal condition for achieving small, stable complexes. The sharp size increase between pH 4 and 7 suggests a critical pH range where substantial structural transformations occur. The ability to harness these pH-dependent properties holds immense promise for advancing drug delivery and biomedical applications, offering opportunities for more precise and effective therapies.

Figure 6b illustrates that the zeta potentials of the (Pec/PAAc)/Ru samples exhibit considerable variation as the pH is adjusted from 1 to 8. Notably, the zeta potential values do not seem to correlate directly with the particle sizes, indicating that the surface charge is largely independent of particle size variations induced by pH changes. The zeta potential values reveal an intriguing pattern. At pH 1, pH 4, and pH 8, the nanogel particles carry positive zeta potentials, with values around 6.96, 11, and 0.5 mV, respectively. Conversely, at pH 2 and pH 6, the zeta potentials are slightly negative, with pH 2 showing the most negative charge (− 4.3 mV) and pH 6 showing the least negative charge (− 0.3 mV).

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One striking observation is that the zeta potential values do not exhibit a discernible trend in relation to pH. Unlike the particle size, which exhibited a systematic increase with increasing pH, the zeta potential values fluctuate within a range of 11 to − 0.3 mV across the pH range of 1 to 8. This suggests that there may not be a clear correlation between pH variations and the surface charge characteristics of the nanogel particles. One possible explanation for the complex surface charge behavior observed in the (Pec/PAAc) rutin nanogel complexes is the dissociation of acrylic acid (AAc) molecules inside the nanogel matrix. AAc is known to be a weak acid, capable of both donating and accepting protons (H + ions) depending on the pH of the surrounding medium. This behavior leads to fluctuations in the surface charge as pH varies, as seen in the experimental results. Understanding the zeta potential behavior of these nanogel/drug complexes is of paramount importance in various applications, particularly in drug delivery and stability assessments. The surface charge can influence interactions with biological systems and other particles in suspension. The observation of both positive and negative zeta potentials across the tested pH range highlights the complexity of these systems. Future research could delve deeper into the molecular mechanisms responsible for the observed zeta potential variations. Additionally, exploring the consequences of these zeta potential changes on the stability, colloidal behavior, and drug release kinetics of the nanogel/drug complexes could provide valuable insights into optimizing their performance for specific biomedical applications. In conclusion, the zeta potential values of (Pec/PAAc)/Ru nanogel/drug complexes exhibit a diverse and intriguing pattern across the pH range of 1 to 8. These surface charge variations are largely independent of changes in particle size induced by pH adjustments. The complex behavior of zeta potential, influenced by the dissociation of AAc molecules within the nanogel structure, underscores the need for a comprehensive understanding of these systems to harness their potential in drug delivery and other biomedical applications.

The cumulative drug content from the nanogel was found to be 86.14 ± 2.61%. This result indicates that the drug was uniformly dispersed throughout the nanogel formulation. This uniformity can be attributed to the effective swelling of the polymeric system at pH 3, which facilitated the entrapment of the drug within the nanogel matrix. Concurrently, spreadability is a vital attribute when assessing nanogels for use in topical use. The chosen nanogel exhibited a spreadability of 55.26 ± 2.67 g·cm/s by weight, indicating good ease of application and optimal consistency for effective spreading on the skin.

Hairless dorsal skin was used in ex vivo permeation investigations to evaluate drug diffusion and compare it with rutin suspension. The findings, shown in Fig. 7, demonstrated that the drug suspension had a lower permeability coefficient (0.019 cm/h) and a smaller cumulative amount permeated over 8 h (358.705 μg) compared to the nanogel formulation (0.046 cm/h and 903.829 μg), yielding an enhancement ratio of 2.389. The marked increase in transdermal permeation indicated that incorporating rutin into a nanogel significantly improved its permeability and drug diffusion. This improvement is likely due to the nanogel’s smaller particle size, which increases surface area, its mucoadhesive properties that prolong skin retention, its ability to enable controlled release, and its enhanced stability (Marwah et al. 2016; Ay Şenyiğit et al. 2021).

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After being stored for 45 and 90 days at 4 °C and 25 °C, the rutin nanogel formulation showed no visible signs of aggregation or any alteration in appearance. The findings presented in Table 1 exhibit the particle size (PS) zeta potential, drug content, and spreadability for both the freshly prepared and stored formulations, revealing no significant variations (p > 0.05 for all parameters). Furthermore, there were no noticeable changes in the physical characteristics of the nanogel, such as color.

Molecular docking studies were conducted using Molsoft software. The experiments employed VEGFR-2 and EGFR^T790M with PDB IDs 4ASD (Khedr et al. 2021; Aziz et al. 2022) and 3W2O (Gandin et al. 2015), respectively.