INTRODUCTION:

According to epidemiological studies, nephrotoxicity is the third most frequent cause of acute kidney disease (AKD)¹. AKD cases were increasing in decades as a result of nephrotoxic drugs uses with a higher risk of kidney damage, studies have shown that up to 20% of critically ill patients use nephrotoxic drugs. The kidneys excretion of drugs and metabolites exposes the kidneys high-energy-requiring structures i.e. the glomeruli and tubules, to large concentrations of drug chemicals, which explains why drug-induced kidney injury rates are so high^2,3,4,5,6,7

Drug-induced nephrotoxicity might manifest as acute or chronic reduced glomerular filtration rate (GFR), nephrotic syndrome, or hydroelectrolytic disorders (HED), which are associated with glomerular and tubular injury. Most studies only take into account an increase in creatinine for epidemiological purposes, which makes it difficult to accurately assess the full scope of the issue⁸. Prioritizing general measures can minimize damage in cases of nephrotoxicity⁹. These include adjusting medication dosages based on renal function, substituting less harmful medications for nephrotoxic ones, correcting hypovolemia and HED, shortening treatment durations, and monitoring serum levels. These are cost-effective methods of lowering the risk of drug-induced renal injury. However, these may cause complications with significant impacts on patient's treatment¹⁰.

In the other side, the use of medicinal plants such as nephroprotective herbs as a fundamental part of complementary medicine has drawn attention¹¹. Considered a strong nephroprotective agent, flavonoids are known to possess strong antioxidant activity that may protect against oxidative stress-mediated kidney disorders. The World Health Organization (WHO) advocates for the advantageous synergy between conventional and modern medicine in primary healthcare.

RUT is a flavonoid glycoside compound found in fruit and vegetables such as apples, sweet potato leaves, passion fruit and tea¹². RUT is often used for its activity as an antioxidant¹³. RUT is lipophilic and are categorized in Biopharmaceutical Classification System (BCS) class II¹⁴. The potential of RUT as an active pharmaceutical ingredient is very much limited by its low solubility in water^15,16. In chronic patients, where the dose was 50-150mg/day for two weeks, plasma levels were only 0.19-0.43µM¹⁷. In addition the poor solubility, RUT also experience hydrolysis in acidic gastric environment (ref). RUT at a dosage of 15mg/kg only produced a Cmax of 46.8±2.3ng/mL, which resulted in bioavailability problems¹⁸.

One approach to address thoses issues was the use of enteric nanoparticles that provides the advantages of increasing the bioavailability of drugs and at the same time avoiding degradation by the acidic gastric fluid^19,20. RUT formulation in form of nanosuspensions increased its solubility leading to in increasing of absorption^15,21,22,23. Selecting polymers is very important in the development of enteric nanoparticle delivery systems. EL100 is one of biocompatible and pH-sensitive polymers that have been widely used in entrapment technology^24,25,26. A part from polymer selection, another important factor in nanoparticle formulation is the use of stabilizer. Poloxamer is a surfactant that has been attractive which composed of a hydrophilic polyoxypropene (POP) unit and hydrophobic polyoxyethylene (POE) chains^13,27.

Considering a potent nephroprotective effect of RUT, its development into enteric nanoparticle delivery system is promising to overcome the solubility and stability issues^28,29. Therefore, research that specifically examines RUT in the formulation of enteric nanoparticles and concentrates on the optimization of the polymer and surfactant combination is crucial for the development of RUT formulation. RUT was created in the current study's enteric nanoparticle delivery system, and its physical properties were examined. Particle size analysis (PSA), differential scanning calorimetry (DSC), x-ray diffractometry (XRD), scanning electron microscopy (SEM), and fourier transform infrared spectroscopy (FTIR) were among the techniques used in the characterizations³⁰.

MATERIALS AND METHODS:

Materials:

RUT, Poloxamer 188 (Kolliphor^® P188), Poloxamer 407 (Pluronic^® F127), ethanol (analytical grade) were purchased from the Sigma-Aldrich (Singapore). Eudragit L100 (Eudragit^®L100) were purchased from Evonik. Aquadest were obtained from PT Brataco (Indonesia).

Method:

Preparation of RENPs Formulations:

The weights of RUT, EL100, P188, and P407 were determined using the amounts listed in Table 1. After dissolving RUT in 5milliliters of 96% ethanol, the mixture was agitated for 15minutes at 1000rpm. Similarly, EL100 was dissolved in 5ml of ethanol. RUT solution was dropwised into EL100 solution under constant stirring at 1000rpm for 15minutes. After that, weighed amounts of P188 and P407 were dissolved in 10milliliters of aquadest and agitated for 15 minutes at 1000rpm. The RUT-EL100 solution was sonicated for 10 minutes at 25°C while being dropwise added to the P188:P407 combination solution. Using the following parameters, freeze drying was used to further solidify the suspension: Overnight at a freezing temperature of -80°C. The frozen sample was then dried at pressure of 0.500 mbar, temperature of −50°C and gradually increased to 25°C for 72hours³¹.

Table 1: Prepared RENPs made with different weight ratio of P188 : P407.

Formulation	RENP-1	RENP-2	RENP-3	RENP-4
RUT	5	5	5	5
ELI 100	50	50	50	50
P188	6	5	4	3
P407	4	5	6	7

Abbreviations: RENP = RUT Enteric Nano Particle

Particle Size Analysis using Dynamic Light Scattering (DLS):

5 milligrams of RENPs were weighed, dissolved in 5 milliliters of aquadest, and agitated for 15minutes. 200 µL of this solution was pippetted into a cuvette after being further diluted in 2mL of distilled water. Samples were analyzed for particle size using the DLS instrument Beckman Coulter Delsa Nano C Particle Analyzer at a temperature of 25°C.

Determination of Drying Yield (%):

In proportion to the initial quantity of materials in the formula, the percentage of drying yield was computed from the total dry powder of RENPs collected after the freeze-drying process. The following formula was used to determine the yield percentage:

Total weight of dry product

% Yield = -------------------------------------------- X 100%

Total weight of initial formula

Differential Scanning Calorimetry (DSC):

The solid condition of the dry RENPs was detected using a DSC equipment. Samples were heated at a rate of 10°C per minute in an aluminum crucible, with temperature ranges ranging from 30°C to 250°C. The empty crucible portion of the aluminium pan is used as a reference blank. The thermogram results obtained were then evaluated for phase miscibility.

X-Ray Diffractometry (X-RD):

RENPs and pure materials were evaluated for their crystallinity using X-ray diffractometer instrument (Philips X'Pert PRO PANalytical, The Netherlands). The sample was levelled on the sample holder and scanned at room temperature with a 2 theta angle range from 5 to 40°. The measurement conditions use a voltage of 40kV and 40kA. Crystalline structure was characterized by the presence of sharp peaks with high intensity. Meanwhile, amorphous structures are characterized by the opposite.

Scanning Electron Microscopy (SEM):

SEM was used to examine the morphology of the dried products of RENPs. Each sample was scattered in sufficient quantity onto the surface of a sample holder. Samples were coated with gold. The shape and surface morphology of the samples were examined under microscope at a magnification of 400 times at several observation spots. The microscopic images of the resulting RENPs were compared to the raw ingredient.

Fourier Transform Infrared Spectroscopy (FTIR):

To verify the chemical structures of the RUT encapsulated and assess any potential drug-carrier interactions in the produced formulation, FTIR analysis was carried out. All of the generated RENPs and the FTIR patterns of the pure materials were examined using FTIR (Bruker, Germany) utilizing the KBr disk method, which covered 400–4000 cm−1 with a resolution of 4 cm−1.

Dissolution Study:

To conduct dissolution tests, phosphate buffer (PB) with a pH of 6.5 was used. Spectrophotometry UV-Vis was used to evaluate the particles that were collected at the early stage of dissolution (0, 5, 10, 15, 30, 60) minutes. The dissolution profile was presented as dissolution efficiency (DE) and dissolution rate (DR), using equations below:

DE = X 100%

This equation is used to calculate the dissolution efficiency of a substance over a given time span. It compares the total amount of dissolution that occurred (calculated from the integral y(t)) with the maximum potential dissolution (y100) that occurred during that time.

Dissolution rate (DR) is the rate or velocity at which a drug substance dissipates in a medium. The DR equation

DR = ------- = k. (C_s – C_t)

where k is the intrinsic dissolution rate constant, dC/dt is the intrinsic dissolution rate (mg cm2/s), and Cs is the drug concentration in saturated solution.

Statistical Analysis:

The software GraphPad InStat (San Diego, CA) was used to analyze the statistical data. The experimental results on solubility were analyzed using a parametric one-way ANOVA test. Statistical significance was established using 95% confidence limits.

RESULTS:

Particle Size Analysis:

DLS is an instrument used to measure the particle size of the samples. The sample in the form of a solid is then dispersed into a liquid medium. The data obtained was in the form of particle size and Poly Dispersed Indeks. The result of four formulation particle size showed in table 2.

Table 2. Particle Size of RENP enteric particle made at different weight ratio of P188 : P407

Formulation	RENP-1	RENP-2	RENP-3	RENP-4
Praticle Size (nm)	401,7 ± 39,00	477,0 ± 10,000	511,0 ± 26,00	438,3 ±12,00
PDI	0,182 ± 0,01	0,208 ± 0,02	0,176 ± 0,01	0,167 ± 0,01

Abbreviations: RENP = RUT Enteric Nano Particle

Based on the results of particle measurements in table 2, it is known that all formulations give results below 600 nanometer. The PDI values were <0.2 which can be interpreted that all formulations were homogeneous. Statistically, the four formulations do not have significant differences. The particle size reduction process was carried out using an ultrasonicator. The small particle size was achieved through applied ultrasonic frequency as energy source in the reduction process.

Characterization of RENPs:

a. Drying Yield (%w/w):

Drying yield is part of the research to determine the percentage comparison between the initial material and the final material after drying. Figure 1. is the result of the drying yield of the study.

Figure 1: Yield percentage of RENPs

Abbreviations: RENP = RUT Enteric Nano Particle

Based on the results of the drying yield calculations in this study, the highest percentage of the drying yield was RENP-3 while the lowest is RENP-2. All the formulations showed yield percentage in a range of 66% to 80%, as presented in figure 1.

b. Differential Scanning Calorimetry (DSC):

thermal analysis called DSC is performed to identify the difference in the amount of heat needed to raise the sample's temperature. The results of the RENP characteristics are compared to the raw material. Figure 2 is the result of RUT enteric analysis with EL100 and a combination of P188:407.

Figure 2: DSC Thermogram of RUT - EL100 enteric particle made at different weight ratio of P188 : P407

Abbreviations: RENP = RUT Enteric Nano Particle

The thermogram results show that there is a difference in peak sharpness between the DSC of the raw RUT and the DSC modified combination of P188: P407. This can be seen in the temperature range of 50°C - 70°C and between temperatures of 210°C - 250°C.

Based on Figure 2 on the results of the characteristics using DSC, it is known that there is a significant difference between the single material and the modified material. It is clear that the changes that occur in the single material P188 and P407 are known to have steep thermograms. But when the material is modified with RUT and EL100, the thermogram changes to a slope.

c. X-Ray Difraction (XRD):

XRD is the change in direction of an X-ray beam due to interaction with electrons around atoms. Diffraction occurs due to elastic scattering, when there is no change in wave energy. The diffractogram of the sample looks very different as seen in figure 3.

Figure 3: XRD difractogram of RUT - EL100 enteric particle made at different weight ratio of P188 : P407

Abbreviations: RENP = RUT Enteric Nano Particle

Figure 3 shows a comparison between the diffractograms of the single material and the developed RENP formulations. The results show that there is a shift and sharpness of the diffractogram peak. Based on Figure 3 regarding the diffractogram, it is known that there is a very significant change in the matrix formed. In the diffractogram of the Single material, it is known that RUT, P188 and P407 have sharp and clear peaks, while EL100 does not have a sharp peak. In the results of the matrix modification, it is known that the diffractogram experiences a change that is clearly visible following the diffractogram in EL100. This happens because the matrix experiences changes in solubility and crystallinity levels.

d. Scanning Electron Microscopy (SEM):

Among the properties of nanoparticle materials is the scanning electron microscope, which creates an image of the sample by using a concentrated electron beam to scan its surface. The figure result the study can see in figure 4.

Figure 4 showed the images formed in the form of porous particles and shows different results with the raw RUT. Based on Figure 4, it is known that the shape of the surface structure of the single material RUT looks like a lump and solid. However, the SEM images of all formulations showed that the matrix has porous structures with a smooth surface.

e. Fourier Transform Infrared Spectroscopy (FTIR):

Figure 5: Spektrogram of RUT - EL100 enteric particle made at different weight ratio of P188: P407

Abbreviations: RENP = RUT Enteric Nano Particle:

Based on the existing spectra images, it is known that the structural groups in each Single material are in the RUT modification matrix. This can be seen in the modified matrices RENP-1, RENP-2, RENP-3, RENP-4 which have the same structural groups with sharp peaks according to the structural groups of the Single material. This means that the newly modified matrix does not provide the existing structure.

f. Disolution Study:

According to Figure 6, the formulation RENP-3 had the highest dissolving rate findings in this investigation at 60 minutes, reaching a percentage of 90.25 percent. According to the current figure, RENP-3 has the best dissolving rate, followed by RENP-4, RENP-2, and RENP-1, in that order. But as compared to raw RUT, all formulations showed better solubility.

Figure 6: Dissolution Rate of RUT - EL100 enteric particle made at different weight ratio of P188: P407

Abbreviations: RENP = RUT Enteric Nano Particle

DISCUSSION:

RUT poor solubility in water affects their absorption in the body. One effective strategy to increase drugs solubility include reducing the particle size up to nano-sized particles. The nanosuspension had particle sizes more than 600nm, according to the developed RENPs formulations (Table 1). This was in accordance to previous study in which RUT nanosuspension showed particle size of 250nm³². Using Eudragit R 100, similar studies found that the average particle size was less than 200 nm.³³. The small sized particles were obtained by the function of poloxamers. In addition, from manufacturing aspect, the use of nanoprecipitation successfully produced small sized particles.

The technique used to maintain the particle size so that it remains at encapsulation is to use nanoprecipitation where the main ingredient is dissolved in an organic solvent, then the main ingredient is absorbed by a suspending agent so that it remains at nanoparticle size, then the trapped main ingredient is stabilized with a combination of poloxamer and an organic solvent. used is evaporated. The nanoprecipitation method was used for the preparation of the encapsulation with slight modification³⁴.

The cause of the rise in particle size was the polymer solution's increased viscosity, which reduced the polymer solution's ability to disperse into the aqueous phase. It is clear that the size of the RUT encapsulation rises regardless of type and concentration. It was observed that the size of RUT encapsulation did not change as the concentration ratio combination P188:P407 remained constant between 6:4 and 3:7. This could be as a result of the fact that more polymer reduces surface tension and facilitates particle partitioning³⁵.

Analysis of the properties of the sample against heat was carried out using a Differential Scanning Calorimetry (DSC) instrument. DSC is a vital tool for obtaining evidence quickly about suitability among the RUT and the carrier by the presence, swing, or vanishing of endothermic or exothermic peaks. Analysis using DSC is related to the phase transition technique which is much in demand because it allows the detection of transitions at various temperatures with a temperature range between -90° - 550°C so that it can be used for qualitative and quantitative analysis³⁶.

The DSC results on the single material of each material used show that the poloxamer compounds both 188 and 407 have sharp endothermic curves at temperatures around 50° - 700°C. Likewise, RUT and EL100 also have an endothermic curve at a temperature of 170° - 1900°C but not as sharp as poloxamer. DSC gives an insight into the melting and recrystallization activities of nanosuspension RUT. The DSC points were attained on raw RUT and physical blend³⁷.

The results obtained show that there is a temperature shift between the raw RUT and RUT combined with EL100. This is the same as research conducted regarding the effect of poloxamer where an endothermic process occurs³⁸. The thermograms of RUT-EL 100 with poloxamer combinations were shifted towards the left, indicating proper of RUT-EL 100 with the carriers used. The research results show that the endothermic process occurs at temperatures between 50-70°C and also occurs at temperatures around 230°C. This is the same as the endothermic curves for RUT-EL 100 and poloxamer alone. The characteristic peaks and stretches of raw RUT and EL 100 were also seen in the RUT-EL 100 with poloxamers combinations, indicating no incompatibility of RUT with the carriers used.

All samples' diffractogram patterns were identified by XRD analysis. Following nanoprecipitation and freeze drying, this method is among the most reliable for examining the crystallographic structure of RUT. Figure 3 displays the XRD data for individual materials. It was discovered that poloxamer 407 and poloxamer 188 had sharp, powerful peaks in their diffraction patterns. Although the intensity peak is not as sharp, the similar phenomenon is also observed in RUT. In contrast, Eudragit L100 had broad patterns devoid of sharp peaks, suggesting that the material was in an amorphous or more disordered crystalline state³⁹.

The main method for characterizing material attributes like strain, size, and crystal structure is X-ray diffraction (XRD). But XRD can take a long time and can need a lot of samples²⁴. The results of XRD measurements on the single compounds used in the research show that each material has a specific curve according to the material. Poloxamer 188 and 407 show sharp curves, and RUT encapsulation also has a specific XRD character. The same thing applies to the Eudragit L100 which has a specific, straighter character. Poloxamer 407 thermosensitivity played a decreasing solubility with temperature rise²⁵.

The research results obtained showed that the diffractogram formed showed that many crystals were formed. This is the same as research carried out regarding RUT comparisons using poloxamer showing almost the same results at almost the same temperature range³⁰. The results in the research show that there is a shift in the diffractogram results between single materials and the results of developing combination formulations. The research results show that the curve formed from the same 4 formulas follows the curve pattern of EL100. This shows that the XRD characteristic reading is the EL100. This shows that the raw RUT is trapped in EL100 so the stability is good.

Figure 4 displays the morphology of RENP as analyzed by SEM. To determine the homogeneity and stability of the RUT encapsulation, the morphology of the final product was examined using scanning electron microscopy (SEM). Surface morphology, cross-sectional morphology, and the formulation's structural integrity have all been assessed using SEM(40). RUT encapsulation SEM results look like chunks of solid crystals that are densely arranged so that it appears that it is very difficult for the water solvent to enter the RUT encapsulation structure. Solid structures need to have their bonds broken so that the water used as a solvent can enter easily, or if possible the crystal chunks are RUT encapsulation broken mechanically to reduce the particle size.

The findings of this investigation revealed that the SEM results in a single RUT changed from RENP-1 to RENP-4. In a single sample, it can be seen that the appearance of RUT shows that it is not porous when compared with the research results. This is the same as research related to the FD solidification process, the interface between solid and liquid forms a colloidal suspension then undergoes sublimation to form a frozen phase which then becomes a porous material³³. In this research, the EL100 mechanism uses the Hot Methyl Extrusion (HME) method which will experience delayed release³². Meanwhile, the poloxamer mechanism acts as a stabilizer, namely because it is hydrophobic for RUT¹².

The results obtained from this research were 4 formulations with nanoparticle sizes (PSA) of less than 600 nanometers. Then the 4 formulas were characterized by SEM where the surface morphology of the encapsulated of RUT was obtained in porous form and DSC and XRD characterization showed the presence of particle crystallization.

The purpose of this study is to ascertain whether adding the suspending agent EL100 and stabilizing it with a mixture of P188 and P407 alters the properties of RUT. This is something new from research related to drug delivery systems because several existing studies use RUT as the main ingredient. which are added with suspending agents and stabilizers, generally in the form of RUT with EL100 or RUT with P188 or RUT with P407. There has never been a combination of RUT with EL100 and P188. The prepared RUT encapsulation is then solidified using the freeze drying (FD) process. The choice of FD Solidification was considered because the FD process provides protection for RUT related to the drying process⁴¹. This is similar to research conducted by Bartos⁴². which states that FD solidification is often used in the pharmaceutical.

RENPs provides advantages in improving the solubility properties of RUT and its bioavailability in the body. It is expected that with the latest formulation of the combination of EL100, P188 and P 4087 can contribute to the discovery of Drug Delivery systems for drugs with low solubility. EL100 is a carrier that has the ability to dissolve in a higher pH. The combination of P188:407 contributes to the hydrophilicity of the active ingredient.

The solid state changes were due to the encapsulation of RUT in E L100 stabilized with a combination of P188:407. The presence of poloxamer at the outer layer of the particles affected its hydrophilicity. The inner layer, was the EL100 that encapsulated RUT where it can dissolve according to the intestine pH 6.8. The dissolved EL100 at pH 6.8 and the changing in the crystal lattice might have increased the dissolution profile of the RUT in the developed RENP formulations by 1.5 times as compared to the raw RUT

CONCLUSION:

From the research conducted, it can be concluded that RUT was formulated in enteric nanoparticle delivery system for aspects of their physical characteristics as particle size, surface morphology, crystal and structure matric of RUT can be designed and engineered using the EL100 enteric particle formulation combined with P188 and P407 and its development into enteric nanoparticle delivery system is promising to overcome the solubility and stability issues.

CONFLICT OF INTEREST:

Regarding this inquiry, the writers have no conflicts of interest.

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Received on 16.06.2025 Revised on 06.10.2025

Accepted on 11.12.2025 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3127-3134.

DOI: 10.52711/0974-360X.2026.00444

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