New method of identifying morphine in urine samples using nanoparticle-dendrimer-enzyme hybrid system

2.1 Materials

TEOS and ethylene diamine were purchased from Merck Co., while β-glucosidase enzyme and lysozyme enzyme were sourced from Sigma Co. Other solvents such as methanol, deionized water, etc., were all provided by Merck Co.

2.2 Synthesis of PAMAM G2

2.3 Replacement of β-glucuronidase in PAMAM dendrimer G2 with lysozyme enzyme

1 mL of Tris buffer (30 mmol L⁻¹, pH = 7) was added to the 10 mg of lysozyme and 0.8 mg of β-glucuronidase. In this case, the concentration of lysozyme amounted to 10 mg mL⁻¹ and the concentration of β-glucosidase was 0.8 mg mL⁻¹. Adding lysozyme improved the functioning of the β-glucuronidase enzyme, but adding β-glucuronidase enzyme caused the MOR- β-glucuronidase enzyme bond to weaken (Song et al., 2014). To the above solution was added 2.5 mg of PAMAM dendrimer G2, which was synthesized in the previous stages, so that the concentration of dendrimer was 20 × 10⁻³. Following this, 7 mL of a pre-hydrolyzed solution of TEOS (diluted 40 times with deionized water) was added to this solution. Then it was incubated at room temperature for 24 h to obtain a white precipitate, and subsequently the solution was centrifuged at 3000 rpm for 5 min. The resulting precipitate was filtered off with a vacuum pump and washed three times with deionized water. Following this it was dried at room temperature.

2.4 Preparation of Tris for enzymatic environment

This phase was marked by dissolving 0.36 g of Tris salt in 50 mL of deionized water and the pH was adjusted to 7 with HCl (1 M). Then the solution was diluted to 100 mL.

2.5 Preparing a urine sample containing MOR

A urine sample was taken from a 38-year-old man with a history of daily consumption of opium syrup (1% opium or opium syrup, an herbal mixture derived from morphine and soluble in water and alcohol). This syrup is sold by the Faran Shimi Pharmaceutical Company and this substance has a concentration of 10 mg in a 250 mL bottle. It is used daily to reduce drug dependence and the prescribed dosage is 0.6 mL or 1 mL four times a day. The presence of MOR in this sample was confirmed by Thin layer chromatography (TLC). After examining the MOR urine strip, the urine sample was acid hydrolyzed and then extracted by the liquid-liquid extraction (LLE) method with the help of CHCl₃ solvent: isopropanol (4: 1). After drying the organic phase with the help of a water bath, TLC was placed on a MN brand glass plate next to the opium standard and then the plate was washed with aqueous solvent. It was dried with the help of an acid iodoplatinate solution, spray plate and in this way the presence of morphine was confirmed. The urine sample was stored in a refrigerator at −4 °C until required for testing. For the experiment, a morphine-containing urine sample was diluted one thousand times with deionized water and the UV–vis uptake of free morphine was observed at 210 nm.

2.6 Release of β-glucuronidase enzyme from PAMAM dendrimer G2 to break the MOR - glucuronidase bond in a real urine sample

2 mL diluted urine sample was added to the 2 mg of enzyme trapped in dendrimer PAMAM dendrimer G2 generation. After dissolving, the sample was incubated for 24 h at 37 °C. UV–vis absorption of free MOR was observed at 210 nm.

2.7 Replacement of β-glucuronidase enzyme in the structure of PAMAM dendrimer G2

To the Tris buffer (1 mL) the following were added: HCl (pH 7, 30 mM L⁻¹), lysosomes enzyme (10 mg) and 0.8 mg β-glucuronidase enzyme. After that, 7 mL of pre-hydrolyzed TEOS solution (40 times diluted with distilled water) was added. The mixture was incubated at room temperature until a white precipitate was formed. From the start of the reaction and for 4 h afterwards, a sample was taken from the reaction vessel every hour with a sampler. 100 μL of the sample was diluted 1:1 with Bradford solution and its absorbance was recorded at 595 nm.

2.8 Replacement of MOR in the structure of PAMAM dendrimer G2

Urine samples contained free MOR in PAMAM dendrimer G2. MOR was placed in dendrimer cavities at room temperature. Every 2 h, a sample was taken from the solution in the container with a sampler and its absorption was measured at 210 nm. Schematic 1 illustrates the preparation of PAMAM dendrimer for trapping MOR by enzyme.

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Scheme 1

Schematic of synthesis of PAMAM dendrimer for trapping MOR by enzyme.

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Scheme 2

General procedure of synthesis of dendrimer for trapping MOR by β-glucuronidase.

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2.9 Release of β-glucuronidase enzyme in the structure of PAMAM dendrimer G2

This study was conducted on a real urine sample containing MOR diluted with distilled water at a ratio of 1/1000. 2 mL of the sample and 2 mg of β-glucuronidase enzyme in the structure of PAMAM dendrimer G2 were added. Due to the complex and crowded matrix of urine, MOR peaks are not visible in real urine samples (UV–vis spectra). MOR absorption was observed when the urine was diluted 1/1000. There was no MOR in the water sample and therefore no absorption was evident. The measurement was done using a spectrophotometer with very high detection power, which was calibrated before it was put into operation. It has the advantage of being able to read low absorption values. The sample was incubated at 37 °C. From 0 to 4 h after the reaction every hour, a sample was taken from the reaction vessel with a sampler. 100 μL of the sample was diluted to a ratio of 1:1 with Bradford solution and its absorbance was recorded at 595 nm. This study was conducted on the urine sample of a 38-year-old man with a long history of heroin use but no history of any underlying disease. This person did have a history of quitting and restarting heroin use several times, and has for one month been using opium syrup. The daily dose is 2.5 mL.

3.1 Bradford's method for determining the amount of protein in a solution

Tris buffer is commonly used to extract proteins (Serafín et al., 2021). However, even a 0.1% sample has a significant effect on UV–vis absorbance compared to control samples (Rae and Jachimska, 2021). In this study, we demonstrated a new method for identifying morphine in urine samples using a nanoparticle-dendrimer-enzyme hybrid system for urine drop applications. Quantitative measurement of total protein value is of great importance in basic sciences and clinical research. Bradford's protein measurement is a colorimetric method, one which is used to measure the concentration of protein in solution. Bradford's law has an index for the values ​​obtained by the variables. Important features are related to size and frequency and one recent approach chosen described the shape of Bradford-type curves (Rae and Jachimska, 2021). The Bradford assay is predominantly used in measuring clinical samples (Rae and Jachimska, 2021). Bradford's approach is based on the formation of a complex between the G-250 blue and proteins in the solution. During this phase there is a shift in maximum absorption from 465 nm to 595 nm. The formation of a complex between color and protein in an acidic solution leads to a change in color from red to blue.

Fig. 1A depicts the UV–vis of the standard of MOR and the urine containing free MOR. Fig. 1 depicts a decrease as shown in the UV–vis absorption diagram. UV–vis of Fig. 1A-E shows the standard of MOR, urine containing free MOR, urine with β-glucuronidase enzyme trapped in PAMAM dendrimer G2 (incubated for 24 h at 37 °C), urine consisting of β-glucuronidase enzyme trapped in PAMAM dendrimer G2 (incubated for 24 h at 24 °C), and urine that contains β-glucuronidase enzyme trapped in PAMAM dendrimer G2 (incubated for 48 h at 37 °C). Fig. 1B-E highlights a reduction in the UV–vis absorption, which suggests the replacement of MOR by the β-glucuronidase enzyme in the dendrimer cavities. Similar UV–vis spectra show that changes are observed for all the other systems. The peak of MOR at 210 nm remains unaltered while the reaction MOR with β-glucuronidase enzyme causes absorption intensity to decline at 210 nm, while a new peak at 220 nm appears (Fig. 1C–E). This peak is attributed to replacement of MOR by the β-glucuronidase enzyme (Esmaeili and Mousavi, 2017). In the 250 nm region, the connection between the donor groups and MOR reduces the peak. So over a long period of time this connection improves and the peak of this area is lost.

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Fig. 1

UV–vis of (A) standard of MOR; (B) Urine containing free MOR; (C) Urine with β-glucuronidase enzyme trapped in PAMAM dendrimer G2 (incubated for 24 hr at 37 °C); (D) Urine consisting of β-glucuronidase enzyme trapped in PAMAM dendrimer G2 (incubated for 24 hr at 24 °C); (E) Urine that contains β-glucuronidase enzyme trapped in PAMAM dendrimer G2 (incubated for 48 hr at 37 °C).

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The measured absorption rate at 595 nm is proportional to the concentration of protein in the solution. The absorption rate in Bradford measurement varies depending on the individual reading to another. Bradford's method served to determine the amount of protein in the sample. As the standard concentration increases, the amount of absorbed by the microliter also increases at the same time.

The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using Eqs. (1) and (2) which are written below:

Herein y_B and s_B denote the blank signal and standard deviations of the blank signal, respectively (Miller and Miller, 2018).

The LOD and LOQ values obtained were 0.027 and 0.073 mg mL⁻¹, respectively.

The BSA calibration curve is shown in Table 1 (A-C) based on Table 2 data. The data are fed by linear regression whose line equation is y = 0.0277x + 0.056 and the value of R² for the data is 0.8194. Y is equal to adsorption and x is the amount of protein in mg mL⁻¹. From this equation and in a particular adsorption, the amount of protein can be calculated. The absorption values were recorded at 595 nm by spectrophotometer, and these values were, respectively, 0.059, 0.070, 0.065, 0.084, 0.089 and 0.09 μg mL⁻¹. The values ​​of protein concentration calculated using the curve line equation and the placement of these values ​​instead of y were 0.108, 0.505, 0.394, 1.0108, 1.191 and 1.227 mg mL⁻¹, respectively. Table 2 and Fig. 1 indicate that the average protein concentration employing this data amounts to 0.739 μg mL⁻¹. Fig. 1 for MOR shows a maximum absorption of 289.0 nm (Amand and Esmaeili, 2020). The loaded nanoparticle has three peaks at 474, 333 and 235 nm (Esmaeili and Ghobadianpour, 2016). My own work makes a comparison to the source material.

3.2 Investigation of urinary pH and MOR concentration as a replacement of dendrimer cavities using SPSS analysis

Statistical analysis utilizing SPSS software was applied to determine the concentration of MOR and urine pH and their effects on the replacement of β-glucuronidase enzyme from PAMAM dendrimer cavities. The output data for the pH were obtained at 24 h according to MOR uptake and concentration. Zero assumption here is that pH changes in terms of adsorption affecting the MOR concentration. The dependent variable changes have been explained by the independent variables.

Table 3 shows the pH and concentration of MOR as established by ANOVA variance statistical analysis. The high F-value (2.423) in ANOVA data indicates that the deviation from the mean data occurred unexpectedly. In addition, the high value of the degree of significance (Sig.) actually reveals the value of p for the data. pH did change in terms of adsorption but does not have a significant effect on MOR concentration (rejection of the null hypothesis. High values ​​of t are confirmed numerically in Table 4. The low standardized error rate confirms the accuracy of the data obtained. The hypothesis (null Hypothesis) for these calculations is that the MOR concentration does not diminish within 24 h. The value of the R² was obtained 0.913 while the adjusted R² value was 0.899 for these data. The values ​​obtained confirm that the regression line was able to describe the data at 91.3%. The standard error value of 0.05885 is a good indication of the accuracy of the data that was generated. ANOVA data are summarized in Table 3. A high value of F = 52.419 represents that deviation from average data occurred unexpectedly. Data is more varied than average and furthermore a very small value of less than 0.05 for the degree of significance (Sig.) is the p-value for the data. It rejects the zero hypothesis so the existing model is reliable with 0.95% confidence and can describe the data. The coefficients (Table 4) confirm this fact with high values ​​of t and a low level of importance. The low standardized error rate (Std. Error) confirms the accuracy of the data obtained. Unlike a previous study conducted in a pH environment, this present one comprised 3 environments and the results were different (Sakr et al., 2011).

3.3 Fourier transform-infrared spectroscopy (FT-IR) spectra investigation of in PAMAM dendrimer G2

Michael addition is in organic chemistry one of the most widely used reactions to carbon-nitrogen bonding. Complete generations of PAMAM dendrimer G2 contain large amounts of amine 1⁰ in the surface and amine 2⁰ in the nucleus. The presence of these potent nucleophilic amines in the dendrimer can react with α-β unsaturated acrylate groups. Dendrimers can accurately control the size, shape, and placement of groups and they are suitable for many life sciences applications. Esmaeili and Mousavi (Esmaeili and Mousavi, 2017) have employed dendrimers to make the oral drug insulin, which is a silica nanoparticle coated with polymer amino acid dendrimer and replaced by chitosan medicine inside its cavities. FT-IR (Thermo Scientific Nicolet 8700, USA) data were consistent with the figures presented in this article. Fig. 2 and the spectrum of the FT-IR illustrate the depleted PAMAM dendrimer G2 of the synthesized. FT-IR spectra regarding PAMAM (Fig. 2A-a) dendrimer G0.5 (Fig. 2A-b), dendrimer G1 (Fig. 2A-c), dendrimer G1.5 (Fig. 2A-d) and dendrimer G2 are shown. As can be seen in Fig. 2A, the absorption at 1580 cm⁻¹ is related to C⚌O (Esmaeili and Khodaei, 2018; Khodaei and Esmaeili, 2019).

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Fig. 2

(A) FT-IR spectrum of PAMAM (a) dendrimer G0.5; (b) dendrimer G1; (c) dendrimer G1.5; (d) dendrimer G2; (B) SEM for β-glucuronidase enzyme trapped in PAMAM dendrimer G2.

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The absorption peak at 1610 cm⁻¹ in the FT-IR spectra of G_0.5 and G₂ shows ester terminated PAMAM dendrimers affirm the presence of —CO₂CH₃ groups. The absorption at 550 and 7500 cm⁻¹ are assigned to the Si—O and O—Si—O vibration in the silica network, respectively. The observed peaks at 1100–1250 cm⁻¹ show that Si—O—Si create the dominant peak. Stretching absorption for OH confirms the existence of the silanol and silanol water groups > 3000 cm⁻¹ (Fig. 2A-a). Vibrations related to the OH group of water and silanol were observed at 3300 cm⁻¹. Si—O absorption is in the 900 cm ⁻¹ range (Esmaeili and Mousavi, 2017). The absorption at 630 cm⁻¹ is related to CH₂ and CH₃ bending vibration. The peak of 1220–1345 cm⁻¹ is attributed to the C—N stretching vibration (Fig. 2A-b) while the bending of CH₂ (vibration sweep) is related to 656 cm⁻¹. C—N bonding is in the area of 1020 cm⁻¹. The peaks at 1200 and 1230 cm⁻¹ represent the stretching vibration of C—O (Fig. 2A-c). The bending vibration at 1442 cm⁻¹ is linked to NH bending and the peak at 1545 cm⁻¹ shows the C⚌O. The amide at 1600 cm⁻¹ is related to C⚌O stretching vibration of ester in the region of 1610 cm⁻¹ and has a strong absorption (Esmaeili and Rafiee, 2015) (Fig. 2A-d). Zhang et al. (Zhang et al., 2019) reported the absorption peak at 1655 cm⁻¹ for the amide group.

The recent peak confirmed the existence of the amide group and what the literature says about it. Other factor groups were consistent with the factor groups reported in this paper. The peak that appears at 720 cm⁻¹ is related to CH₂ bending vibration sweep; the band in the range 1000–1350 cm⁻¹ is linked to C—N stretching vibration. The strip of C—O stretching vibration can be seen in the 1000–1300 cm⁻¹ range. Also evident were the swing bending vibration of warped CH₂ band at 1257 cm⁻¹, the CH₂ scissors vibration at 1400 cm⁻¹, and C⚌O stretching vibration of the ester group with a strong peak at 1735 cm⁻¹. Moreover, symmetrical and asymmetrical CH₂ stretching vibration peak can be seen at 2831 and 2954. These peaks are characteristic of the half generation polyamide amine (Esmaeili and Khoshnevisan, 2016). The absorption at 1330 cm⁻¹ is related to the third type of amine. The absorptions at 3363 and 3281 cm⁻¹ correspond to NH₂ while the peaks at 1200 and 1161 cm⁻¹ are related to the vibration of the first and third types of amine traction, respectively.

3.4 Scanning electron microscopy (SEM)

Establishing the morphology and size of nanoparticles containing β-glucuronidase enzyme from PAMAM dendrimer G2 was studied using SEM (model TESCAN, VEGA/XMU, Czech Republic). A drop of dendrimer-containing solution containing the synthesized enzyme was placed on a laboratory slide and dried. In vacuum conditions, the slide was coated with a layer of gold for further clarity and surface electron excretion and analyzed at a voltage of 20 kV. Fig. 2B shows the morphology of the SEM for β-glucuronidase enzyme trapped in PAMAM dendrimer G2. Miller et al. (Miller et al., 2006) explained that the amount of silica particles is directly related to the amount of enzyme. The results of this study show that the electrostatics of an enzyme silica complex and dendrimer have a wide range of chemical and biological activities to composite materials. Esmaeili and Khodaei (Esmaeili and Khodaei, 2018) investigated enzyme targeted magnetism for rifampin simultaneously. They showed that nanoparticles increased the amount of enzymes in the drug.

According to the histological study, drug injection can reduce damage done to the liver. Most NPs with a spherical morphology were dispersed on porous surfaces which is a benefit of higher absorption. The average nanoparticle size of MCM-41 for single dispersed particles is <100 nm. According to SEM information, the nanoparticles were spherical in shape but their surfaces were porous due to the presence of PAMAM dendrimer. In a study done on PAMAM dendrimers, the surface contained SEM micrograph enclosures within the structure of the PAMAM dendrimer (Cavalcanti et al., 2011). This supported our findings. Amand and Esmaeili (Amand and Esmaeili, 2020) analyzed the properties of NPs made from hybrid polymer containing anticoagulants. The morphology (average size of the spheres created) shows that the NCs produced were between 136 and 183 nm in diameter, the average being 176 nm. A comparison of current results with other research demonstrates that a plant extract is added to the NPs system and is not used in particles to recreate the force. Nugraheni et al. (2016) investigated PVA/CS/TiO₂ NPs using the electrospinning method with a diameter of 112 nm.

3.5 Bradford protein assay

Protein quality was improved by adding 200 µL from Nadaford solution (1:4 diluted with ionized water) to 20 µL of dendrimer polyamide amine sample, which added to β-glucuronidase enzyme. The sample was subjected to room temperature for 10 min. The standard curve for different enzyme concentrations existed in the 0.2–1 µg mL⁻¹ range. Enzymatic activity was initially observed in the prepared solution while the solution obtained from washing the dendrimer containing the enzyme was not observed. After the reaction lasting 24 h at 37 °C, the enzyme concentration of 0.0626 µg mL⁻¹ was measured (Amand and Esmaeili, 2020).

3.6 Investigation of MOR replacement in PAMAM dendrimer G2

After diluting the actual urine sample by $\frac{1}{1000}$ with ionized water, the UV–vis of the MOR uptake spectrum was obtained in the range of 200–380 nm. After the reaction and release of the enzyme from the dendrimer cavities in the urine sample, UV–vis spectra MOR was taken again. According to UV–vis spectra, MOR uptake increased at this stage and the urine sample was incubated at room temperature for 24 h. Following the replacement of free MOR within the dendrimer cavities of PAMAM dendrimer G2, a reduction was noticeable and finally, after 48 h of incubation at ambient temperature, the absorption of MOR reached zero.

Quality sample was reported to be good, S_d was suitable and a comparison-based recent study indicated high selectivity for the process. Ward et al. (Ward et al., 2013) evaluated the efficacy of the MOR formula with PAMAM dendrimer in vitro and in vivo. The release of MOR can play a useful part in a pharmaceutical strategy as part of the therapeutic process.

3.7 Investigating the effect of urine pH

Since the urine sample employed in this study had a normal pH = 6, it was adjusted using ammonia and diluted HCl in areas 4, 5, 7, and 8 to investigate its effect. The samples were then diluted to $\frac{1}{1000}$ using deionized water. The preparation steps were completed according to the previous procedure and finally the protein concentration in urine samples containing β-glucuronidase enzyme trapped in PAMAM dendrimer G2 was measured using the Bradford method. As shown in Table 4, pH = 7 is the best pH for releasing MOR from the urine sample.

3.8 Optimizing the method and statistical study of the loaded enzyme using response surface methodology (RSM)

Optimization and a statistical study of the loaded enzyme using the RSM method is described here. The importance and impact of parallel parameters were evaluated. The accuracy of the experimental model and ANOVA results for the experimental parameters according to the Surface Methodology (RSM) model are documented in Table 1A. The high p-value in comparison with the F-value indicate the high response rate of the studied model. The higher the R² value then the better the correlation model performed. The value of R² obtained from this system is 0.8328. An acceptable result is a statistical response. According to ANOVA calculations and the model used in this quadratic test, the amount of P value is less than 0.05, which is very important for sample regression. The value of the correlation coefficient (R²) is 0.8328. The response level method of the quadratic equation shows well the relationships between the variables. The adjusted coefficient of determination (Adjusted R²) was 0.7175 and the regression line describes the data relatively well. The effective experimental variables are included in β-glucuronidase-enzyme (X₁), MOR (X₂), time (X₃), temperature (X₄), and pH (X₅). After the experiments, ANOVA analysis was performed using quadratic and polynomial (Y) response model with Eqs. (1) and (2), respectively.

Eqs. (1) and (2) show the relationship between the variables defined in Table 1B. The Eq. (1) can be evaluated for the response for certain levels concerning each factor. By default, the low and high levels of factors are (+1 and −1). Comparing the coefficient of factors is useful for reorganizing the impact these factors make. ANOVA method for 3D surface is the statistical process required to measure MOR in this project (Fig. 3A). Furthermore, the ANOVA method helped to investigate the coefficient of different models, and the relationship between MOR and temperature as illustrated in Fig. 3B. Also, the relationship of MOR with time is presented in Fig. 3C. It shows a diminishment in time compared to temperature when all other factors are constant. The coefficient of RSM is expected to change per unit according to each factor’s value.

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Fig. 3

(A) Based distribution of RSM trapped β-glucuronidase enzyme trapped in PAMAM dendrimer G2; (B) ANOVA comparison of different standard concentrations of MOR with temperature changes in real urine samples; (C) ANOVA comparison of different standard concentrations of MOR with times changes in real urine samples; (D) BRAD FORD trapped β-glucuronidase enzyme trapped in PAMAM dendrimer G2; (E) Diagram BRAD FORD of loading trapped β-glucuronidase enzyme trapped in PAMAM dendrimer G2; (F) Diagram BRAD FORD of relaese trapped β-glucuronidase enzyme trapped in PAMAM dendrimer G2; (G) Diagram of UV–Vis uptake of free MOR in real urine samples after exposure to the released β-glucuronidase enzyme;

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The coefficients are adjustments around that average based on the factor settings. When the factors are orthogonal the VIFs are (1); VIFs greater than (1) indicate multicollinearity. Hence, with higher VIF the correlation of factors will be more severe. As a general rule of thumb, VIFs less than 10 are tolerable. In this study, a change was noted in the percentage of polymers. In addition, the nanoparticles were exposed to nitrogen plasma under varying conditions of power and time. The most appropriate concentration of polymers for nanoparticles was obtained from 6% solution of polymers in formic acid. With reference to the p-value, a higher F-value means that the response level model is highly significant (Esmaeili and Singh, 2017). Positive values suggest a positive effect on enzyme release while negative values ​​indicate the opposite effect on the enzyme release process.

The experiment design was based on the finding of ideal conditions and the results of the experimental variables included time, amount of nanoparticles and concentration of the drug (Tadayon et al., 2015). These equations prove there is a direct relationship between plasma and scaffold conditions. The experiment set out to discover the best conditions while the results of the experimental variables included time, amount of nanoparticles and concentration of vancomycin (Tadayon et al., 2016).

3.9 Loading of MOR in PAMAM dendrimer G2

The standard MOR sulfate powder is used in aqueous base. The solutions contained free MOR at 100, 200, 300, 400, 500, and 600 µg L⁻¹ and then the solutions were diluted to $\frac{1}{1000}$ . The above solutions of β-glucuronidase enzyme trapped in PAMAM dendrimer G2 were added. Fig. 3D shows Bradford trapped β-glucuronidase enzyme in PAMAM dendrimer G2. Afterward, the sample was incubated for 24 h at 37 °C and then incubated for 48 h at room temperature. Fig. 3E indicates the synthesis and replacement of β-glucuronidase enzyme trapped in PAMAM dendrimer G2 cavities. According to the Bradford method, it is observed that over time the amount of β-glucuronidase enzyme in the environment shrinks and replaced in PAMAM dendrimer G2 cavities. MOR uptake was measured with a conventional spectrophotometer, while the Bradford method serves to measure enzyme concentrations at 595 nm. Wei et al. (Wei et al., 2021) explained that the enzyme was replaced in the dendrimer cavities containing silica particles. In the current study, free morphine was replaced by β-glucuronidase enzyme in the PAMAM dendrimer G2 cavities containing silica particles. After 48 h, the concentration of free morphine in the real urinary tract shrank. Montalbo R, Marquez (Montalbo and Marquez, 2017) used the UV–vis test which compared white cell viability. 79% of the drug was released in the first 12 h.

3.10 Release and replacement of β-glucuronidase enzyme from PAMAM dendrimer G2

Fig. 3F and 3G show the replacement and release of β-glucuronidase enzyme and MOR investigated for PAMAM dendrimer G2. In Fig. 3F, the real urine sample contains MOR and diluted at a ratio of 1/1000. Over time, the β-glucuronidase enzyme escapes from the PAMAM dendrimer G2 cavities and breaks the MOR- β-glucuronidase enzyme bond in real urine. As the β-glucuronidase enzyme is released from the PAMAM dendrimer G2 cavities (Fig. 3F), the concentration of the β-glucuronidase enzyme in the real urine environment increases over time. The UV–vis absorption of free MOR after effect of the β-glucuronidase enzyme is shown in Fig. 3G. So, it breaks the MOR- β-glucuronidase enzyme bond. Free uptake of MOR wanes over time and this indicates the replacement of MOR in the PAMAM dendrimer G2 cavities.

The Bradford method of the supernatant in the first and second cases shows that its enzymatic activity diminishes over time. The enzyme in solution is replaced over time in the dendrimer cavities. The second stage was determined after replacing the enzyme inside the polyamide dendrimer cavities and the PAMAM dendrimer G2 in real and diluted urine samples containing MOR, under the influence of 37 °C. The enzyme was released from the dendrimer cavities over time and in this way, it entered the urine system. In the final stage, after the effect of the enzyme released from the dendrimer cavities, the MOR- β-glucuronidase enzyme bond was broken.

Subsequently, the free MOR concentration increased and the released MOR was then replaced inside the dendrimer cavities. The decline in UV–vis absorption of free MOR in the solution confirms this scenario. Esmaeili and Mousavi (Esmaeili and Mousavi, 2017) explained that the best release rate is closely linked to pH 7.4. In other studies, the maximum amount of drug release is 300 min after the start of the release process but drug release of the synthesized structure in our research is more controlled. This is due to loading the drug in a multiple layer structure that prevents rapid degradation (Sahu, 2013). In other research the maximum amount of the drug release is 300 min after the release process commences but drug release of synthesis structure in this study is more controlled. This is due to loading the drug in the multiple layer structure which prevents the rapid degradation (Sahu, 2013).