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Article Cite This: ACS Omega 2019, 4, 555−562
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Optimization of Rutin-Loaded PLGA Nanoparticles Synthesized by Single-Emulsion Solvent Evaporation Method Kadriye Kızılbey*
ACS Omega 2019.4:555-562. Downloaded from pubs.acs.org by 146.185.203.163 on 01/10/19. For personal use only.
̇ ̇ Biomedical Engineering Department, Faculty of Engineering and Architecture, Istanbul Yeni Yüzyıl University, 34010 Istanbul, Turkey ABSTRACT: Bioactive rutin molecule has high-volume applications in food and pharmaceutical products. A considerable problem is its poor water solubility and low bioavailability. In this study, propylene glycol was first used as in the literature to dissolve rutin molecule while entrapping it into poly(D,L-lactide-co-glycolide) nanoparticles by oil-inwater single-emulsion solvent evaporation method. The reason of using propylene glycol is to improve rutins’ low bioavailibility and to determine the optimized nanoparticles. For this reason, various encapsulation parameters were tested and their effects were analyzed. Then, NP4 (hydrodynamic particle size: 252.6 ± 2.854 nm)-optimized nanoparticle with 47% reaction yield and 81% encapsulation efficiency was determined. In vitro % cumulative releases of rutin from optimum NP4 in two different concentrations (0.5 and 1 mg/mL) were examined. NP4 with 0.5 mg/mL concentration reached 100% release on the 5th day (120 h). Optimum rutin-loaded nanoparticles are expected to be a suitable candidate for further multidisciplinary studies. structure whether it is glycoside or aglycone.9 Rutin was absorbed very slowly10 at the terminal ileum or even the large intestine.11 It has cardioprotective, anti-inflammatory, cholesterol-lowering, anticancer, and antioxidant activities.12 It is a major bioactive ingredient with potential applications in pharmaceutical products. But the considerable problem of the molecule is its poor water solubility13 that heavily limits its applications. Solving the solubility problem of rutin improves the usage of it.12 Solubility and gastrointestinal permeability are significant factors for the bioavailability and absorption of drugs.14,15 Nanotechnology provides some benefits to the drug delivery systems at this point. Nanosystems and nanomaterials are general terms assigned to any presence having sizes ranging in the nanometric scale with different properties, such as nanoemulsions, nanoparticles (NPs), polyplexes, dendritic structures, micelles, and liposomes, among others.16 These systems improve the delivery of poorly water-soluble drugs and macromolecules, the targeting of drugs, and the transition of molecules through the intestinal barrier.17 Polymeric micelles first came out as potential carriers in 1984,18 and after recognition of the importance of particle size in drug delivery, polymeric micelles were applied in nanotechnology.15 Due to their advantages, such as (a) ability to reach to any human organ because of their nanometric size, (b) protection of drug molecules from enzymatic degradation, (c) controlled and sustained drug release, and (d) preparation using easily scalable and cost effective methodologies, polymeric nanoparticles are a promising alternative in drug
1. INTRODUCTION Flavonoids are a large group of natural compounds. These variable phenolic structures that are found in plants are anthocyanidins, flavan-3-ols, flavonols, flavanones, flavones, and isoflavones.1 They are identified as pigments2 and are found in fruits, vegetables, grains, barks, roots, stems, and flowers,3 which recently has been the subject of considerable scientific and therapeutic interest.4 The flavonoids have an important role in successful medical treatments3 from past times to present.4 Many flavonoids have free radical-scavenging capacity, antioxidative activity,2 anti-inflammatory effects, antitumor effects, antithrombogenic effects, antibacterial activity,5 and antiviral activity.1 Rutin (Figure 1), also known as rutoside, quercetin-3-Orutinoside, and sophorin,6 is a glucoside flavanoid found in red wine, buckwheat, red pepper, and tomato.7 Rutin needs no prescription as a dietary supplement.6 The activities of flavonoids depend on their molecular size, configuration, and solubility.8 The absorption of flavonoid depends on the
Received: October 11, 2018 Accepted: December 25, 2018 Published: January 8, 2019
Figure 1. Chemical structure of a rutin molecule. © 2019 American Chemical Society
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delivery systems.19 Polymeric nanoparticles can be obtained from nanoemulsions either by monomer polymerization or using a polymer as the dispersed phase of the nanoemulsion followed by solvent evaporation. The use of biocompatible preformed polymers is superior to monomer polymerization because the presence of reactive substances like initiators or byproducts is avoided, thus improving the biocompatibility of the system and reducing purification steps. Both natural polymers (collagen, chitin, chitosan, keratin, silk and elastin, starch, cellulose, pectin etc.)20 and synthetic polymers (poly(lactic acid), poly(glycolic acid), poly(lactic acid-coglycolic acid), and poly(ε-caprolactone) etc.)21 are used as biomaterials. Among preformed polymers, poly(lactic-coglycolic acid) (PLGA) is beneficial due to its features.22 It has been the most appealing copolymer used for drug delivery systems,23 nanovaccines, and nanoparticles (NPs).24 It is a biodegradable, biocompatible, nontoxic, and FDA-approved polymer.25 In the literature, emulsification−diffusion,26 solvent emulsion−evaporation,27 interfacial deposition,28 and nanoprecipitation methods have been used to prepare PLGA nanoparticles.24 The purpose of the present study was to synthesize the rutin-loaded PLGA nanoparticles in optimum formulation by oil-in-water (o/w) single-emulsion solvent evaporation method and their detailed characterization. In accordance with this purpose, different amounts of rutin and PLGA, stabilizer concentration in aqueous phase, and oil/aqueous phase ratio were examined to determine the optimum NP formulation. Effects of variables were analyzed by reaction yield (RY), encapsulation efficiency (EE), hydrodynamic particle size, polydispersity index (PDI), and Zeta (ζ)-potential. An optimized NP was determined after evaluation of analysis results, and then in vitro release of rutin from optimum NP was examined.
Table 1. Reaction Parameters in Preparation of RutinLoaded NPs NP no
Rutin amount (mg)
PLGA amount (mg)
PVA concentration w/v (%)
PVA (mL)
10 20 30 40 50 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 0
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 150 250 300 100
3 3 3 3 3 0.1 0.5 1 2 3 5 3 3 3 3 3 3 3 3 3 3
4 4 4 4 4 4 4 4 4 4 4 2 4 6 8 10 4 4 4 4 4
NP1 NP2 NP3a NP4 NP5 NP6 NP7 NP8 NP9 NP3a NP10 NP11 NP3a NP12 NP13 NP14 NP3a NP15 NP16 NP17 NP18 (empty NP) a
Common in all groups.
were stored at −80 °C.29 In this study, a series of experiments with different process parameters (rutin and PLGA amount, PVA concentration, and volume (ratio of aqueous-to-organic phase volume) ratio) were conducted. Their effects on reaction yield (RY), encapsulation efficiency (EE), the mean particle size (Z-ave), PDI, and ζ-potential were examined. 2.3. Characterization of Rutin-Loaded PLGA NPs. 2.3.1. Calculation of Reaction Yield and Encapsulation Efficiency. Reaction yield (RY) was calculated gravimetrically using formula (1) with the ratio of the amount of produced NP (mg) to the sum of rutin (mg) and PLGA amounts (mg).30
2. EXPERIMENTAL SECTION 2.1. Materials. PLGA (lactide/glycolite = 50:50; viscosity: 0.45−0.60 dL/g, Mw ∼ 38−54 kDa), rutin, poly(vinyl alcohol) (PVA), and propylene glycol were purchased from SigmaAldrich (St. Louis), and dichloromethane (DCM) was purchased from Ridel de Haen. All chemicals and solvents are in analytical purity, with no need for extra further purification. Ultrapure water was obtained from Millipore Milli-Q gradient system in the laboratory. In vitro release measurements were performed at 37 °C in phosphate buffered saline (PBS) at 7.4 pH. 2.2. Preparing Rutin-Loaded PLGA NPs. Rutin-loaded PLGA nanoparticles were prepared by modified oil-in-water (o/w) single-emulsion solvent evaporation method.19 Different amounts of PLGA and rutin (Table 1) were dissolved into the organic phases consisting of DCM and propylene glycol, respectively; then, they were mixed. These organic solutions were emulsified with aqueous solution of PVA with diverse concentrations and volumes (Table 1) by sonication (output power of 70 W, power of 80%, and 2 min) using a microtip probe sonicator (Bandelin Sonopuls, Germany) over an ice bath. The free nanoparticles were prepared by the same method without rutin. They all were stirred overnight on a magnetic stirrer at room temperature for evaporation of organic phase. After centrifugation at 9.000 rpm (4 °C) for 40 min (Hettich-Universal 32 R), collapsed particles were collected, washed three times with ultrapure water to remove excess PVA, and then lyophilized. All lyophilized nanoparticles
RY% =
produced NP amount (mg) × 100 rutin amount (mg) + PLGA amount (mg) (1)
Encapsulation efficiency (EE) was calculated with the ratio of the encapsulated rutin amount (mg) to the total rutin amount (mg) by using formula (2).30 EE% =
total rutin amount (mg) − free rutin amount (mg) total rutin amount (mg) × 100
(2)
The total amount of rutin (mg) is the rutin amount used to prepare the nanoparticle. The free rutin amount was determined using the supernatant obtained after nanoparticle production. A series of standard rutin solutions of known concentrations was prepared, and a standard rutin UV calibration curve was drawn from the absorbance values at 354 nm.31 The amounts of rutin in the upper phases were determined after centrifugation by using that calibration curve. 2.3.2. Dynamic Light-Scattering Hydrodynamic Particle Size and Zeta (ζ) Potential Analysis. Hydrodynamic particle sizes (Z-ave), polydispersity indexes (PDI), and ζ-potentials of 556
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Figure 2. Effects of parameters on RY% and EE%: (A) rutin amount (mg), (B) PVA concentration w/v (%), (C) PVA volume (mL), and (D) PLGA amount (mg).
attenuated total reflectance mode. The FTIR spectrum was performed with 16 scans at a resolution of 4 cm−1 at the wavelength range of 4000−750 cm−1 per sample. 2.3.4. Scanning Electron Microscopy (SEM) Analysis. Surface morphology of rutin-loaded nanoparticles was visualized using a scanning electron microscope (Zeiss EVO LS 10, Germany). Powder NP samples were affixed to the metal surface with double-sided adhesive tape for this analysis. All samples were gold−palladium (Au−Pd) coated, and they were analyzed at 5 kV under vacuum. 2.3.5. In Vitro Rutin Release Analysis. In vitro release of rutin from optimum nanoparticles was studied by direct dissolution method33 via minor modification at pH 7.4 phosphate buffer solution (PBS).30 This pH value was selected due to the simulation of physiological pH.34 Optimized NPs (2.5 and 5 mg) were added separately to 5 mL of PBS (containing 0.01% sodium azide), and those suspensions were incubated at 37 °C in a shaking incubator (60 rpm). Supernatants were collected by centrifugation at 9000 rpm for 20 min at selected time intervals (1 h, 2 h, 3 h, 1 day, 2 days, 3 days, 4 days, 7 days, 8 days, 9 days, 10 days, 11 days, and 14 days), and the pellets were suspended in 5 mL of fresh PBS. Rutin concentrations in the supernatants were determined by a standard calibration curve drawn using a series of previously known concentrations at a wavelength of 354 nm with UV−vis spectroscopy.
NPs were determined by photon correlation spectroscopy using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.) equipped with mean 4.0 mV He−Ne laser (633 nm).30 Dynamic light scattering (DLS) gives a hydrodynamic diameter based on the diffusion of the particles. The Z-average is a harmonically weighted intensity mean. Fresh NP solutions prepared in ultrapure water at 25 ± 0.1 °C were used for the measurements, and each measurement was repeated three times. For solutions with a dielectric constant of 79, 0.8872 cP viscosity and 1.330 refraction index were used; f(ka) was 1.50 (Smoluchowski value). All samples were filtered with a 0.20 μm regenerated cellulose-membrane filter (Sartorius) before measurements. The hydrodynamic diameter (dH) is calculated from the diffusion coefficient (D) at the Stokes−Einstein equation by using formula (3), k is the Boltzmann constant (1.38 × 10−23 N m/K), T is the absolute temperature (°K) and η is the solvent viscosity.32 dH =
kT 3πηD
(3)
ζ-Potential (ζ) of a sample in a solution is calculated from the Henry equation by using formula (4) from the electrophoretic mobility (UE) (ε is the dielectric constant of the medium, f(ka) is the Henry function, and η is the viscosity of the medium) (Manual, Z.N.S.U., 2003, Malvern Instruments Ltd. Worcestershire).32 UE =
2εζf (ka) 3η
3. RESULTS AND DISCUSSION 3.1. Effects of Parameters on Reaction Yield and Encapsulation Efficiency. Some efficient methods of preparing nanoparticles prevent material loss, improve particle production, and lower production costs. In this study, all formulated NPs achieved a wide reaction yield between 23 and
(4)
2.3.3. Fourier Transform Infrared (FTIR) Spectrometry Analysis. An IR-Prestige 21 FTIR spectrophotometer (Shimadzu, Japan) was used for chemical analysis of functional groups of the nanoparticles. Measurements were performed in 557
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Figure 3. Effects of parameters on hydrodynamic particle size (nm) and PDI: (A) rutin amount (mg), (B) PVA concentration w/v (%), (C) PVA volume (mL), and (D) PLGA amount (mg).
Table 2. RY%, EE%, Z-ave (nm), PDI, and ζ-Potential Values of NPs Formulation NP1 NP2 NP3a NP4 (optimum NP) NP5 NP6 NP7 NP8 NP9 NP3a NP10 NP11 NP3a NP12 NP13 NP14 NP3a NP15 NP16 NP17 NP18 (empty NP)
RY% 23 31 40 47 55 50 47 29 44 40 67 42 40 33 37 41 40 41 42 32
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3 3 5 4 6 5 4 4 3 5 3 2 5 5 4 7 5 7 7 6
ζ-average (nm)
EE% 38 55 54 81 84 80 81 55 75 54 87 78 54 78 71 75 54 83 84 85
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5 7 4 5 8 6 4 6 7 4 6 5 4 3 2 6 4 7 6 4
229.4 214.8 212.4 252.6 935.5 646.1 514.3 338.6 356.8 212.4 570.3 404.1 212.4 348.5 349.3 383.1 212.4 338.1 341.9 326.4 203.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.179 2.921 9.408 2.854 232.6 52.69 96.26 2.055 23.89 9.408 66.13 5.766 9.408 7.695 10.17 20.15 9.408 9.959 2.052 14.05 2.974
PDI 0.236 0.268 0.293 0.209 0.775 0.568 0.450 0.189 0.415 0.293 0.524 0.290 0.293 0.337 0.322 0.365 0.293 0.314 0.259 0.343 0.054
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.011 0.042 0.024 0.008 0.071 0.043 0.124 0.031 0.130 0.024 0.046 0.028 0.024 0.012 0.004 0.018 0.024 0.003 0.006 0.03 0.036
ζ-potential −26.7 −26.1 −27.5 −23.7 −16.9 −25.8 −22.4 −29.8 −25.5 −27.5 −1.80 −32.2 −27.5 −23.9 −24.1 −25.0 −27.5 −24.0 −29.4 −20.2 −6.88
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.513 0.964 0.500 1.36 1.14 1.20 0.872 1.82 0.404 0.500 0.912 1.59 0.500 3.23 1.08 0.361 0.500 0.666 0.781 0.693 0.972
a
Common in all groups.
67%. The results showed that the reaction yield was largely dependent on the process parameters of the reactions. The increase in the amount of rutin and PLGA triggered the increase of reaction efficiency. Conversely, an increase in PVA volume and concentration reduced reaction yield. Figure 2A,D shows the rise in reaction yield according to the amount of rutin from 10 to 50 mg and PLGA from 100 to 300 mg. These results are consistent with the literature.35 Additionally, in parallel with the reaction yield,
it is clearly seen in Figure 2D that the encapsulation efficiency increased by increasing of the amount of polymer and active substance. Increment in the PVA concentration reduced the reaction yield and encapsulation efficiency from 50 to 29 and 80 to 55%, respectively. This decrease occurred when the PVA concentration was between 0.1 and 1%, consistent with the literature. A linear increase was observed when the concentration of PVA rose from 1 to 5%. The graph can be 558
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Figure 4. Graphics for optimized NP4: (A) hyrodynamic particle size−intensity graph, (B) SEM image, (C) FTIR analysis, and (D) in vitro cumulative release (%) (0.5 and 1 mg/mL).
separated into two sides; it can be said that there is a linear decrease between 0.1 and 1% and a linear increase between 1 and 5% PVA, as seen in Figure 2B. Figure 2C shows the effect of PVA volume in the aqueous phase on the reaction yield and encapsulation efficiency. As shown in the figure, there was no steady increase or decrease between 2 and 10 mL. No significant difference was observed in RY and EE between the first and last volume applications. 3.2. Effects of Parameters on Average Particle Size (Zave), PDI, and ζ-Potential. The effects of dissimilar rutin and PLGA amounts, PVA concentration, and volume on mean hydrodynamic particle size, PDI, and ζ-potential of nanoparticles were investigated (Figure 3). Particle size is an important parameter in nanoparticle-based drug delivery systems because it affects the pharmaceutical properties of the drug.36 The homogeneity of the size of the formulation is said to be an indicator of stability.37 The amount of PLGA was fixed at 100 mg in the reactions wherein the amount of the rutin was changed (Figure 3A). There was no significant difference in nanoparticle hydrodynamic size (229.4 ± 4.179 and 252.6 ± 2.854 nm) in the range of 10−40 mg of the rutin molecule added. However, when a starting amount of 50 mg of rutin was added, the particle size increased to 935 ± 232.6 nm with a 0.775 ± 0.071 PDI value. It can be considered that the small-sized nanoparticles formed aggregations and agglomerations. Also, PDI and ζ-potential values did not show significant differences when the rutin amount was between 10 and 40 mg. When the polymer ratios were increased in the study (Figure 3D), the other variables were kept constant. The amount of polymer was gradually increased from 100 mg to
300 mg. According to the increase in polymer concentrations from 150 to 300 mg, their EE% were between 82 ± 5 and 85 ± 4%. While evaluating polymer concentration effect, it was seen from the results that the nanoparticles that had compatible EE % had also similar dimensions ranging between 326.4 ± 14.05 and 341.9 ± 2.052 nm. But unlike others, for NP3, wherein 100 mg of PLGA was used, the EE% (54 ± 4%) was quite low compared to that of the others and also its particle size (212.4 ± 9.408 nm) was lower than that of the others in this group. Similar results were reported previously with polymer composition for hydrophobic compound entrapment by a similar synthesis method and emulsifier.38,39 In reactions where increasing volumes of PVA were used (Figure 3C), the particle sizes increased from 212.4 ± 9.408 to 383.1 ± 20.15 nm in reactions except for the reaction using 2 mL of PVA. PDI and ζ-potential values were also found to be harmonious when examined in Table 2. PDIs of nanoparticles prepared in this sequence were between 0.290 ± 0.028 and 0.365 ± 0.018. ζ-Potentials were between −23.9 ± 3.23 and −32.2 ± 1.59. The amount of stabilizer used also had an effect on the properties of NPs (Figure 3B). If the concentration of the stabilizer is too low, aggregation of the polymer could occur, whereas if too much stabilizer is used, drug incorporation could be reduced as a result of the interaction between the drug and stabilizer.40 In the solvent extraction/ evaporation technique, the concentration of PVA in nanoparticle formation should be normally at least 1%, as mentioned in the literature.41 3.3. Optimization and Detailed Characterization of Rutin-Loaded PLGA NPs. Although single-emulsion method 559
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3.6. Cumulative % in Vitro Release of Rutin from Optimum NP4. Figure 4D demonstrates cumulative % in vitro release of rutin from NP4. In this study, 0.5 and 1 mg/mL concentrations of NP4 were studied. The graphics of the two NP samples showed a similar characteristic release curve but different numerical values in release due to their concentration difference. The NPs were also abruptly released after 24 h in the first place. At immediate releases, the values reached 30 and 55% for 1 and 0.5 mg/mL, respectively, at stage 1. At stage 2, the values of 0.5 mg/mL NP4 and 1 mg/mL NP4 reached 95 and 47%, respectively, with continued breakage of the polymer chain and diffusion of the drug. In the last stages (4− 14 days), the % release stayed constant at these values. NP4 with 0.5 mg/mL concentration reached 100% release on the 5th day (120 h). NP4 with 1 mg/mL concentration reached 50% release on the 5th day (120 h). Consistent with the literature, the cumulative rutin release percentage was more than that of the NP4 solution with lower concentration. Lowconcentration NPs contact with a greater amount of water molecules in PBS,46 take water quickly, and begin to degrade more rapidly.47 On evaluating the obtained result, it can be said that longer periods should be tried for high-concentration nanoparticles. As a result, a major bioactive ingredient, rutin, with potential applications in pharmaceutical products was used in this study. The most important problem of the molecule is its poor water solubility13 that heavily limits its applications. Several strategies, such as complexation, microemulsification, and novel drug delivery systems (nanoparticles, lipid-based vesicles, and micelles) have been recommended for poorly watersoluble materials that are beneficial in terms of health.48 Nanotechnology-based delivery systems from such methods for poorly water-soluble drugs have recently gained great importance. Due to their nanosize and ability to solubilize hydrophobic drugs and achieve targeted delivery, nanoparticles hold promise of obtaining desirable biopharmaceutical and pharmacokinetic properties of drugs and improve their bioavailability.15
is the most commonly used method, it requires a cautious optimization while preparing the NPs with the desired size, narrow PDI, and high encapsulation efficiency.42 Process parameters, such as the amount of encapsulated material, polymer concentration, oil/water ratio, stabilizer concentration, sonication time, and speed, affect the NP characteristics.43 Results indicated that rutin molecules were successfully encapsulated into the PLGA nanoparticles by this method. To optimize as the best nanoparticle with the lowest particle size and the highest entrapment efficiency by this method, rutin-loaded PLGA optimum nanoparticles (NP4) were prepared as follows: 40 mg of rutin and 100 mg of PLGA were dissolved into 0.5 mL of propylene glycol and 1.5 mL of DCM, respectively. The mixed organic phase was emulsified with 4 mL of aqueous PVA solution (3% w/v) by using a microtip probe sonicator (output power 70 W, power of 80%, and 2 min) on ice bath. The organic phase was evaporated by stirring overnight. The particles were collected and lyophilized. After lyophilization, the detailed characterization of all nanoparticles was studied, and then the optimum nanoparticles decided. Reaction yield, encapsulation efficiency, and loading capacity were calculated for all NPs. The selected optimized NPs had 81% encapsulation efficiency, 252.6 ± 2.854 nm particle sizes, and 0.209 ± 0.008 PDI. The hydrodynamic particle size−intensity graph of optimized NP4 is shown in Figure 4A. 3.4. SEM Analysis for Optimum NP4. The surface morphology of the optimized nanoparticle NP4 was determined by SEM. Figure 4B shows uniform distribution of smooth and spherical nanoparticles of NP4. SEM results of rutin-loaded nanoparticles were similar to the size distributions obtained from the DLS results. Unlike DLS analysis (hydrodynamic size in solution form), SEM analysis is applied to the solid form of nanoparticles. This results in small differences in the size measured by both techniques. 3.5. FTIR Analysis for Optimum NP4. Figure 4C shows the FTIR analysis of rutin, NP4, and NP18 (empty PLGA). As known from the literature, PLGA’s typical CO ester bonds and C−O bonds were expected to be seen in the infrared spectrum. Characteristic bands of symmetrical and asymmetric stretches of CH2 and CH3 groups were presented between 2980 and 2850 cm−1. The bands at 2993 and 2989 cm−1 were the C−H stretching of CH2 and the C−H stretching of C− H−, respectively. In the FTIR spectra of PLGA and NP4, the band at 1751 cm−1 corresponds to the strong and narrow stretching of CO of the ester bond and C−O stretching occurs at 1165−1087 cm−1. These bands were considered to be the characteristics of the PLGA molecule.44 In the IR spectrum of the free rutin molecule examined, a strong OH stretching observed at 3600−3200 cm−1 was seen at 3327 cm−1; a weak CH stretching (2950−2850 cm−1) was determined at 2920 and 2974 cm−1; a strong CO ketone band was seen at 1745 cm−1, a weak aromatic CC band (1600−1400 cm−1) was seen at 1595 cm−1, and characteristic peaks were seen between 1200 and 1020 cm−1 45 of strong C− O−C stretching and strong C−OH stretching. When the spectra of free NPs and NP4 were compared, it was seen that specific functional groups on the surface of NP4 have almost the same chemical characteristics as those of the free nanoparticles. The FTIR spectra show that rutin was successfully encapsulated to PLGA nanoparticles without having any physical or chemical interaction.
4. CONCLUSIONS In this study, biodegradable, biocompatible, nontoxic, and FDA-approved copolymer PLGA was used to obtain rutinloaded PLGA NPs by o/w single-emulsion solvent evaporation method. The novelty of the study was using propylene glycol to dissolve rutin molecule. To determine the optimum nanoparticle, four preparation parameters involved in nanoparticle formation were changed. These parameters were rutin amount, PLGA amount, PVA concentration, and volume. After preparation of NPs, determination of reaction yield, encapsulation efficiency, hydrodynamic particle size, poly dispersity index, ζ-potential, and FTIR analysis were performed and effects of the parameters on the analyzed data were discussed. The nanoparticle NP4 with high reaction yield (47%) and encapsulation efficiency (81%), small size (252.6 ± 2.854 nm), and PDI value (0.209 ± 0.008) was determined as optimized NP. SEM images of the optimum nanoparticle, NP4, were taken, and spherical structures were observed. In vitro % rutin release for two different concentrations of NP4 was subsequently investigated. NP4 with low concentration (0.5 mg/mL) successfully had 100% release on the 5th day (120 h). Results show that encapsulation with PLGA could be useful for poorly soluble but beneficial-to-health extracts like rutin as 560
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delivery systems to enhance their biological activities. It can be concluded that determination of an optimum formulation can also be applied as a model study to prepare nanoparticles for clinical application of other drugs. This study could provide a vision for different approaches and could be used as a platform in design and optimization of different nanoparticle formulations.
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[email protected]. ORCID
Kadriye Kızılbey: 0000-0002-0297-0057 Notes
The author declares no competing financial interest.
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ACKNOWLEDGMENTS The author would like to acknowledge Assoc. Prof. Serap Derman and Asst. Prof. Zeynep Mustafaeva Akdeste from Yıldız Technical University, Bioengineering Department for providing facility required for the research undertaken.
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