Preparation of poly (lactic-co-glycolic acid)-loaded pentoxyfilline by nanoparticipation technique

Saad Saleem Raheem; Huda Falah Hasan

doi:10.4103/MJBL.MJBL_59_20

ORIGINAL ARTICLE

Year : 2021 | Volume : 18 | Issue : 1 | Page : 12-17

Preparation of poly (lactic-co-glycolic acid)-loaded pentoxyfilline by nanoparticipation technique

Saad Saleem Raheem, Huda Falah Hasan
Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq

Date of Submission	17-Sep-2020
Date of Acceptance	18-Nov-2020
Date of Web Publication	17-Mar-2021

Correspondence Address:
Saad Saleem Raheem
Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Baghdad, Baghdad
Iraq

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/MJBL.MJBL_59_20

Abstract

Background: Nanomaterial concerned to an incidental, natural, or synthesized material containing particles utilized for curing several diseases and keeping for human with animals health; it is either in an unbound condition or as a combination in which one or more external measurements in the size range of 1–100 nm. Objectives: The study intended to assess the possible mitigating outcome of the reference pentoxifylline by progressed a new manner of poly (lactic-co-glycolic acid) (PLGA) nanoparticles loading with pentoxifylline on induced thin layer endometrial in female rats. Materials and Methods: PLGA nanoparticle was made by employing two procedures included nanoprecipitation technique and double emulsion solvent evaporation method. The experiment firstly was achieved numerous of diagnostic tests to the knowledge of PLGA nanoparticle characteristics that involved ultraviolet (UV) spectrophotometer test. Results: UV spectrophotometer test shown for Pentoxyfilline absorbance (0.300124), at wave length (2979) and PLGA- loaded Pentoxyfilline absorbance (3.07877), at wave length 1565), while other tests (scanning electron microscope, cumulative %; encapsulation drug efficiency loading, and zeta) of PLGA-nanoparticle appeared (28.35 nm-35.45 nm, (8.48, 93.3 at maximum wavelength 275 nm, (−13.44 mV), with mobility (−1.05 [μ/s]/[V/cm]) respectively. Conclusion: Regarding the characterization of PLGA displayed in this study, it can be concluded that DMSO related organic phase alone gives the shape, particle size with a small diameter, negatively charged in addition to suitable LD, EE with suitable stabilizer. Also, PLGA-Pentoxyfilline regimen treatment with different doses had the ability in recovery of female rats that exposed experimentally induced thin layer endometrium in the uterine horn by ethanol.

Keywords: Nanoparticipation, poly (lactic-co-glycolic acid)-pentoxifylline, scanning electron microscope, ultraviolet spectrophotometer

How to cite this article:
Raheem SS, Hasan HF. Preparation of poly (lactic-co-glycolic acid)-loaded pentoxyfilline by nanoparticipation technique. Med J Babylon 2021;18:12-7

How to cite this URL:
Raheem SS, Hasan HF. Preparation of poly (lactic-co-glycolic acid)-loaded pentoxyfilline by nanoparticipation technique. Med J Babylon [serial online] 2021 [cited 2023 May 28];18:12-7. Available from: https://www.medjbabylon.org/text.asp?2021/18/1/12/311448

Introduction

Poly (lactic-co-glycolic acid) (PLGA) is a copolymer manufactured by means of ring-opening copolymerization of two different monomers, the cyclic dimers (1,4-dioxane-2,5-diones) of glycolic acid and lactic acid, and polymers are synthesized as either random or block copolymers herewith cooperating additional polymer properties.^[1] The definition for nanotechnology, nanomaterial, nanoscale, or other linked terms are ingredients that have at least one dimension in the size extent about approximately 1 nanometer (nm) to 100 nm. Focused on the current scientific and technical compassion of nanomaterials and their characteristics, the Food and Drug Administration counsels that estimations of effectiveness, safety, public health influence, or regulatory status of nanotechnology products should consider any unique properties and manners that the application of nanotechnology may communicate^{[2],[3],[4],[5],[6],[7]} certified that the PLGA nanoparticles supplied. The particle size is more beneficial in the field of medical purposes in which the polydispersity depends on its typical characterization, so the smaller particle size and high polydispersion leading to good interesting in the medical field such as lesser minimum inhibitory concentration, higher entrapment efficiency for augmented drug release, minimum bacterial concentrations meaning that a better antibacterial activity is accomplished with a smaller amount of drug, vaccine delivery systems, and they were progressed as gene carriers. Nanotechnology can be planned to progress the pharmacokinetics and biodistribution of the drug.^[8],[9]

Materials and Methods

Preparation of poly (lactic-co-glycolic acid)-pentoxifylline nanoparticles

Nanoparticles were prepared by the nanoprecipitation method^[10] [Figure 1] and [Figure 2] according^{[11],[12],[13]} with mild modification. All steps including stabilization, organic solvents, sonication, centrifugation, magnetic stirrer, etc.. can be listed by the following points:

Figure 1: Nanoprecipitation method for preparing poly (lactic-co-glycolic acid) –pentoxifylline polymer nanoparticles

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Figure 2: PVA O/ W emulsion for preparing poly (lactic-co-glycolic acid) - Pentoxifylline polymer nanoparticles

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The organic solution included 10 mg of PLGA was placed in a glass test tube and then transferred 1 ml of each following solvent (ethyl acetate, dichloromethane, dimethyl sulfoxide [DMSO], acetone, and chloroform) to choose the suitable one of the solvents and then each one was added to the PLGA, after that the top of the tube was closed with a small piece of aluminum foil, and parafilm. The parafilm was put tightly opposite to the top edge of the tube
The level of each solvent was tagged on the outside of the test tube and the dissolving PLGA was incubated overnight, and then, the vortex on high speed until all PLGA is entity dissolved (~10 min)
Twenty (20 μl) of pentoxifylline was immediately added to the polymer solution (PLGA), the tube was put in the vortex at 800 rpm for 5 min in order to encapsulate it and make it homogenously dispersed as emulsified polymer solution
The emulsified polymer solution was put in the small beaker 20 ml and then directly transferred to the ultrasonicator and immersed in the ice water and the emulsion was sonicated for 9 min, pulse on time 15 s with pulse off time 15 s, and 50% amplitude
An aqueous phase was prepared by adding 100 ml of 0.03% w/v Vitamin E-tocopheryl polyethylene glycol succinate (TPGS) to a 200 ml glass beaker for overnight, after that it was put on a magnetic stir bar at stirring speed 500 rpm. And then, 10 ml of 0.03% w/v Vitamin E-TPGS was added to a glass test tube of the emulsified polymer solution. Then, the test tube was stirred at 500 rpm stirring speed
The emulsified polymer solution nanoparticle was put in a beaker 100 ml and then wrapped in aluminum foil; the top of the beaker was left open to facilitate solvent evaporation
The suspension obtained was filtered (Whatman filter paper 1, diameter: 9 cm, pore size: 11 μm) to discard any precipitated and then centrifuged at 14,000 rpm at 4°C. The supernatant containing the unbound drug was discarded; the pellet obtained was washed 2–3 times with distilled water
The empty nanoparticles were prepared according to the same procedure that was mentioned during the preparation of pentoxifylline – PLCA nanoparticles without added any drug.

Instrumentation

A double beam ultraviolet (UV)-visible spectrophotometer (Shimadzu, model 1800) having two matched quartz cells with 1 cm light path length and loaded with UV probe software was used for the recording of spectra and measuring absorbance for method development and validation study.

Selection of diluents such as methanol, acetone, and DMSO was used as diluent for pentoxifylline sulfate drug substances based on the solubility characteristics of the drug substances; excellent solubility was recorded in DMSO so that chosen as preferable diluent. Each 1 mg of pentoxifylline was dissolved in 0.2 ml of DMSO and completed to 10 ml with deionized water. After that spectra for pentoxifylline sulfate were measured from 100 to 3000 nm for wavelength by recording the UV-visible spectrum of the standard solution. Maximum absorbance (λ max) was shown at 2979 nm for standard solution pentoxifylline sulfate with absorbance 0.300124.

Preparation of standard solutions for pentoxifylline sulfate

Pentoxifylline sulfate (1 mg) exactly was moved for working standard into a 50 ml volumetric flask and then was auxiliary (DMSO) 0.2 ml then complete to 10 ml of de-ionized water and sonicated for 5 min to dissolve with intermittent shaking. Then, 1 ml of diluent DMSO (100 μg) was transferred to 50 ml beaker and the diluent DMSO from 50 μg/ml was made to concentration 1 μg/L, as well as the absorbance was verified at each concentration to fix the unknown concentration occasioned from loading drugs by dimension the first supernate of nanoparticle of pentoxifylline after centefugated at 14,000 rpm for 20 min.

Characterization of pentoxifylline loaded poly (lactic-co-glycolic acid) nanoparticles

This step was conducted using apparatus zeta potential measurement, UV-visible spectrometry, and scanning electron microscope (SEM). The cumulative drug release and drug loading (DL) at different solvent and emulsifier were studied. Characterization of nanoparticles yield (DL) and encapsulation efficiency (EE) were quantified by measuring the absorbance of 275 nm.[14] DL and EE of the prepared nanoparticles were quantified by measuring the absorbance of 275 nm^[14] using a SHIMADZU UV-1700 UV-Vis spectrophotometer (EE = 93.3%, DL = 8.48%), respectively. The freeze-dried nanoparticle samples (1 mg) were dissolved in 0.2 ml of DMSO and completed to 10 ml of deionized water, for spectrophotometric measurement.

Pentoxifylline solutions were dissolved within DMSO at various concentrations (1–100 mg/ml), and the absorbance at 275 nm was measured at different concentrations to generate a standard calibration curve (R2 = 0.8533). Nanoparticle yield, DL, and EE were calculated from the following three equations.^[13],[15]

Encapsulation efficiency (E. E).

Drug loading (D. L).

Preparation of standard and sample solutions for pentoxifylline sulfate

Pentoxifylline sulfate (1 mg) exactly was moved for working standard into a 50 ml volumetric flask and then was auxiliary (DMSO) 0.2 ml then complete to 10 ml of de-ionized water and sonicated for 5 min to dissolve with intermittent shaking. Then, 1 ml of diluent DMSO (100 μg) was transferred to 50 ml beaker and the diluent DMSO from 50 μg/ml was made to concentration 1 μg/L, as well as the absorbance was verified at each concentration to fix the unknown concentration occasioned from loading drugs by dimension the first supernate of nanoparticle of pentoxifylline after centefugated at 14,000 rpm for 20 min.

Scanning electron microscope

The SEM of PLGA loading pentoxifylline was done in the Qualitative Research Department of the Ministry of Science and Technology as follows:

Samples for SEM were equipped on the day of making images; a slip of double-sided carbon tape was put on an SEM stub. A perpetual marker was done to label the metal percentage of the stub for later reference
A metal spatula was used together a small amount of lyophilized nanoparticles and softly feast them through the surface of the stripe, the ram surface was downy with tissue
The specimens with gold-palladium were coughed and covered for 30–120 s. A longer splutter time was created a regular surface for imaging
The parameters for visualizing particles were done in variety from 5 mm to15 mm, the ray intensity of 5–12 KV, and a spot size of 1–3. Microparticles are exposed at ×100 magnification and nanoparticles were renowned at ×3000 magnification
The samples were contained a uniform covering of particles across the ram flat surface.

Zeta sizers

The zeta potential measurement was attained using a Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, United Kingdom); this parameter was done in the Ministry of Science and Technology in the province of Baghdad. Temporarily, I ml of PLGA loading pentoxifylline suspension was equipped in MilliQ water by sonication for 30 s. The previously suspension (10 μl) was completed to 1 ml with MilliQ water and the analysis was done. As well as, the mean diameter of three determinations was calculated for each sample.

Ethical consideration

The study was conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki. The study protocol and the subject information and consent form were reviewed and approved by a local ethics committee.

Results

In [Figure 3], the SEM test of pentoxifylline loaded PLGA result showed the particle size about (28.35-35-45 nm) with a circle shape, zeta potential measurement was (−13.44 mV) with mobility [(−1.05 (μ/s)/(V/cm)] [Figure 4], herein negatively charged PLGA particles, that was induced a specific zeta potential.^[16]

Figure 3: The scanning electron microscope properties and particle sizes of pentoxifylline loaded poly (lactic-co-glycolic acid)

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Figure 4: The zeta properties (-13.44 mV) and mobility ((-1.05 [μ/s]/[V/cm]) of poly (lactic-co-glycolic acid)-pentoxifylline

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The potential at the slipping plane is called the zeta potential. Zeta potential is an important property of the particle in a dispersion as it has exerted a significant, UV analysis displayed absorbance of pentoxifylline and pentoxifylline loaded PLGA in which it was (3.07877 at wavelength λmax 1565 nm and 0.300124 at wavelength λmax 2979 nm) [Figure 5]a and [Figure 5]b. The ratio of 1:10 drug: polymer was used in the current study. The percentage cumulative of it appeared at maximum wavelength 275 nm, the encapsulation (EE = 93.3), and the loading was (LD = 8.48) [Figure 5].

Figure 5: Calibration curve of determination reference pentoxifylline against balance, encapsulation efficiency = 93.3, loading = 8.48 at maximum wavelength of 275 nm. (a) The ultraviolet absorption spectrum of reference pentoxifylline dissolved in dimethyl sulfoxide. The curve referred to the maximum wavelength of pentoxifylline at 1565. Nm. (b) Ultraviolet absorption spectrum of reference of free pentoxifylline. The curve referred to the maximum wavelength of pentoxifylline at 2979. nm

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Discussion

In this study, the characteristics of pentoxifylline loaded PLGA gave it the ability to increase drug-releasing which may be caused by (an extensive) polymer degradation, resulting in increased permeability of the drug in the polymer matrix. These results were compatible with the previous study reported by Stevanovi et al.^[16] As well as, these properties indicated to physicochemical properties of PLGA included degradation by hydrolysis of its ester in the presence of water.^[17]In addition, these results are in agreement with^[18] who proved the flexibility of PLGA leading to use of it in medical devices such as prosthetic devices, surgical sealant films, grafts, sutures, implants, micro- and nanoparticles, and also leading to minimizing the side effect to treatment with any drug that loaded with PLGA.^[19] Furthermore, The PLGA and its characterization and correlation with the mechanism of action had proved by,^[20] and^[21] that demonstrated the control release of PLGA and classified it according to the mechanism of action and employed it as a therapeutic agent due to practical solubilization of drugs, easy fabrication of nano-sized drug delivery systems, and intelligent manipulation of drug release. On the other hand, the properties of PLGA were compatible with previous studies that showed the principle characteristics of nanoparticles in numerous of applications in medicine.^[22],[23] They also used the term nanomedicine for describing the nanotechnology for medical purposes and defined this science as the use of nanomaterials for diagnosis, monitoring, control, prevention, and treatment of diseases. So, using the PLGA came from the concept of its specific possession as polymer established continued-release delivery systems to keep therapeutic drug concentrations for longer periods of time is accepted for decades.^[24] These results were noticed as same as with the study of Kashi et al.,^[5] who demonstrated the safety in using of nanoparticles in treatment of groups that induced with alcohol (ethanol) for 10 days and then treated with it (empty PLGA, pentoxifylline loaded PLGA) for 20 days and compare them with other groups documented the many of properties that included providing a wide range of advantages such as smaller particle size which facilitates the penetration into the cells, higher entrapment efficiency for increased drug release, lower minimum inhibitory concentration, and minimum bacterial concentrations meaning that a better antibacterial activity is achieved with a smaller amount of drug. The idea of performing this trail was unique about using of type of nanoparticle in the surgery and reproductive behavior disorder in the female reproductive system and presenting of varying troubles that were caused infertility that involved thin layer in the endometrium of uterine horn in rats by taking advantage of the properties of nanoparticles and this study had been well matched with previous projects earlier proven capability of PLGA in treatment of endometriosis where it was and still a common gynecological disorder affecting almost 10% of the women in their reproductive age.^[25]

Conclusion

Regarding the characterization of PLGA displayed in this study, it can be concluded that DMSO related organic phase alone gives the shape, particle size with a small diameter, negatively charged in addition to suitable LD, EE with suitable stabilizer. Also, PLGA-Pentoxyfilline regimen treatment with different doses had the ability in recovery of female rats that exposed experimentally induced thin layer endometrium in the uterine horn by ethanol.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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