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The effectiveness of prostaglandin nanoparticles in corpus luteum regression

Research examined the formation of prostaglandin nanoparticles and their effect on corpus luteum (CL) regression carried out at IRIAP. The nanoparticles formation was carried out using the ionic gelation method. The nanoparticles have a particle size of 316.80±0.14 nm, polydispersion index of 0,453±0,001, zeta potential of +17,40±0,85 mV with 69,69±8.81% hormone entrapment. The effectiveness of nanoparticle in CL regression was observed (prostaglandin vs prostaglandin nanoparticles) using ultrasound observation, hormone profile and estrus response. Further, the size of the ovulating follicle, the time of ovulation, the size of the CL and the onset of estrus after the administration of the prostaglandins were observed. The observation showed that the intramuscular administration of prostaglandin and prostaglandin nanoparticles did not significantly differ on the onset of estrus, time of ovulation, the ovulating follicle size, size of CL and progesterone concentration. The onset of estrus occurred on 2.50 ± 0.58 and 2.33 ± 0.52 days, the ovulation time after hormone administration was on days 3.50 ± 0.55 and 2.83 ± 0.75 with the ovulation follicle size of 16, 62 ± 0.96 and 17.03 ± 1.13 mm, while the CL measures at H-3 were 13.56 ± 2.28 and 10.45 ± 0.88, the progesterone H-2 concentrations were 0.299 and 0.395, respectively for prostaglandin and prostaglandin nanoparticles. It can be concluded that the formation of nanoparticles did not impair the effectiveness of hormones in CL regression so that it can be used to increase the effectiveness of estrus synchronization


Introduction
Nanoparticles (NPs) are substances/material with one final dimension generally ranging from 1-100 nm (10-9-10-7) which are referred to as particles. This size provides them with different physical and chemical properties from materials normally found in the environment [1] hence some materials are considered toxic. Some researchers agree the size of nanoparticles is not more than 1,000 nm [2,3,4]. Meanwhile, in drug delivery, nanoparticles diameter of 200 nm [5,6], 200-400 nm [7] are recommended for drug delivery systems.
The application of nanoparticles has the opportunity to increase drug efficiency [8]. Some of the advantages of nanoparticles in drug delivery system are their ability to penetrate the intercellular spaces that can only be penetrated by colloidal particle size. The ability to penetrate the cell wall is higher and increases the affinity due to the increased contact surface area by the same amount [9].
Estrus synchronization is a reproductive manipulation technology commonly applied to increase the success of the artificial insemination (AI) program. Application of prostaglandin is usually done to shorten the luteal phase by lysing the corpus luteum [10,11]. The effects of prostaglandins depend on the phase of the estrus cycle. Prostaglandins given in the middle of the cycle (5-16 days after ovulation) will efficiently induce luteolysis, while if given when no active CL is found (day -3 to +4 from the time of ovulation), estrus synchronization will not work [12]. The study aimed to determine the responses of prostaglandins nanoparticles to CL regression in the estrus synchronization program.

Formation of nanoparticle
Prostaglandin nanoparticles were formed using ionic interactions [13] with modifications. Chitosan 0.2% w/v in acetic acid 1% v/v as a source of cations and tripolyphosphate/TPP (0.1% w/v) in sterile water as a source of anions were used in ion exchange reactions. Prostaglandin nanoparticles (0.25 mg/ml) were formed by adding prostaglandins in a chitosan solution and were homogenized using a 1500 rpm magnetic stirrer for 10 minutes. The final pH of the solution formed was adjusted to reach a pH of 5.5 using 1 N NaOH.

Analysis nanoparticle
The analysis of the particle size and zeta potential was carried out to determine the physical properties of the nanoparticles formed. The mean and distribution of the particle size were measured based on photon correlation spectroscopy (PCS, dynamic light scattering and DLS). Zeta potential was determined based on the electrophoretic light scattering (ELS) technique. The entrapment efficiency (EE) process was determined by centrifugation at 12,000 rpm, and 10ºC for 10 minutes to obtain a hormone that was not bound to the nanoparticles (supernatant) and measured using UV spectrophotometry at λ= 260 nm. Entrapment efficiency (EE%) of prostaglandins was calculated as follows.

EE% = Total number of prostaglandin -free prostaglandin in supernatant x 100
Total Prostaglandin

In vivo study
This study was conducted to determine the effectiveness of the application of prostaglandin and prostaglandin nanoparticles in regressing the CL. Treatment 1: Prostaglandin injection (D-11) followed by prostaglandin injection (D0) Treatment 2: Prostaglandin nanoparticles injection (D-11) followed by prostaglandin nanoparticle (D0) PGF2α blood collection 3 day/week USG -estrous detection Monitoring of CL development, follicles, time of estrus (ultrasound observation), concentration of progesterone (blood collection) and onset of estrus was carried out in vivo study. Prostaglandins were injected on day -11 and day 0, and ultrasound (USG) observation was performed every day (start from D0) until an ovulating follicle was found. Blood sampling was carried out every two days until ovulation was found. Ovulation was determined by ultrasound observation characterized by the loss of the dominant follicle. Observation of estrus was carried out after the injection of the 2nd prostaglandins (D-0) to obtain data on onset of estrus. Blood samples were collected to obtain blood plasma for 3 determination of progesterone concentrations. Blood plasma was collected and frozen until it was ready to be evaluated using the ELISA method.
All data in the in vivo study were analysed using a completely randomized design and the differences were tested using LSD.

Results and discussion
The physicochemical characteristics of prostaglandin nanoparticles in the form of particle size, polydispersity index (PDI), zeta potential and entrapment efficiency are presented in Table 1. Chitosan in acid media can interact with negatively charged TPP, thereby forming inter-and intramolecular cross-ionic chitosan nanoparticles [14,15]. The particle size obtained in this study is still in the nanoparticle size range as reported by [4] that nanoparticles are dispersed or solid particles with sizes ranging from 10-1,000 nm, 200-400 nm [7] are recommended for drug delivery systems. Likewise, the polydispersity index value is a measure of the molecular mass distribution in a particular sample. The results showed a PDI value of 0.453 where this value indicates the calculation of the average molecular weight divided by the average molecular weight. The closer to zero, the better the distribution.
Zeta potential is a surface charge that can affect the stability of particles in suspension due to electrostatic repulsion between particles [16]. The results showed that the zeta potential and entrapment efficiency of prostaglandin nanoparticles were +17,00±0,85 mV and 69,69±8,81%. The positive zeta potential (+) a is characteristic of nanoparticles formed from natural polymer chitosan/TPP. In the process of nanoparticle formation, the positively charged amine groups are neutralized by their interaction with the negative charge on the tripolyphosphate molecule and the remining amino groups result in a positive zeta potential [17]. The higher the zeta potential value in a certain range, the more stable the chitosan nanoparticles formed. The results showed that the prostaglandin nanoparticles formed were quite stable (>20) with fairly good hormone entrapment (69,69±8,81%).
The results of in vivo studies in the form of ovulating follicle size, ovulation time, size of the corpus luteum and onset of estrus after administration of prostaglandin and prostaglandin nanoparticles are presented in Tables 2 and 3 and Figure 2.
The application of prostaglandin nanoparticles did not cause a significant difference in CL size and onset of estrus (Table 2). CL size decreased from day 0 to day 4 after injection of prostaglandin and prostaglandin nanoparticles. CL size decreased from 21.83 ± 2.85 to 7.66 ± 1.45 mm for prostaglandin nanoparticles and 22.80 ± 2.11 to 9.72 ± 0.86 mm for prostaglandin (control). CL regression causes a decrease in progesterone in the blood, resulting in a negative feedback that stimulates gonadotropin release which causes growth and maturation of follicles.
Application of prostaglandin and prostaglandin nanoparticle showed that there was no significant difference between prostaglandin (control) and prostaglandin nanoparticles on the ovulating follicle size and ovulation time (Table 3). Ovulation occurred on day 3.50 ± 0.55 and 2.83 ± 0.75 with an ovulating follicle size of 16.62 ± 0.96 and 17.03 ± 1.13 mm respectively on the application of prostaglandin (control) and prostaglandin nanoparticle. The timing of ovulation is related to the diameter of the dominant follicle. The diameters of the ovulating follicles were 11.1-14.4 mm [18,19] and 10.6-17.6 mm [20]. : Prostaglandin nanoparticles D-0 : Day of the second injection of prostaglandin or prostaglandin nanoparticle D1-D4 : Day 1-4 after the second injection of prostaglandin or prostaglandin nanoparticle : Day of the second injection of prostaglandin or prostaglandin nanoparticle D1 : Day 1 after the second injection of prostaglandin or prostaglandin nanoparticle Progesterone concentrations decreased after application of prostaglandin and prostaglandin nanoparticles. There was no significant difference in progesterone concentrations in the two treatments ( Figure 2). Corpus luteum plays a role in producing progesterone hormone. Application of prostaglandins causes CL lysis so that the progesterone concentration will decrease to 0.5 ng/ml within 24 hours. CL is responsible for the heat cycle phenomenon [21,11]. The use of prostaglandin hormones  in estrus synchronization is only effective if livestock have CL or are in the luteal phase. PGF2α will reduce the concentration of progesterone in the blood so that the ratio between the concentration of estrogen and progesterone increases thus cattle will show a pattern of sexual behaviour and ovulation [22].

Conclusion and suggestion
The formation of prostaglandin nanoparticles at a particle size of ±300 nm did not change its function and role in regressing CL.
There were no significant changes in parameters (CL size, follicle, onset of estros , ovulation time and progesterone concentration) that supported the estrus synchronization program thus the formation of prostaglandin nanoparticles at a particle size of 300 nm could be developed to increase the effectiveness of estrous synchronization.
It is necessary to improve the nanoparticle formation method (to increase stability and hormone entrapment) and to test the effectiveness of the application of prostaglandin nanoparticles in large scale of estrus synchronization.