Melatonin Nanoparticles Adsorbed to Polyethylene Glycol Microspheres as Activators of Human Colostrum Macrophages

The effectiveness of hormones associated with polymeric matrices has amplified the possibility of obtaining new drugs to activate the immune system.Melatonin has been reported as an important immunomodulatory agent that can improvemany cell activation processes. It is possible that the association of melatonin with polymers could influence its effects on cellular function. Thus, this study verified the adsorption of the hormone melatonin to polyethylene glycol (PEG) microspheres and analyzed its ability to modulate the functional activity of human colostrum phagocytes. Fluorescence microscopy and flow cytometry analyses revealed that melatonin was able to adsorb to the PEG microspheres. This system increased the release of superoxide and intracellular calcium. There was an increase of phagocytic and microbicidal activity by colostrum phagocytes when in the presence of melatonin adsorbed to PEGmicrospheres.Themodified delivery ofmelatonin adsorbed to PEGmicrospheresmay be an additional mechanism for itsmicrobicidal activity and represents an important potential treatment for gastrointestinal infections of newborns.


Introduction
Polyethylene glycol (PEG) microspheres are polymeric particles that have the capacity to adsorb organic compounds and are considered major drug carriers [1].The adsorption capacity of microspheres for organic compounds can be modified to improve their biological function [2] as well as their ability to modulate the immune response [3,4].The microspheres are made of materials that have been developed as controlled release systems for drugs.These synthetic polymers allow for controlled cell recognition and communication, trigger immune responses, cell adhesion, or signal transduction [5], and are important in pathological processes.
Clinical and experimental evidence supports the hypothesis that colostrum is important during infection because it contains soluble and cellular components, such as lipids, carbohydrates, proteins, viable leukocytes (1 × 10 9 cells mL −1 in the first days of lactation), especially neutrophils and macrophages [6], and hormones, which are important for immune defense [7,8].The literature has reported the action of hormones associated with PEG microspheres, which together act as immunomodulators [4].
Melatonin, one of the hormones contained in milk, is produced by the pineal gland and plays an important protective role for infants [7].Many of the benefits of melatonin and its metabolites are related to their antioxidant, antiinflammatory [9,10], and prooxidative effects [11].Melatonin has been shown to act on human phagocytes [12,13] as well as rat splenic macrophages [14,15].
However, the oral bioavailability of melatonin is less than 20% due to extensive first-pass hepatic metabolism and variable rates of absorption [16][17][18].Thus, PEG microspheres are a promising agent for the delivery of the hormone melatonin, as they can prevent its degradation and increase its bioavailability within the organism.
It is possible that melatonin affects colostrum by modulating the microbicidal mechanisms of phagocytes, attracting cells to the site of infection, and reducing the possibility of infection.The aim of this study was to verify the adsorption of the hormone melatonin to PEG microspheres and to analyze the effect of this material on the functional activity of human colostrum phagocytes.

Materials and Methods
2.1.Subjects.Upon informed consent, approximately 8 mL of colostrum was collected from clinically healthy women aged 18-35 years at the Health System Program of Barra do Garc ¸as, MT, Brazil ( = 70).All of the mothers had given birth to healthy full-term babies.The colostrum samples were collected in sterile plastic tubes between 48 and 72 hours postpartum.All of the procedures were submitted for ethical evaluation and received institutional approval.

Polyethylene Glycol (PEG)
Microsphere Preparation.The microspheres were obtained from polyethylene glycol (PEG) 6000 using a modification [3,4] of a previously described protocol [2].Briefly, 20 g of PEG 6000 was resuspended in 100 mL of a 2% sodium sulfate solution in phosphate-buffered saline (PBS) and incubated at 37 ∘ C for 45 min.After incubation, the PEG microspheres were diluted 3 : 1 in PBS and washed twice in PBS (500 ×g, 5 min).The PEG microspheres were resuspended in PBS.The formation of the microspheres was thermally induced by subjecting the solution to 95 ∘ C for 5 min.For adsorption, the suspensions of PEG microspheres in PBS were incubated with melatonin (Sigma, St. Louis, MO, USA; concentration 100 ng mL −1 ) at 37 ∘ C for 30 minutes.The PEG microspheres with or without adsorbed melatonin were fluorescently labeled overnight at room temperature with a solution of Dylight 488 (Pierce Biotechnology, Rockford, IL, USA; 10 g mL −1 ) in dimethylformamide at a 100 : 1 molar ratio of PEG : Dylight.The samples were then analyzed by fluorescence microscopy.

Flow Cytometry.
Immunofluorescence staining was performed with phycoerythrin (PE, Sigma, St. Louis, MO, USA) to compare the abilities of the PEG and polymethylmethacrylate microspheres (CaliBRITE-BD, San Jose, CA, USA) to bind fluorescent markers.The PEG microspheres were incubated with 5 L of PE (0.1 mg mL −1 ) for 30 min at 37 ∘ C.After the incubation, the PEG microspheres were washed twice in PBS containing BSA (5 mg mL −1 ; 500 ×g, 10 min, 4 ∘ C).In all of the experiments, the PEG microspheres were analyzed by flow cytometry.The study was performed on a FACSCalibur (BD, San Jose, CA, USA).The PEG microspheres sizes were compared to those of the BD microspheres (6 m CaliBRITE 3 Beads, BD cat.no.340486, San Jose, CA, USA) and those bound or unbound to PE.The fluorescence intensity of the PEG microspheres was expressed as the geometric mean fluorescence intensity, and the size was calculated according to the geometric mean of the Forward Scatter (FSC).

Obtaining Supernatants from Human Colostrum.
Colostrum supernatant samples of different mothers were obtained by centrifugation (10 min, 160 ×g, 4 ∘ C), the upper fat layer was discarded, and the aqueous supernatant was stored at −70 ∘ C to isolate the posterior melatonin hormone.

Separation of Colostrum Cells.
Approximately 15 mL of colostrum was collected in sterile plastic tubes from each woman.The samples were centrifuged (160 ×g, 4 ∘ C) for 10 min, which separated the colostrum into three different phases: a cell pellet, an intermediate aqueous phase, and a lipid-containing supernatant, as described by Honorio-Franc ¸a et al. [19].The cells were separated by a Ficoll-Paque gradient (Pharmacia, Uppsala, Sweden), producing preparations with 98% pure mononuclear cells, and they were analyzed by light microscopy.The purified macrophages were resuspended independently in serum-free medium 199 at a final concentration of 2 × 10 6 cells mL −1 .

Melatonin Hormone Dosage by Immunoenzymatic
Method.Melatonin was extracted by affinity chromatography, concentrated in a speed vacuum, and then quantified by ELISA (Immune-Biological Laboratories, Hamburg).The reaction rates were measured by absorbance at 405 nm in a spectrophotometer.The results were calculated by a standard curve and shown in pg/mL.
2.8.Release of Superoxide Anion.Superoxide release was determined by cytochrome C (Sigma, St. Louis, MO, USA) reduction [19,20].Briefly, mononuclear phagocytes and bacteria were mixed and incubated for 30 min to allow phagocytosis.The cells were then resuspended in PBS containing 2.6 mM CaCl 2 , 2 mM MgCl 2 , and cytochrome C (Sigma, St. Louis, MO, USA; 2 mg mL −1 ).The suspensions (100 L) were incubated for 60 min at 37 ∘ C on culture plates.The reaction rates were measured by their absorbance at 630 nm, and the results were expressed as nmol/O 2 − .All of the experiments were performed in duplicate or triplicate.
2.9.Bactericidal Assay.Phagocytosis and microbicidal activity were evaluated by the acridine orange method [21].Equal volumes of bacteria and cell suspensions were mixed and incubated at 37 ∘ C for 30 min under continuous shaking.Phagocytosis was stopped by incubation on ice.To eliminate extracellular bacteria, the suspensions were centrifuged twice (160 ×g, 10 min, 4 ∘ C).The cells were resuspended in serum-free 199 medium and centrifuged.The supernatant was discarded, and the sediment was dyed with 200 L of acridine orange (Sigma, St. Louis, MO, USA; 14.4 g L −1 ) for 1 min.The sediment was resuspended in cold 199 medium, washed twice, and observed under an immunofluorescence microscope at 400x and 1000x magnification.The phagocytosis index was calculated by counting the number of cells ingesting at least 3 bacteria in a pool of 100 cells.To determine the bactericidal index, the slides were stained with acridine orange, and 100 cells with phagocytized bacteria were counted.The bactericidal index was calculated as the ratio between orange-stained (dead) and green-stained (alive)

Characterization of Microspheres with Melatonin.
The fluorescence microscopy image (Figure 1(a)) shows the PEG microspheres that were produced in PBS.This result confirmed that the method produces different sizes of microspheres that are easily separated in suspension.The microspheres retained their ellipsoid structures, had regular sizes, and were homogeneous (Figure 1(a)).The PEG microspheres were able to adsorb the melatonin.Furthermore, the melatonin was distributed throughout the surface (Figure 1

Superoxide Release by Colostrum Phagocytes in the Presence of PEG Microspheres Adsorbed to Melatonin.
In the absence of bacteria, melatonin increased the release of superoxide by colostrum phagocytes compared to the spontaneous release (PBS = 1.32 ± 0.3-melatonin = 3.47 ± 0.69; Table 1).(PBS = 1.01 ± 0.24-melatonin = 2.74 ± 0.37; Table 1).The phagocytes incubated with bacteria and melatonin also displayed higher superoxide release than the controls (bacteria plus melatonin = 2.74±0.37;PBS = 1.01±0.24).Additionally, the phagocytes exposed to melatonin that had been adsorbed to PEG microspheres displayed increased superoxide release when compared to the phagocytes exposed to the PEG microspheres alone (bacteria plus PEG microsphere = 1.41 ± 0.31, bacteria plus PEG microsphere plus melatonin = 1.96 ± 0.55).The effect of melatonin stimulation alone was higher than that of bacteria and melatonin (melatonin = 3.47 ± 0.69; bacteria plus melatonin = 2.74 ± 0.37).Furthermore, the release of superoxide decreased significantly in the presence of PEG microspheres with adsorbed melatonin compared to the phagocytes exposed to melatonin alone, and this was independent of bacteria (PEG + MLT = 2.35 ± 0.42; bacteria plus PEG + MLT = 1.96 ± 0.55; Table 1).

Phagocytosis of Colostrum Mononuclear Cells in the
Presence of PEG Microspheres Adsorbed to Melatonin.The colostrum phagocytes displayed some phagocytic activity in response to EPEC (34.4 ± 4.6).Phagocytosis increased significantly in the presence of melatonin (48.8 ± 12.8).A comparison of the PEG microspheres adsorbed to melatonin and the PEG microspheres alone showed that phagocytosis was similar (melatonin = 82 ± 5.1; PEG microspheres = 71.5 ± 3; PEG microspheres plus melatonin = 78.5 ± 3; Figure 3).

Bactericidal Activity of Colostrum Phagocytes in the
Presence of PEG Microspheres Adsorbed to Melatonin.The colostrum mononuclear phagocytes that were not stimulated had some bactericidal activity against EPEC (34.9 ± 9.5).The mononuclear phagocytes incubated with melatonin showed increased bactericidal activity (66.9 ± 10).The bacterial killing by colostrum mononuclear phagocytes mediated by PEG microspheres adsorbed to melatonin is shown in Figure 4.The mononuclear phagocytes incubated with PEG microspheres adsorbed to melatonin showed increased microbicidal activity in response to EPEC (61.5 ± 5.8).5).Table 2 shows the rate of intracellular Ca 2+ release of colostrum phagocytes treated with PEG microspheres adsorbed to melatonin or PEG microspheres alone using Fluo-3 to assess the fluorescence intensity (PBS = 13.97 ± 2.2; PEG + MLT = 30.98± 5.03; PEG = 21.24±1.2).The highest intracellular Ca 2+ release was found in phagocytes treated with melatonin (87.9 ± 8.3), whereas melatonin adsorbed to PEG microspheres decreased the release of intracellular Ca 2+ by colostrum phagocytes (30.98 ± 5.03; Table 2).

Discussion
In this study, microspheres adsorbed to melatonin were produced, and this material was found to stimulate the functional activity of colostrum phagocytes as evidenced by the release of superoxide and intracellular calcium.
Microsphere-based polymeric substances can be used for controlled drug delivery.The release of drugs from a microsphere may be due to the leaching process of the polymer or by the degradation of the polymer matrix, and it is therefore important to understand the physical and chemical properties of the release medium [22].In this study, the analyses by fluorescence microscopy and flow cytometry showed that the PEG microspheres had ellipsoid shapes and were easily separated from the suspension.The literature has reported the use of flow cytometry as an alternative method for analyzing and visualizing particles [23,24].It was observed by flow cytometry that the PEG microspheres had a size of approximately 5.8 m.The adsorption of melatonin reduced the size to approximately 5.35 m, suggesting that melatonin may bind at the same site as the marker.These data are in agreement with previous studies, which found that the PEG microspheres changed in size or in the ability to bind fluorescent substances after the adsorption of bioactive molecules [3,4,25].
The literature reports that polyethylene glycol in microsphere formulations can allow for the control and development of pores by the molecular weight, and the concentration can modulate the speed at which the drug is released from the polymer matrix [26].There are currently several drugs associated with PEG that are widely traded, including interferon alpha (PEGASYS, PEG-Intron) growth hormone (Somavert), asparaginase (Oncaspar), camptothecin, and insulin, and the PEG has been able to prolong the bioavailability of the drugs [27].
The effectiveness of hormones associated with polymer matrices has expanded the possibility of obtaining new drugs to activate the immune system [4].According to the literature, the generation of free radicals is an important mechanism of protection against infection [28][29][30].
The results of this work confirm the importance of the superoxide anion in activating colostrum phagocytes in association with modified drug release systems.Melatonin increased the production of these radicals independently of the presence of bacteria.Moreover, when melatonin was adsorbed to PEG microspheres, superoxide anion release decreased, but it remained higher than that found during spontaneous release.These data suggest that the PEG microspheres are able to modify the release of melatonin while maintaining cellular activation.This can minimize the adverse effects that may occur with high doses of superoxide anion.
The beneficial actions of melatonin are associated with its ability to remove free radicals and increase the enzymatic activity of antioxidants [31][32][33][34].Furthermore, melatonin has immunostimulatory effects [35] and can stimulate cells of the immune system [36,37].Phagocytes produce large amounts of superoxide radicals during oxidative stress, an important protective mechanism during infection.Controlling this release is fundamental for appropriate immune responses against infection.
Soluble components present in colostrum interact with cells to increase superoxide release, and this can increase the phagocytic and microbicidal abilities of macrophages [30,37].In the present study, phagocytosis was increased independently of the stimulus used.The highest rates of phagocytosis were observed when the cells were directly stimulated by melatonin, which is in agreement with previous studies [13,38].
Interestingly, the superoxide release by cells exposed to the melatonin-adsorbed PEG microspheres, although in lower concentration, was sufficient to activate the microbicidal mechanisms of phagocytes.This demonstrated that the microsphere-mediated release of melatonin may be important for the modification of cellular activation because elevated levels of free radicals cause cellular damage that eventually culminates in the activation of cell death pathways [15,38,39].Microbicidal activity promoted by melatonin and the resulting oxidation products may have important clinical applications [40,41].Alterations in superoxide anions modify the responses of intracellular calcium and phosphorylation events during oxidative metabolism [42].Furthermore, melatonin has been reported to increase intracellular calcium in human cells [43].
In the present study, melatonin stimulated the release of intracellular calcium by colostrum phagocytes.The adsorption of melatonin to PEG microspheres decreased this release, suggesting that the PEG microspheres can modify the effect of melatonin on intracellular calcium influx.This delivery system provided by melatonin and PEG microspheres may be useful for various diseases because the excessive release of intracellular Ca 2+ can induce apoptosis [44,45].
In this study, we found that melatonin is present in colostrum, and the interaction between hormones and cells generates natural protection for the newborn.Due to the immaturity of the newborn digestive system, digestive enzymes and other factors do not destroy the cells received through colostrum.Therefore, the cells likely remain intact throughout the upper portions of the intestine and can interact with each other to protect the mucosa.Some studies have suggested that the cells from the colostrum remain viable within the newborn intestinal mucosa for a period of 4 hours [46,47] and may exert microbicidal activity and produce antibodies [48].Importantly, the interaction of hormones associated with modified delivery systems is critical for the newborn, and such systems can utilize colostrum in concentrations that best promote cellular activity.

Conclusion
The results indicate that melatonin-adsorbed PEG microspheres modify the release of superoxide and intracellular Ca 2+ by colostrum phagocytes and increase the microbicidal activity of these cells.The modified delivery system of melatonin via PEG microspheres may be an additional mechanism to improve the immune responses of colostrum phagocytes and represents a fundamentally important mechanism for the protection and treatment of gastrointestinal infections of newborns.

Figure 1 :
Figure 1: Fluorescence microscopy image of the PEG microspheres stained with Dylight 488.(a) represents the PEG microspheres (100x), and (b) represents the PEG microspheres adsorbed to melatonin.The experiments were repeated five times, and the results were comparable.
(b)), indicating its interfacial deposition under the surface of the microspheres.Figure 2(a) compares the fluorescence intensity of PEG microspheres, PEG microspheres adsorbed to melatonin, and the BD microspheres (standard).The PEG and BD microspheres had the highest geometric mean fluorescence intensities.The adsorption of melatonin to the PEG microsphere altered the geometric mean fluorescence intensity.The sizes of the PEG microspheres were similar to the standard microspheres (Figures 2(a) and 2(b)).Analysis by flow cytometry showed that the PEG microspheres had a size of approximately 5.8 m, the polymethylmethacrylate BD microspheres had a size of 6 m, while the microspheres adsorbed to melatonin had a mean size of 5.3 m (Figure 2(c)).

Figure 2 :
Figure 2: The PEG microspheres were stained with phycoerythrin (PE) as described in Section 2. PE-labeled polymethylmethacrylate microspheres were used as a standard.Immunofluorescence and size analyses were carried out by flow cytometry.(a) represents the fluorescence intensity (FL2), (b) represents the size based on Forward Scatter (FSC), and (c) represents the geometric mean fluorescence intensity and the geometric mean of size.

Figure 5 :
Figure 5: Colostrum mononuclear cells stimulated with melatonin adsorbed to PEG microspheres were stained with Fluo-3 to assess intracellular Ca 2+ release as described in Section 2. Immunofluorescence analyses were carried out by flow cytometry.
Fluo-3 was detected using a 530/30 nm filter for intracellular Ca 2+ .The ratio of intracellular Ca 2+ release was expressed as the geometric mean fluorescence intensity of Fluo-3.The experiments were repeated on several occasions, and the data presented in the figures are from single representative experiments.

Table 1 :
Superoxide anion release from phagocytes in human colostrum mononuclear cells.

Table 2 :
Release of intracellular Ca 2+ by colostrum phagocytes in the presence of melatonin adsorbed to PEG microspheres.The intracellular Ca 2+ release is represented by mean fluorescence intensity and was obtained by flow cytometry.The results represent the mean and SD of five experiments with cells from different individuals.*  < 0.05: comparing the treated cells with non-treated cells (PBS); #  < 0.05: comparing the different treatments (MLT and PEG microspheres).