The aim of the present work was to compare in vitro the possibility of application of unmodified silica nanoparticles (UMNPs) and modified by aminopropyl groups silica nanoparticles (AMNPs) for topical delivery of immunomodulatory drug GMDP to the peritoneal macrophages of women with endometriosis. The absence of cytotoxic effect and high cellular uptake was demonstrated for both types of silica nanoparticles. The immobilization of GMDP on the UMNPs led to the suppression of the stimulatory effect of GMDP on the membrane expression of scavenger receptors SR-AI and SR-B, mRNAs expression of NOD2 and RAGE, and synthesis of proteolytic enzyme MMP-9 and its inhibitor TIMP-1. GMDP, immobilized onto AMNPs, enhanced the initially reduced membrane expression of SRs and increased NOD2, RAGE, and MMP-9 mRNAs expression by macrophages. Simultaneously high level of mRNAs expression of factors, preventing undesirable hyperactivation of peritoneal macrophages (SOCS1 and TIMP-1), was observed in macrophages incubated in the presence of GMDP, immobilized onto AMNPs. The effect of AMNPs immobilized GMDP in some cases exceeded the effect of free GMDP. Thus, among the studied types of silica nanoparticles, AMNPs are the most suitable nanoparticles for topical delivery of GMDP to the peritoneal macrophages.
Nowadays the immunomodulators are widely used for the treatment of different diseases with proved immune etiology. It was demonstrated that immune mechanisms are directly involved in the endometriosis pathogenesis [
Despite the intensive work in the field of new immunomodulatory drugs development, the list of clinically approved immunomodulators is still rather short. Muramyl dipeptide (MDP), the minimal active fragment of peptidoglycan of the cell wall of Gram-positive and Gram-negative bacteria, has gained much attention in the last years due to its significant immunomodulatory effect upon phagocytes [
In the literature various materials are proposed as drug carriers [
So, in the present work we attempted to immobilize GMDP on the different types of silica nanoparticles by adsorption from isotonic solution with subsequent estimation of the effects of obtained GMDP/silica nanocomposites as well as free GMDP upon macrophage expression and synthesis of: (i) several PRRs molecules (scavenger receptors SR-AI (CD204) and SR-B (CD36), NOD2 receptors, RAGE (receptor for advanced glycation end products)), (ii) suppressor of cytokine signaling 1 (SOCS1), which is known as a negative-feedback regulator of PRRs-induced signaling, and (iii) proteolytic enzyme MMP-9 (matrix metalloproteinase-9) and its tissue inhibitor TIMP-1, which are the important enzymes, providing interaction of phagocytes with its targets during phagocytosis process.
Tetraethoxysilane (TEOS) (high purity grade, Russia), 3-aminopropyl triethoxysilane (APTES) (Aldrich, 99%), sucrose (ICN Biomedicals, >99% purity), trypan blue solution (0.1%; Sigma-Aldrich, USA), nitrotetrazolium blue (Sigma-Aldrich, USA), zymosan (Sigma-Aldrich, USA), sheep IgG, labeled by fluorescein isothiocyanate (FITC) (Sorbent, Moscow), FITC-conjugated monoclonal antibodies anti-HLA-DR, CD14, CD11b, CD95, CD36, CD204 (Beckman Coulter), and immunomagnetic beads charged anti-CD14 mouse IgG (Dynabeads CD14, Invitrogen by Life Technologies AS, Oslo, Norway) were used in the work. N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) was kindly given by “Peptek” (Institute of Bioorganic Chemistry of RAS).
Sodium chloride (high purity) and double distilled water were used for preparation of isotonic solution. Potassium bromide (Acros, 99+%, IR grade) was dried at 250°C before use.
Commercial kit “Oncoscreen” (“GenoTechnology”, Moscow, Russia) was used to perform the reaction for total RNA isolation and reverse transcription. Sets of primers, probes, and enzymes solutions for estimation of MMP-9 and TIMP-1 mRNAs expression (Fractal Bio, St.-Petersburg, Russia) and for detection of NOD2, RAGE, and SICS1 mRNAs expression (Sintol, Moscow, Russia) by peritoneal macrophages were used in our study.
The unmodified silica was synthesized via HCl-catalyzed sol-gel procedure of TEOS in the presence of pore forming agent (sucrose) as described elsewhere [
The aminopropyl modified silica was synthesized via hydrolysis and cocondensation of precursor mixture (TEOS: APTES = 3 : 1 v/v) as described earlier [
To indentify the synthesized silica materials, the FTIR spectra of the samples were recorded on an Avatar 360 FTIR ESP spectrometer in a range of 4000–400 cm−1. The powders were milled and pressed into discs with KBr. The FTIR spectra of the sucrose and fructose containing materials are also recorded.
Small-angle X-ray scattering (SAXS) study is used to measure the periodicity of materials’ structure. The synthesized silica powders were investigated by SAXS. The experiments were performed with a diffractometer DRON-2 (Russia) operating at 40 V (Cu-K
In order to study effects of the silica nanoparticles on functional state of peritoneal macrophages, the suspensions of the silica nanoparticles with an average radius of 50 nm were prepared in isotonic solution as described earlier [
The immobilization of GMDP on the silica nanoparticles was carried out by addition of the drug solution (isotonic solution) to the suspensions of the nanocarriers (UMNPs or AMNPs with a radius of 50 nm). The concentrations of the UMNPs and AMNPs in suspensions were found to be 3.8 · 10−10 mM and 1.5 · 10−10 M, respectively. Molar ratio of the nanoparticles to GMDP was 1 : 10.
Peritoneal fluid of 40 women with endometriosis (32 with mild endometriosis (stage II of disease according to the classification of American Society for Reproductive Medicine) and 8 with severe endometriosis (III-IV stages of disease)) who underwent laparoscopic examination for pelvic pain or infertility was used as the material. Informed consent was given from each woman participating in our study, according to local Ethic Committee protocol. All patients ranged in age from 20 to 40 years and were not taking any hormone therapy at least 3 months prior to collection of the samples. Samples of peritoneal fluid were aspirated into the sterile tubes from the Douglas pouch during laparoscopic surgery and immediately used for the investigation. The standard procedure of isolation of the peritoneal mononuclear cells (MNC) by centrifugation in density gradient of Ficoll-Urografin (d-1,078) was performed. According to flow cytometry data obtained fractions of peritoneal MNC contained macrophages with phenotype CD45+CD14+ (70–80%) and lymphocytes with phenotype CD45+CD14− (20–30%).
Peritoneal MNC in concentration of 2 × 106 cells/mL was incubated in RPMI 1640 medium in the presence of the synthesized silica nanoparticles for 24 h at 37°C and 5% CO2. According to the literature data, concentration of the nanoparticles in the culture media was 400
To estimate the level of cells, reacting with the silica nanoparticles, the peritoneal MNCs (100
The peritoneal MNCs were incubated in vitro in 0,5 mL RPMI 1640 medium either in the presence of free GMDP (2
One hundred thousand cells per tube were used for immunofluorescence staining. Cells were incubated with 5
For RT-PCR assays we used pure CD14+ population of peritoneal macrophages. After incubation of peritoneal MNC in the presence of free GMDP or GMDP, immobilized onto nanoparticles, the positive separation of CD14+ macrophages using immunomagnetic beads, charged anti-CD14 antibodies (Invitrogen by Life Technologies AS, Oslo, Norway), was performed according to manufacturer’s protocol. Total RNA was isolated from pure fraction of peritoneal macrophages using the standard acid guanidinium thiocyanate-phenol-chloroform method. RNA was converted to complementary DNA (cDNA) using commercial kit “Oncoscreen” (GenoTechnology, Moscow, Russia). Reverse transcription was performed at 70°C for 3 min and 37°C for 90 min.
For quantitative estimation of the mRNA expression of
Sequences of corresponding genes cloned were used as controls. For each sample, the amount of copy numbers of
Results of the immunologic study were presented as the mean ± standard error. Data were analyzed using STATISTICA 6.0 software. All variables were checked for normal distribution with the Kolmogorov-Smirnov test. Student’s
Figure
FTIR spectra of unmodified silica
The materials were studied by small angle X-ray scattering (SAXS) method. The spectra showed a broad peak corresponding to amorphous silica (
According to trypan blue dye exclusion assay data, the viability of macrophages after 24 h incubation with studied silica nanoparticles did not change significantly. It was found to be about 85%–95% after incubation of cells with both UMNPs and AMNPs.
The data presented in Table
The influence of 24-hour incubation of peritoneal macrophages with different types of silica nanoparticles upon functional activity of macrophages.
Parameters, % | Control (RPMI 1640 medium)
|
AMNPs |
UMNPs |
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HLA-DR+ |
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CD11b+ |
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CD95+ |
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CD36+ |
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CD204+ |
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NBT sp |
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NBT st |
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The amount of HLA-DR+, CD11b+, and CD95+ macrophages after its incubation with the silica nanoparticles of all investigated types was similar to that in the control (
We estimated the cellular uptake of UMNPs and AMNPs by peritoneal macrophages after its cocultivation for 1 h and 24 h with FITC-labeled silica nanoparticles using flow cytometry. The data characterizing the intensity of interaction between the peritoneal macrophages and studied silica nanoparticles are presented in Table
Characteristics of the intensity of interaction between peritoneal macrophages and different silica nanoparticles after 1-hour and 24-hour incubation.
Incubation time | AMNPs |
UMNPs |
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1 hour |
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24 hours |
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After incubation of the macrophages with the UMNPs for 1 h, the amount of the immune cells interacting with the nanoparticles was found to be about 82%. After incubation for 24 h, these values were found to be approximately 94% for this type of nanoparticles, and these results were statistically higher than analogous data obtained after 1 h incubation (
These results have been compared with those for the AMNPs (Table
Representative dot plot of macrophages, reacting with FITC-labeled silica nanoparticles after 24 h incubation. (a) Negative control, cells were incubated in the presence of sheep FITC-labeled immunoglobulin, (b) distribution of macrophages according to FITC-negative (low left corner) and FITC-positive cells (low right corner) after 24-hour incubation of macrophages with AMNPs, (c) distribution of macrophages according to FITC-negative (low left corner) and FITC-positive cells (low right corner) after 24-hour incubation of macrophages with UMNPs.
Negative control
Mph + AMNPs-FITC
Mph + UNPs-FITC
At the next step of our work we immobilized the GMDP on unmodified mesoporous silica nanoparticles (UMNPs) and the aminopropyl modified silica nanoparticles (AMNPs) as nanocarriers. The formation of the composites of GMDP with the silica nanoparticles is confirmed by FTIR spectroscopy. Figure
FTIR spectra of free GMDP
The bands at 1658 cm−1 and 1549 cm−1 are the characteristic for GMDP (spectrum 1). They are assigned to amide I band (mainly C=O stretch) and amide II band (C–N stretch coupled with N–H bending mode) [
The effects of free GMDP and the GMDP, immobilized on UMNPs and AMNPs on the membrane expression of scavenger receptors SR-AI (CD204) and SR-B (CD36) by peritoneal macrophages of women with endometriosis, were studied. The obtained results are presented in Table
Comparative characteristics in vitro of effects of free GMDP and GMDP immobilized on UMNPs and AMNPs on spontaneous expression of functional and SR molecules by peritoneal macrophages of women with endometriosis.
Parameter, % | Macrophages + RPMI 1640 medium (control) |
Macrophages + free GMDP |
Macrophages + GMDP immobilized on AMNPs |
Macrophages + GMDP immobilized on UMNPs |
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CD36 + (SR-B) |
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CD204 + (SR-AI) |
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xStatistically significant differences between data received for free GMDP and GMDP, immobilized on AMNPs (x
yStatistically significant differences between data received for GMDP, immobilized on AMNPs and UMNPs (yy
As we can be seen from Table
The influence of GMPD, immobilized upon the different silica nanoparticles, and free GMPD on the NOD2, RAGE, and SOCS1 mRNAs expression by peritoneal macrophages of women with endometriosis. (a) The influence of GMPD on NOD2 mRNA expression. (b) The influence of GMDP on RAGE mRNA expression. (c) The influence of GMPD on SOCS1 mRNA expression. (Notes: C—control (incubation in RPMI 1640 medium only), GMDP—free form of GMDP, AMNPs-GMDP, immobilized onto the AMNPs, UMNPs-GMDP, immobilized onto the UMNPs, results are presented as the mean ± standard error; *differences in comparison to the control values are statistically significant,
We found that after 24 h incubation of macrophages with free GMDP the insignificant increase of NOD2 mRNA expression by macrophages was observed comparing to the control values. After immobilization GMDP onto the AMNPs, the significant increase both of NOD2 and RAGE mRNAs expression by peritoneal macrophages of women with endometriosis was seen (
We also studied the action of GMDP upon synthesis of SOCS1 by peritoneal macrophages (Figure
Our experiments in vitro showed that after 24-hour incubation of the peritoneal macrophages with free GMDP the level of MMP-9 mRNAs expression slightly increased in comparison to the control values (
The influence of free GMPD and GMPD, immobilized on different silica nanoparticles, upon the MMP-9 and TIMP-1 mRNAs expression by peritoneal macrophages of women with endometriosis. (a) The influence of GMPD on MMP-9 mRNA expression. (b) The influence of GMDP on TIMP-1 mRNA expression. (Notes: C—control (incubation in RPMI 1640 medium only), GMDP—free form of GMDP, AMNPs-GMDP, immobilized onto the AMNPs, UMNPs-GMDP, immobilized onto the UMNPs, results are presented as the mean ± standard error; *differences in comparison to the control values are statistically significant,
No significant effect of free GMDP on the expression of TIMP-1 was observed. Incubation of the peritoneal macrophages with GMDP immobilized on AMNPs resulted in significantly increase of the MMP-9 mRNA expression in comparison to that for the unstimulated macrophages (Figure
On the contrary, incubation of the macrophages with GMDP, immobilized on UNPs, led to the decrease of TIMP-1 mRNA expression by the macrophages (
The obtained results have demonstrated the possibility of application of hybrid silica nanomaterials for topical delivery of GMDP to the peritoneal macrophages. Our studies in vitro have showed that the immunomodulatory effect of GMDP can be intensified by the immobilization of GMDP on silica nanoparticles. Two types of the silica nanoparticles have been investigated as nanocarriers for the drug: unmodified silica nanoparticles (UMNPs) and modified by aminopropyl groups silica nanoparticles (AMNPs). In vitro studies have showed that although UMNPs, prepared by sol gel synthesis using sugar as template, exhibit a high ability to interact with the peritoneal macrophages, they cannot be applied as nanocarriers for topical delivery of GMDP to the peritoneal macrophages of women with endometriosis. The immobilization of GMDP on the nanoparticles of this type led to the complete suppression of the stimulatory effect of GMDP on the expression of PRRs molecules and synthesis of proteolytic enzymes from the matrix metalloproteinases (MMPs) family. The topical delivery of GMDP to the peritoneal macrophages by AMNPs led to the enhance the initially reduced membrane expression of SRs by the macrophages of women with endometriosis, increased NOD2 and RAGE mRNAs expression, and also promoted an increase of the expression of MMP-9 mRNA. Simultaneously elevated synthesis of factors, preventing undesirable overactivation of peritoneal macrophages (SOCS1 and TIMP-1) was observed after the action of GMDP, immobilized onto AMNPs. The effect of the AMNPs immobilized GMDP in some cases exceeded the effect of free GMDP. Thus, comparison of two types of silica nanoparticles as possible nanocarriers for topical delivery of immunomodulatory drug GMDP in peritoneal macrophages has demonstrated that AMNPs are the most suitable nanocarrier for the drug.
The authors of the paper do not have a direct financial relation with the commercial identities mentioned in the paper that might lead to a conflict of interests for any of the authors.
This work was supported by Grant of Russian Fond of Basic Research 12-04-97528-r_center_a.