The formulation parameters were designed to produce stable monodisperse particles of 100 to 200 nm and with high drug loading. The excipients used in the formulation were selected after careful consideration based on previous data from our lab, literature, and most importantly based on the technical requirements of the International Council for Harmonization (ICH) guidelines for pharmaceuticals for human use. Acetone was used as organic phase for the following reasons: It is a good solvent for both polymers (i); it is miscible with water, which is a pre-requisite for solvents used in nanoprecipitation (ii) [21]; it can be easily removed from the formulation by evaporation at room temperature (iii); and according to the ICH, acetone is a Class 3 solvent with a low toxicity [22]. Next, for the solubilization of the drug, due to the high lipophilicity of BRP-187, the choice of the solvent was limited only to dimethylsulfoxide (DMSO) and dimethylformamide (DMF). Thus, considering that DMF is more toxic than DMSO (residual concentration limit 880 ppm vs. 5000 ppm, respectively), the latter was selected for the formulation [22]. Meanwhile, the residual amount of DMSO in our NP formulations was < 250 ppm. Furthermore, the volumetric ratio of organic-to-aqueous phase was kept at 1:8 to produce particles < 200 nm with an encapsulation efficiency EE > 50%, higher volumetric ratios produce NPs > 200 nm [23]. Partially-hydrolyzed poly(vinyl alcohol) (PVA) was used as surfactant and cryoprotectant to prevent aggregation during purification and lyophilization, respectively. We have previously demonstrated that PVA provided a superior stability of PLGA NPs than poloxamers and polysorbates at concentrations < 0.5%, and no toxicity was evident even at 100-fold higher concentrations [10].
2.1 Characterization of NPs
Initial experiments revealed that NPs prepared by the nanoprecipitation method had a higher EE than NPs prepared by the emulsion-evaporation method. In addition, the nanoprecipitation method is favored because it is a low-energy method with an easy operation that can be easily adapted to large-scale production batches [24]. The size and polydispersity (PDI) of BRP-187-loaded NPs as well as unloaded control NPs were analyzed after purification and after lyophilization, and the zeta potential (ζ) was measured after lyophilization (Table 1). The average hydrodynamic diameter (dH) of the final NPs was between 130 to 211 nm with PDI values of 0.09 to 0.28. PLGA NPs were in average up to 50 nm smaller than Acdex NPs.
Table 1
Overview of the NP properties.
NP formulation | After purification | After lyophilization | EE [%] | LC [%] | PVA [%] |
dH [nm] | PDI | dH [nm] | PDI | ζ [mV] |
Acdex | 210 ± 26 | 0.17 ± 0.07 | 211 ± 35 | 0.26 ± 0.09 | -12 ± 2 | / | / | 0.01 ± 0.000 |
Acdex[BRP-187] | 196 ± 51 | 0.16 ± 0.11 | 178 ± 26 | 0.13 ± 0.07 | -13 ± 8 | 59 ± 23 | 1.7 ± 0.6 | 0.01 ± 0.000 |
Acdex-RhodB[BRP-187] | 163 ± 15 | 0.20 ± 0.06 | 189 ± 37 | 0.28 ± 0.11 | -24 ± 2 | 67 ± 10 | 2.0 ± 0.3 | n.m. |
PLGA | 124 ± 6 | 0.06 ± 0.03 | 130 ± 2 | 0.09 ± 0.03 | -20 ± 2 | / | / | 0.02 ± 0.002 |
PLGA[BRP-187] | 153 ± 41 | 0.17 ± 0.14 | 158 ± 35 | 0.12 ± 0.06 | -15 ± 3 | 76 ± 22 | 2.2 ± 0.6 | 0.03 ± 0.002 |
PLGA-DY635 | 143 ± 3 | 0.08 ± 0.03 | 154 ± 5 | 0.15 ± 0.02 | -20 ± 1 | / | / | 0.02 ± 0.006 |
PLGA-DY635[BRP-187] | 153 ± 2 | 0.12 ± 0.01 | 168 ± 9 | 0.19 ± 0.05 | -19 ± 1 | 87 ± 6 | 2.5 ± 0.2 | 0.02 ± 0.002 |
dH = Hydrodynamic diameter obtained by DLS measurements. EE = encapsulation efficiency. LC = loading capacity. Concentration of NPs used for the PVA assay was 3 mg mL− 1. SD for all measurements n ≥ 3. n.m. – not measured. |
SEM imaging showed a spherical morphology of the NPs and smaller NP sizes compared to the results acquired by DLS, a common phenomenon when using orthogonal characterization techniques (Fig. 1) [25]. The size of the NPs measured by SEM was as follows: Acdex 95 ± 11 nm, Acdex[BRP-187] 73 ± 8 nm, PLGA 96 ± 11 nm, and PLGA[BRP-187] 84 ± 6 nm. Furthermore, EE of all NPs is given in Table 1 and was roughly 60% for Acdex particles and 80% for PLGA particles. Based on previous experiments, a drug-to-polymer content > 3% (w/w) fed in the formulation resulted in problems with the stability of the suspension (data not shown), a phenomenon that was also reported by others [26–28]. Meanwhile, the conditions used in this protocol (3%, w/w) were effective to encapsulate more than 60% of the drug without compromising the stability of the NPs. In addition, BRP-187 is a highly potent drug (IC50(FLAP) = 8 nM and IC50(mPGES-1) = 200 nM) [7], and a loading capacity of 1.7 to 2.5% corresponded to 37 to 55 µM of BRP-187 in 1 mg mL-1 NP suspension. Here, it was observed that Acdex formed larger particles but encapsulated less drug compared to PLGA, which is probably due to different drug-polymer interactions [26].
2.2 Degradation profile of the nanoparticles
In DLS, the count rate corresponds to the number of the light photons detected in kilo-count per seconds (kcps), which is a good indicator of the quality of the measured sample [29]. A decreasing count rate indicates that less photons are detected (i.e. less light is scattered) [29] and, thus, less particles are present in a sample. In such a measurement, the NPs should show stable size and PDI values (100% intact NPs) at time point 0 of NPs incubated with buffer (Fig. 2A and C). As the NPs start to degrade, they steadily increase in size and polydispersity, which is a result of the degraded products dissolved in water (Fig. 2B and D). Consequently, the count rate decreases over time as the degradation of the NPs proceeds.
The release of a molecule from a NP polymer matrix depends on several factors, i.e. the structure-property relationship between the drug and the polymer, hydrophobicity of the drug and the polymer, as well as the degradation rate, melting point and crystallinity of the polymer [30]. The degradation rate of the polymeric NPs in a cell-free environment was studied since it directly influences the release kinetics of the drug from the NP core. According to the literature, Acdex is an acid-labile polymer with a considerably higher pH sensitivity compared to the PLGA polymer [17, 31]. Our results revealed that after
10 h of incubation Acdex NPs exhibit good stability at pH 7.4 showing only swelling of the NPs, whereas at the same pH value the Acdex[BRP-187] NPs degraded to a degree of about 25%. Furthermore, at pH 4.8 after 50 min, the Acdex NPs degraded by only about 30%, whereas the Acdex[BRP-187] NPs degraded by 75% (Fig. 2A). This degradation behavior is suitable since NPs maintain 75% stability at physiological conditions. However, once internalized by the cells, the acidic environment of the endolysosome would trigger the degradation of Acdex, thereby releasing the drug. In case of PLGA, a complete degradation of NPs was observed at pH 4.8 within 40 days, whereas at pH 7.4, an 80% degradation was observed after 60 days (Fig. 2C), which is in line with previous studies [32–34]. Considering the degradation profile of the polymers in an acidic medium, the release of BRP-187 from Acdex NPs is expected to be fast due to the rapid degradation of the polymer, whereas the drug release from PLGA NPs is expected to be slower due to the diffusion of drug from the polymer matrix, since degradation of this polymer is very slow [35]. The degradation studies of the NPs at different pH values were investigated to obtain a first impression on the release of the drug from the NPs. However, it should be noted that such degradation profiles differ from the more complex environment of the endolysosomes [36].
2.3 Residual amount of PVA
Previously, we described effects of surfactants on the stability of drug-loaded NPs, where we demonstrated that concentrations of ≤ 1% (w/v) PVA are desirable to formulate stable particles [10]. Here, we formulated NPs using 0.3% (w/v) PVA to obtain both suspension- and cryo-stability. The amount of residual PVA in the final NPs is listed in Table 1. The enzymatic- and pH-dependent degradation of the NPs relies not only on the properties of the polymer itself but is also strongly influenced by the digestibility of the surfactant. PVA coating can protect from enzymatic hydrolysis of the NPs by decreasing the wettability of the NPs [37], thus influencing the rate of degradation and drug release.
2.4 Fluorescence dye-labeled nanoparticle uptake in PMNL
PMNL are pro-inflammatory innate immune cells that are abundant in the blood and produce substantial amounts of LTs and also PGE2 as targets for BRP-187 [7, 38]. Therefore, we used human PMNL as relevant cells to study the uptake of BRP-187-loaded NPs that were covalently labeled with fluorescent dyes (i.e., PLGA-DY635, Acdex-RhodB) for visualization in the cells. The dye-labeled NPs (loaded with BRP187) were efficiently taken up by PMNL, which depends however on the nature of the polymer, being superior for PLGA over Acdex (Fig. 3). Within 10 min, 40% of the PMNL digested PLGA-DY635[BRP-187] NPs. After 180 min, approx. 85% of the PMNL took up these NPs along with a concomitant increase of the mean fluorescence intensity (MFI) per PMNL up to 1937 ± 283. Thus, PLGA NPs display an excellent cellular uptake. In contrast, Acdex-RhoB[BRP-187] NPs were taken up by only 23% of the PMNL after 120 min, with an even slightly lower uptake after 180 min, correlating with the MFI per cell over the entire time course (Fig. 3). According to the degradation profile of Acdex NPs (Fig. 2), the lower abundance might be a consequence of the concomitant degradation and elimination of the polymer inside the cell. After 2 h, the Acdex NPs are mostly degraded at the endolysosomal pH of approx. 4.8. Therefore, the fluorescence signal may not increase further, even though NPs are still taken up, because the elimination of the labeled monomers from the cell is ongoing. However, both PLGA- and Acdex-based NPs are rapidly taken up by PMNL, even though PLGA NPs are ingested by a higher fraction of cells as compared to Acdex NPs. Additionally, we used confocal laser scanning microscopy to confirm the cellular internalization of NPs into PMNL (Fig. 4). PLGA-DY635[BRP-187] NPs show a time-dependent accumulation within the cells which appears as an increase of the intracellular fluorescence signal over time that is most prominent after 3 h. For Acdex-RhoB[BRP-187] NPs a comparable signal was already observed after 30 min. Remarkably, the NP-uptake after 30 min, as measured by the corresponding fluorescence intensity signal, does not further increase upon longer incubation up to 3 h.
2.5 Encapsulation of BRP-187 into PLGA or Acdex NPs is not detrimental for target cells
Next, we evaluated whether or not the NPs may cause detrimental effects upon long-term incubation (≥ 24 h) towards relevant target cells. Since PMNL are short-lived cells upon isolation being not suitable for long-term cytotoxicity tests, human monocyte-derived macrophages were used since they also possess the capability to produce PGE2 and LT [39]. None of the NP formulations (nor free BRP-187) showed detrimental effects on the viability of macrophages (with M1 or M2 phenotype) in terms of damage of the cell membrane over 24 h as measured by LDH assay (Fig. 5). In supportive experiments using a RAW264.7 macrophage cell line that was incubated for 72 h with the NPs, an MTT assay revealed also no significant cytotoxic effects (data not shown). Thus, the NPs exert no detrimental effects against relevant target cells at concentrations used in functional assays.
2.6 Effect of encapsulated BRP-187 on 5-LO product formation in PMNL
BRP-187 (1 µM) efficiently suppressed 5-LO product formation in isolated PMNL upon short preincubation periods ≤ 2 h (Fig. 6A,B,C), which is in agreement with our previous data [7]. However, after prolonged preincubation (5 h) with PMNL, the efficiency of BRP-187 (1 µM) was clearly reduced and suppression of 5-LO product formation was only 37 ± 5% of the control (Fig. 6D). Of interest, BRP-187 encapsulated into PLGA or Acdex NPs (corresponding to 1 µM BRP-187 as well) potently and consistently inhibited 5-LO product formation in PMNL by 80 to 92%, even after a preincubation period of 5 h. Note that NPs devoid of BRP-187 did not suppress 5-LO activity in PMNL. Therefore, we conclude that encapsulation of BRP-187 into PLGA or Acdex NPs generally accomplishes efficient inhibition of 5-LO product formation in PMNL, and, moreover, allows to overcome the loss of potency of BRP-187 upon prolonged exposure (i.e. 5 h) of PMNL.
2.7 Effect of encapsulated BRP-187 on PGE2 formation in human M1 macrophages
Human M1 macrophages express high levels of mPGES-1 [40] and, upon exposure to pathogenic E. coli, produce high amounts of pro-inflammatory PGE2 [39]. Thus, E. coli-stimulated M1 macrophages are a suitable cell model to study the efficiency of mPGES-1 inhibitors. Note that many mPGES-1 inhibitors are highly potent in cell-free assays but markedly loose efficiency in cellular assays or in vivo [41], which necessitates technological approaches to overcome these hurdles. BRP-187 potently inhibited mPGES-1 in a cell-free assay (IC50 = 0.2 µM) [7], however, pretreatment of human M1 with 1 µM BRP-187 caused only moderate inhibition (30–37%) of E. coli-induced PGE2 formation regardless of the preincubation period (15 min, 5 or 20 h). In our study, encapsulation of BRP-187 into PLGA NPs strongly suppressed PGE2 levels at short (15 min) and prolonged (20 h) preincubation periods (Fig. 7). Also, Acdex [BRP-187] NPs caused strong reduction of PGE2 formation when M1 were preincubated for 20 h, while 15 min pretreatment was not effective. These data are in line with the cellular uptake pattern of the NPs, where PLGA NPs surpassed the uptake efficiency of Acdex NPs. Note that again, as for 5-LO product formation in PMNL, the empty NPs did not suppress PGE2 biosynthesis.
In summary, encapsulation of BRP-187 in PLGA and Acdex NPs overcomes the loss of effectiveness against mPGES-1 in intact cells versus cell-free assay conditions and confers the drug marked potency, highlighting this technological approach for efficient interference with pro-inflammatory PGE2 and LT formation in human cells. The beneficial effect of encapsulation of BRP-187 especially after prolonged incubations up to 20 h might be related to better stability and delayed release inside the cell. Intriguingly, encapsulation of BRP-187, particularly in PLGA-based NPs, accomplished efficient mPGES-1 inhibition in intact M1 macrophages, which was not the case for the free drug. It is conceivable that PLGA is cleaved in close proximity to the endoplasmic reticulum where mPGES-1 is located, thus, enabling unhindered access of BRP-187 to its target protein without being bound to other cellular membranes or cell compartments.