Modulation of immunosuppressive effect of rapamycin via microfluidic encapsulation within PEG-PLGA nanoparticles

Materials

All chemicals were purchased as reagent grade from Aladdin (China) and used as received unless stated otherwise. PEG-PLGA was purchased from Chongqing Yucai (China). Rapamycin was purchased from Fujian Kerui (China). All of chemicals, solvents, and reagents used for HPLC analysis were HPLC grade. All reagents were used without further purification. CCK-8 reagent was purchased from Dojindo (Japan). Membrane protein extraction kits were purchased from Proteintech (China). All ELISA kits were purchased from Adamas-life (China).

Design and fabrication of PDMS microfluidic channels

AutoCAD software was applied to design microfluidic channels. The silicon wafer was micro-nano-processed to obtain a channel mold. PDMS (30 g) and glidant (3 g) were weighed and mixed uniformly. After the bubbles were exhausted with the freeze dryer, the degassed mixture was poured onto the mold silicon plate and degassed again using a freeze dryer. After that, the mixture was placed on a 60°C oven for 2 h to cure to obtain a PDMS channel layer. The PDMS plates and glass slides were bonded by plasma treatment. A steel needle was inserted into the channel and fixed to obtain PDMS chip Figure 1.

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Figure 1.

(a) A graphic of a microfluidic channel. The side-channels are the aqueous phase, and the middle channel is the organic phase. (b) Flow-chart of PDMS chip processing. The designed channel pattern is fabricated onto the PDMS chip. (c) Illustration of Rapa and P nanoparticles formation.

Formulation of Rapa&P nanoparticles by nanoprecipitation

PEG-PLGA only or with rapamycin were dissolved in acetonitrile at concentrations of 25 mg/mL to obtain the organic phase. It was sonicated for 2 min to dissolve. The PEG-PLGA with different PLGA block were used. The MW of PEG block was 5 k, while the MW of PLGA block was 20 k, 50 k, and 95 k, respectively. The organic phase was drawn with a 10 mL syringe for later use. Poloxamer 188 was dissolved in ultrapure water under magnetic stirring to obtain a final concentration of 0.5% (w/v). The aqueous phase was drawn with a 30 mL syringe for later use.

The organic phase as the dispersed phase and the aqueous phase as the continuous phase were injected into the channel of the inner phase and the outer phase by a needle tube and PTFE tubing, respectively. The volume flow rates of organic phase (QI) and aqueous phase (QII) were set to be 5 mL/h and 100 mL/h by syringe pumps with glass syringes, respectively. Nanoparticles were formed in the microfluidic chip. The rapamycin encapsulated nanoparticles Rapa&P-20 k, Rapa&P-50 k, Rapa&P-95 k and empty nanoparticles P-20 k, P-50 k and P-95 k were collected, concentrated and washed with deionized (DI) water by ultrafiltration (MW = 30,000, 6000 r/min, 15 min) and stored for further use.

As for the preparation of nanoparticles by the bulk method, the aqueous and organic phases were directly mixed in a beaker in the same proportion as described above, and the nanoparticles were collected through the same post-treatment.

Characterization of nanoparticles

The hydrodynamic diameter, polydispersity index (PDI) and Zeta potential were measured by using dynamic light scattering technique with zeta sizer (Nano-ZS90, Malvern Instruments) and analyzed by Zetasizer software, using a material refractive index of 1.590 and dispersant (water at 25°C) refractive index of 1.330. All the formulations were properly diluted with deionized water and shaken to get sufficient count rate. The morphological features of the nanoparticles were observed with a scanning electron microscope (SEM, Nova Nano SEM 450, FEI, USA) and a transmission electron microscope (TEM, JEM-2100, JEOL, Japan). The absorption spectra in the infrared region for the samples, within the range of 4000 cm⁻¹ to 500 cm⁻¹, were obtained in a Nicolet 6700 FTIR spectrometer (Thermo Fisher SCIENTIFIC). The spectra were obtained at room temperature (20°C) with the sample added directly into the device.

Encapsulation efficiency and drug loading

A certain amount of harvested nanoparticle solution were dried with a freeze dryer. The nanoparticles were weighed and then dissolved with acetonitrile. The concentration of rapamycin in the solution was determined by HPLC.

A high performance liquid chromatography based method was developed to measure the amount of loaded rapamycin in nanoparticles using an automated HPLC system (LC 20AT, SHIMADZU, Japan) containing a pump and an auto sampler. Samples (20 μL) were injected into a C8 column (250 × 4.6 mm², Agilent, USA), and heated to 50°C by a column heater. The mobile phase (70/30 acetonitrile in water) was pumped at a flow rate of 1 mL/min. Rapamycin was detected at 278 nm by a UV detector (SPD 20A, SHIMADZU, Japan). Rapamycin concentration was obtained from the regression line relating the peak-area to rapamycin concentrations.

The drug loading and encapsulation efficiency were calculated using the formula equations:

Rapamycin loading (w/w%) = [(amount of loaded rapamycin in mg)/(amount of polymer in mg)] X100

Encapsulation efficiency (%) = [(amount of loaded rapamycin in mg)/((amount of rapamycin added in mg)] X100

In vitro release of rapamycin from Rapa&P nanoparticles

In vitro release of Rapamycin from Rapa&P nanoparticles was conducted using dialysis tubes. Briefly, 2 mL of concentrated Rapa&P nanoparticles suspension (400 μg/mL) was sealed in dialysis tubes (MWCO = 3500 Da) and immersed in 20 mL of PBS solution (pH = 7.4), respectively, in an incubator shaker at 37°C. At predetermined time intervals, 1 mL of release medium in each vial was withdrawn and replenished with an equal volume of release medium. Finally, the drug concentration in the release medium was determined by the HPLC and the cumulative release was calculated.

Cell culture

Murine dendritic cells (DCs) primary cultures were generated from bone marrow precursors obtained from femurs of C57BL/6 mice following the method described by Lutz et al. ³⁹ Bone marrow derived DCs were cultured for 7 days in EMEM with 10% FBS, 1% P/S at 37°C in a humid atmosphere of 5% CO₂. The 2-4 generations of DCs were used for in vitro experiments. Cell culture medium was refreshed every 3 days.

Cell viability and toxicity studies

The cytotoxic effects of Rapa&P nanoparticles were conducted in DCs by CCK-8 assay. DCs were seeded in a 96-well plate (Corning Costar Co., USA) at 5 × 10³ cells/well. The cells treated with regular culture medium or medium contain LPS were used as negative and positive control respectively. 24 h after LPS stimulation, culture medium was added with free rapamycin or rapamycin-loaded nanoparticles, respectively and cultured for another 48 h. At predetermined time points, 100 µL fresh medium and 10 µL CCK-8 agent were used to replace the conditioned medium. After incubation in darkness for 2 h, the optical density (OD) at the wavelength of 450 nm (OD 450) was measured following the manufacturer’s instructions.

Detection of costimulatory molecules by protein extraction

DCs were seeded in a 12-well plate at a density of 5 × 10⁵ cells/mL. The cells treated with regular culture medium or medium contain LPS which were used as negative (briefed as Ctrl group) and positive controls (briefed as LPS group), respectively. At 24 h after LPS stimulation, culture medium containing 50 μg/mL nanoparticles or 500 ng/mL free rapamycin was added (briefed as Rapa group), respectively. After 48 h incubation, the supernatant was collected for cytokine detection (section 2.10), and the remaining cells were washed with PBS buffer solution to remove excess drugs and medium for detection of costimulatory molecules. Protein extraction kits were used to extract membrane proteins following the manufacturer’s instructions. CD40 and CD80 ELISA kit were used to detect the level of costimulatory molecules.

Cytokine detection

DCs were seeded in a 12-well plate at a density of 5 × 10⁵ cells/mL. The cells treated with regular culture medium or medium contain LPS which were used as negative (briefed as Ctrl group) and positive controls (briefed as LPS group), respectively. At 24 h after LPS stimulation, culture medium containing 50 μg/mL nanoparticles or 500 ng/mL free rapamycin was added (briefed as Rapa group), respectively. After 48 h incubation, the supernatant was collected for cytokine detection. Cytokine detection was performed according to the manual of the ELISA kit. Briefly, supernatant was collected from the culture medium of the pretreated DCs. Three parallels were set for each concentration, and each cytokine standard curve was prepared according to the instructions. After incubating the samples with IL-1β, IL-12 and TGF-β1 ELISA kit, respectively, the samples were detected at a wavelength of 450 nm to obtain the OD values. The standard curves were fitted to calculate the corresponding sample concentrations to analyze the concentrations of IL-1β, IL-12 and TGF-β1.

Statistical analysis

All the experiments were performed at least in triplicates. The results were expressed as mean value ±standard deviation (SD). Analysis was conducted using Graphpad Prism 9 software. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test. Differences between each group and the control group which were statistically significant were indicated by “*” and those which were not statistically significant were indicated by “ns”. The significant level was established at *p < .05, **p < .01, ***p < .001, respectively.

Characterization of Rapa&P nanoparticles

For this study, the acetonitrile and PEG-PLGA system was used for the nanoparticle formulation via microfluidics, and the traditional bulk method was also performed as a control. A comparison of particle size, PDI and Zeta potential properties of empty PEG-PLGA nanoparticles and rapamycin-loaded nanoparticles obtained by microfluidics and bulk method was made in Table 1. It was shown that the average size of the pristine nanoparticles prepared by the microfluidics was ranged from 62.01 nm to 104.00 nm, with Zeta potential ranging from −17.6 mV to −22.2 mV. The size of the pristine nanoparticles prepared by the bulk method was ranged from 74.13 nm to 141.90 nm, with Zeta potential ranging from −15.9 mV to −23.0 mV. Upon the encapsulation of rapamycin within the PEG-PLGA nanoparticles via microfluidics, the size of the respective nanoparticles was larger than the one of pristine nanoparticles (i.e. 99.95 nm for Rapa&P-20k vs 62.01 nm for P-20 k). The same trend was observed for those prepared by bulk method. Moreover, the size of the same type of nanoparticles obtained via microfluidics was smaller than the one via the bulk method, that is 62.01 nm (P-20 k by microfluidics) versus 74.13 nm (P-20 k by bulk method). The results by microfluidics were more stable than those by the bulk method.

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Table 1.

Properties of nanoparticles obtained by microfluidics or bulk method.

	Microfluidics			Bulk method
	Size (nm)	PDI	Zeta potential (mV)	Size (nm)	PDI	Zeta potential (mV)
P-20k	62.01 ± 1.07	0.181	−17.6 ± 0.82	74.13 ± 2.11	0.139	−15.9 ± 0.50
P-50k	104.00 ± 0.50	0.093	−19.7 ± 0.60	122.90 ± 6.48	0.058	−16.9 ± 0.78
P-95k	83.08 ± 0.76	0.149	−22.2 ± 0.20	141.90 ± 7.21	0.117	−23.0 ± 0.80
Rapa&P-20k	99.95 ± 1.61	0.247	−14.5 ± 1.80	115.80 ± 2.18	0.270	−13.9 ± 0.67
Rapa&P-50k	123.30 ± 5.27	0.128	−19.1 ± 1.86	149.10 ± 8.63	0.120	−15.7 ± 0.89
Rapa&P-95k	116.0 ± 2.07	0.122	−20.3 ± 1.06	176.40 ± 9.01	0.143	−18.5 ± 0.35

As to the current acetonitrile and PEG-PLGA system, nanoprecipitation was the main mechanism for particles formation. During the nanoprecipitation process, the nuclei developed first when the polymer reached oversaturation due to the diffusion of the solvent into the non-solvent, and then the nuclei grew larger with molecular deposition of polymer chains on the surface.^41,42 Microfluidics utilized micron-level channels that allowed the self-assembly of nanoparticles to occur in tiny and fixed spaces. The fluids mixed rapidly, and then rapid mass transfer allowed the nucleation and growth of nanoparticles to occur in a very short period. The growing of nanoparticles into larger particles was limited due to the presence of micro-channels, leading to the reduction in nanoparticle sizes. Consequently, the particle size of nanoparticles prepared by microfluidics was relatively smaller than that of nanoparticles prepared by bulk method.

It was shown that the particle size of nanoparticles prepared by bulk method was enlarged along with the increase in the molecular weight of PLGA block. The increase in hydrophobic block made the polymer more hydrophobic and thus the molecules aggregated more easily, which was consistent with the previous studies. ⁴³ Surprisingly, however, the nanoparticles prepared by microfluidics performed differently. The particle size of P-50 k nanoparticles was the largest among the three. It might be caused by the fast mass transfer in microfluidics. Due to the extremely fast diffusion of molecules, the nanoparticle cores grew in a very short time. When the molecular weight was low, it might be the growth of nanocore that acted as the main factor to control the size of nanoparticle nucleation due to the relatively slow growth of nanocore. The size of nanoparticles prepared from polymers of higher molecular weights showed greater sensitivity to the mixing time. ⁴⁴ For molecules of higher molecular weights, molecules nucleated faster with higher hydrophobicity and had smaller particle sizes during the early stages of nucleation. ⁴⁵ The growth process was constrained by the mixing time in microfluidics, ⁴⁶ making it difficult to grow into larger particles relying on high hydrophobicity. In such case, the rate of nucleation would mainly limit the particle size. Due to excessive oversaturation, the nucleation rate was expected to be enhanced, directly leading to a decrease in size. These results were based on the conditions of high microfluidic flow rate and high non-solvent to solvent flow rate ratio in our study, which enhanced the yield and facilitated the large-scale production of nanoparticles. High microfluidic flow rate and flow rate ratio could allow a very short mixing time, which led to a very quick nanoparticle growth process and constrained the growth of nanoparticles. Hence, the nucleation process of nanoparticles limited the particle size. On the other hand, at lower flow rate and flow rate ratios, the nanoparticle particle size would increase with the increase in molecular weight of PLGA block, which was demonstrated in previous studies.^47,48

Briefly, due to the better controllability of microfluidics than the bulk method, the following experiments were thus focused on the microfluidics to prepare nanoparticles.

The DL and EE of nanoparticles prepared by microfluidics were shown in Table 2. It could be seen that P-20k had the lowest amount of rapamycin loaded with the lowest encapsulation efficiency. P-50 k had the highest amount of rapamycin loaded with the highest loading efficiency. P-20 k molecules had relatively stronger hydrophilicity and weaker hydrophobicity, leading to the poorer performance of encapsulating the hydrophobic drug rapamycin, while P-50 k and P-95 k had higher hydrophobicity, thus allowing the better encapsulation of rapamycin with encapsulation efficiency higher than 80%. However, P-95 k did not exhibit a better effect than P-50 k, probably due to the very short mixing time aforementioned. The fast nucleation rate and inadequate growth time of P-95 k nanoparticles did not allow it to encapsulate more drug than P-50 k nanoparticles. Additionally, the ability to load rapamycin might be weaker due to the reduced size of the nanocores, as there was less free space within the particle core for rapamycin. ⁴⁹ Overall, the ability of nanoparticles to encapsulate rapamycin was influenced by hydrophobicity, particle size, and mixing time. In this study, it was shown that the highest rapamycin encapsulation efficiency was achieved by P-50 k nanoparticles and the overall drug loading performance of all the current chosen PEG-PLGA molecules was maintained at a high level.

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Table 2.

DL and EE of Rapa&P nanoparticles.

	DL (%)	EE (%)
Rapa&P-20k	10.45 ± 1.15	69.70 ± 7.68
Rapa&P-50k	12.68 ± 1.66	84.55 ± 11.05
Rapa&P-95k	12.24 ± 1.25	81.62 ± 8.32

It was shown in Table 1 that Zeta potential of nanoparticles either by microfluidics or by bulk method was increased with the increase in the PLGA segment. It was reported that the Zeta potential of nanoparticles decreased with the increase in the PEG content, which was due to the shielding effect of PEG molecular layer. ⁵⁰ PEG covers the surface of nanoparticles, and nanoparticles with high PEG content have a higher PEG covering density, resulting in the formation of charge shielding layer, which reduces Zeta potential. This makes the nanoparticles invisible, preventing them from being recognized by the immune system and thus extending the cycle time. ⁵¹ Therefore, in the current study, with the increase in PLGA segment, the relative PEG content was reduced, which led to the increase in the Zeta potential of nanoparticles. The encapsulation of rapamycin would further decrease the Zeta potential of nanoparticles.

The morphology of nanoparticles was observed by TEM as shown in Figure 2a. All the rapamycin-loaded nanoparticles had a clear corona structure, formed most probably by PEG covering.^52,53 There was no significant difference in the morphology of different groups of nanoparticles. It was then shown by SEM images (Figure 2(b)) that nanoparticles were well distributed without serious aggregation. Morevoer, the FT-IR spectrum of Rapa&P-50 k was similar to that of P-50 k, with only a change at 1643 cm-1, which might be attributed to the stretching vibration of amide C = O in rapamycin (Figure S1).

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Figure 2.

Morphology of Rapa&P nanoparticles by (a) TEM and (b) SEM.

In vitro release of rapamycin from Rapa&P nanoparticles

In vitro drug release was performed by dialysis tubes (MWCO = 3500 Da) in PBS solution. The usage of dialysis tubes could allow migration of rapamycin and release medium but not nanoparticles. The corresponding release profiles of drugs from the Rapa&P nanoparticles were shown in Figure 3. The drug release curves of the three types of nanoparticles were all quite smooth, indicating that there were no obvious initial burst release, which could make the drug release processes more controllable. The whole duration could be lasted for more than 1 month.

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Figure 3.

In vitro release of rapamycin from Rapa and P nanoparticles in the PBS solution (pH = 7.4) at 37°C.

Comparing the release profiles of the three Rapa&P nanoparticles, Rapa&P-20 k nanoparticle was the one with the fastest release rate. At day 30, the rapamycin cumulative release of Rapa&P-20k reached around 75%, while both Rapa&P-50k and Rapa&P-95k reached around 60%. The release of rapamycin by Rapa&P-50k and Rapa&P-95k were almost synchronized from the beginning to the 34th day of release, indicating that with the increase in the molecular weight of PLGA from 20 k to 50 k, the release was slowed down, whereas the further increase from 50 k to 95 k did not render significant effect on the release pattern.

The molecular weight of hydrophobic block of P-20 k was the smallest among the three, thereby its interaction force with the hydrophobic drug, rapamycin, was the weakest. In the free space of the nanoparticle core, rapamycin was subject to weaker binding force and was more likely to undergo free motion. Hence, its release could be fastened in comparison to the others. Rapa&P-95 k should had the stronger hydrophobic force than Rapa&P-50 k, but its size was smaller than Rapa&P-50 k (the size of Rapa&P-95 k was 116 nm, while the size of Rapa&P-50 k was 123 nm). Therefore, the specific surface area for Rapa&P-95 k was larger, which might enhance the diffusion and hydrolysis mediated degradation of nanoparticles. Consequently, the two had the comparable drug release performance.

Cell viability and toxicity studies

According to ISO 10,993-5, materials designed for biomedical applications that show cell viability above 80% are classified as slight cytotoxicity, above 50% as mild cytotoxicity and below 50% as moderate toxicity, respectively. ⁵⁴

Cytotoxicity assay of these nanoparticles on DCs was performed by CCK-8 method. The viabilities of cells after being treated with nanoparticles or free rapamycin for 48 h were shown in Figure 4, and the cells treated with regular culture medium or medium containing LPS were used as negative and positive controls, respectively. When the concentrations were at 100 µg/mL or lower, the nanoparticles were classified as slight cytotoxicity (Figure 4(a)–(f)), while the safe concentration of free rapamycin was 0.5 µg/mL (Figure 4(g)).

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Figure 4.

Cell viability and toxicity studies by CCK-8 assay. In vitro cytotoxicity assay of Rapa&P, P (empty nanoparticles) and free rapamycin on DCs. DCs were incubated with different treatments for 48 h, and the respective viability of cells was determined by CCK-8 assay.

As shown in Figure 4(a)–(c), the toxicity of all the drug-loaded nanoparticles increased with the increase in the nanoparticle concentration. At around 500 µg/mL, the viability dropped to around 70%. The empty nanoparticles were also subjected to the same assay and it was shown that there was almost no cytotoxicity observed at concentrations at 100 µg/mL or lower (Figure 4(d)–(f)). However, at 500 µg/mL, the viability dropped significantly and reached to the same level as that of the respective rapamycin loaded nanoparticles. Free rapamycin exhibited high toxicity to the DCs and was highly dependent on its concentration. At a concentration of 1 µg/mL of free rapamycin, the viability was already dropped to around 70% (Figure 4(g)). Hence, it was suggested that the cytotoxicity of rapamycin loaded nanoparticles might be controlled by two factors. At concentrations at 100 µg/mL or lower, the main factor was the presence of rapamycin, while at 500 µg/mL, both the nanoparticles and rapamycin affected the viability of DCs. However, rapamycin encapsulated within nanoparticles was slowly released from nanoparticles, while the corresponding free rapamycin had effects at once. Hence, the safe concentration of rapamycin for single-dose was actually improved when it was used after being encapsulated within nanoparticles. The drug loading of nanoparticles was around 10%–12% as shown in Table 2, and these nanoparticles were added at a concentration of 100 µg/mL. Hence, the concentration of loaded rapamycin was around 10 µg/mL. The viability of Rapa&P-20 k, Rapa&P-50 k and Rapa&P-95 k was higher than 80%, whereas the one of free rapamycin was below 60%. Therefore, the loading of rapamycin in PEG-PLGA nanoparticles attenuated the cytotoxicity of rapamycin, and sustained its release, thereby facilitating single high-dose, less-frequent administration. Due to the low oral availability of rapamycin, it was often prepared as suspensions or injections. ⁵⁵ The sustained release and reduced toxicity properties shown in this study would therefore greatly facilitate its application.

Expression of costimulatory molecules

The expression of co-stimulatory molecules by LPS-stimulated DCs treated with free rapamycin and rapamycin-loaded nanoparticles was compared. Membrane proteins of DCs were extracted by protein extraction process, and the expression of several key co-stimulatory molecules, such as CD40 and CD80 were detected by quantitative ELISA kits. The comparison was shown in Figure 5.

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Figure 5.

Expression of costimulatory molecules quantified by ELISA assay. “Rapa” represents free rapamycin group. (a) CD40; (b) CD80.

The expression of CD40 and CD80 in LPS-stimulated DCs was enhanced as expected. Upon the further treatment with free rapamycin, expression of CD40 and CD 80 were downregulated significantly to a comparable level with the Ctrl, indicating that rapamycin could reduce the degree of maturity of DCs. This was consistent with the results by Rama et al. ⁵⁶ The expression of co-stimulatory molecules in LPS-stimulated DCs treated with Rapa&P nanoparticles, especially Rapa&P-20k and Rapa&P-50k, was even lower than those of free rapamycin group. Most probably, the nanoparticles had continued release of rapamycin which then had a sustained stimulating effect, making the inhibitory effect stronger than that of free rapamycin. As to Rapa&P-95 k group, it also had an inhibitory effect on CD40 and CD80, but the effect was similar to that of the free rapamycin.

Among all, Rapa&P-50 k nanoparticles had the strongest inhibiting effect and Rapa&P-95 k nanoparticles was the weakest, which was not consistent with data of in vitro release. It might be related to the release rate of rapamycin in vitro and the stimulating effect of nanoparticles as foreign antigens. ⁵⁷ The Rapa&P-95 k nanoparticles released rapamycin slowly and caused weak immunosuppressive effect due to the dose-dependent nature of rapamycin.^56,58 Moreover, the steric hindrance generated by PEG of Rapa&P-95 k nanoparticles was low with lowest PEG content, resulting in easy interaction between nanoparticles and cells and triggering immunogenicity, which led to the maturation of DCs and promoted the expression of costimulatory molecules. The PEG coverage density of Rapa&P-20 k nanoparticles and Rapa&P-50 k nanoparticles was higher, so the shielding effect was strong and the immunogenicity was weak. Theoretically, Rapa&P-20 k nanoparticles released rapamycin the fastest and had the strongest immunosuppressive effect. However, since the drug loading of Rapa&P-20 k nanoparticles was lower, a higher concentration of nanoparticles was required to obtain the same amount of rapamycin. More nanoparticles had the opposite effect because of the stimulating effect of nanoparticles as foreign antigens. The immunosuppressive effect of Rapa&P-20 k nanoparticles was then weaker than that of Rapa&P-50 k nanoparticles. Therefore, the immunosuppressive effect of Rapa&P-50 k nanoparticles was the strongest among the three groups. LPS treatment induced maturation of DCs, and increased expression of CD40 and CD80 as well as other costimulatory molecules, which helped DCs to play a signal transduction role in the later immune processes.

Rapamycin was an immunosuppressive macrolide with a high affinity for FK506 binding protein (FKBP12). The Rapa-FKBP12 complex was an allosteric inhibitor of the mammalian target protein of Rapamycin (mTOR), binding to mTOR and affecting immunity and inflammation. ⁵⁹ It was thus demonstrated that upon encapsulation of rapamycin within PEG-PLGA nanoparticles, the therapeutic effect could be enhanced and it could be further adjusted by the chain length of the PLGA segment.

Secretion of cytokines

LPS-stimulated DCs were treated with free rapamycin or rapamycin loaded by PEG-PLGA to compare their effects on cytokine production. Immature DCs and LPS treated alone group were established as negative and positive control respectively.

IL-1β acts as an inflammatory cytokine and promotes the inflammatory response.^60,61 Activated DCs secrete IL-12. ⁶² Rapamycin probably regulates the immune function of DCs by regulating IL-12 secretion. ⁶³ The secretion of IL-1β and IL-12 in DCs stimulated by LPS was up-regulated, and the DCs initiated an inflammatory response. As shown in Figure 6(a), (b) rapamycin treatment attenuate the upregulation of IL-1β and IL-12 which triggered by the LPS stimulation, indicating the immunosuppressive effect of rapamycin in this in vitro model. And intriguingly, the rapamycin-loaded nanoparticle groups, especially Rapa&P-20 k and Rapa&P-50 k, exhibited a significant downregulation of both cytokines secretion even more efficient than those of the rapamycin only group (Figure 6 a, b), while in Rapa&P-95 k rerated group only the IL-1β was significantly downregulated but not the IL-12 compare with the free rapamycin treated group (Figure 6 a, b). Those data were consistent with the results of previous evaluation about costimulatory molecules. The nanoparticles with faster release rate had a stronger stimulus, and the slower ones had a weaker stimulus. Moreover, the difference in PEG content led to the difference in shielding effect, which affected the stimulation effect of nanoparticles on DCs. The higher the PEG content, the stronger shielding effect, resulting in weaker promoting immunity and stronger suppressing immunity. The low drug loading would be compromised with a high nanoparticle concentration, which also then promoted immune stimulation. Therefore, those results indicating that among the three groups of nanoparticles, Rapa&P-20 k and Rapa&P-50 k nanoparticles performed the highest inhibitory effect on IL-1β and IL-12 secretion.

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Figure 6.

Secretion of cytokines by DCs quantified by ELISA. “Rapa” represents free rapamycin group. (a) IL-1β; (b) IL-12; (c) TGF-β1.

TGF-β1, as an immunosuppressive cytokine, ⁶⁴ its secretion upon different treatments in the current cell model was also analyzed. As shown in Figure 6 (c), Rapamycin treatment elevated the expression of TGF-β1 by DCs as expected, indicating its immunosuppressive effect. Furthermore, in consistent with previous data, nanoparticle encapsulation enhanced the immunosuppressive effect of rapamycin, as the expression of TGF-β1 was significantly upregulated in Rapa&P-20 k and Rapa&P-50 k treated group compared to the rapamycin treated group, while in the Rapa&P-95 k group only a minor increase in TGF-β1 secretion was observed (Figure 6 (c)).

It was shown that Rapa&P-20 k nanoparticles had the highest amount of TGF-β1 secretion and thereby the strongest immunosuppressive effect among the three nanoparticle groups. However, no significant difference between any of these two groups of nanoparticles was observed. It was possible that the sensitivity of TGF-β1 secretion to the rapamycin concentration might be different or lower than those of IL-1β and IL-12.

After all, the immunosuppressive effect of Rapa&P nanoparticles was stronger than that of free rapamycin. PEG-PLGA nanoparticles delivery caused rapamycin to be released continuously, so as to stimulate the DCs efficiently. Moreover, rapamycin, as a macrolide antibiotic, is structurally unstable and easily hydrolyzed. Nanoparticle encapsulation could protect the hydrolysis of rapamycin and ensure its function. Among the three groups designed for rapamycin delivery, Rapa&P-50 k would be the first choice based on the overall performance. The chain length of PLGA within the PEG-PLGA molecules could render significant effect on rapamycin encapsulation and loading, the subsequent release and immunosuppressive effect.