Preparation, Properties and Therapeutic Effect of a TPL Nanoparticle Thermosensitive Gel for Intra-Articular Injection

2.1. Characterization of TPL-NS-Gel

Freeze-dried TPL-NS were white loose powder. When the microspheres were dispersed in water, they were in the state of a uniform emulsion with blue opalescence (Figure 1A). The particle size of the microspheres measured by TEM was approximately 100 nm (Figure 1B). The size distribution of the microspheres measured by a Malvern laser particle size analyzer showed that the particle size was fairly uniform (Figure 1C). The encapsulation efficiency (EE%) and loading efficiency measured by the UPLC-MS method was approximately 83.55 ± 2.67% and 4.73 ± 1.05%, respectively. The poloxamer gel was a flowing liquid at a low temperature (below 20 °C) and formed a soft semisolid gel at 37 °C (Figure 1D). The internal structures of TPL-NS-Gel at 4 °C and 35 °C were detected with a laser capture microdissection system, which showed a radiation micellar shape of more than 30 μm at 35 °C (Figure 1E).

2.2. Effect of PLGA on the Phase Transition Temperature of Poloxamer Gel

The hydrogen NMR spectra of poloxamer thermosensitive gel (298 K, Figure 2A) showed that the chemical shifts of EO-CH₂-, PO-CH₂- and PO-CH₃ were approximately 3.6, 3.4–3.6 and 1.1 ppm, respectively. The protons on poloxamer molecules all moved to the low field (high ppm) with increasing temperature (28–32 °C); that is, the protons moved in the direction of decreasing polarity, indicating that the chemical environment of protons changed from hydrophilic to hydrophobic, and the hydrophobic effect between poloxamer molecules was enhanced. However, the chemical shifts of different protons were different (Figure 2B), indicating that the sensitivity to temperature was different. The chemical shift of protons on PO-CH₂- can reach 1.23 × 10⁻² ppm/°C, and the chemical shift of protons on PO-CH₃ can reach 1.04 × 10⁻² ppm/°C. TPL-NS-Gel containing 1%, 5% and 10% TPL was placed at different temperatures (18 °C, 20 °C, 22 °C, 24 °C, 26 °C, 28 °C, 30 °C and 32 °C) to monitor the chemical shift of protons (Figure 2C). The results show that the chemical shift of the proton on PO-CH₂- and PO-CH₃ increases in temperature before the gelling temperature, and the chemical shift of the proton on PO-CH₂- and PO-CH₃ changes obviously near the phase transition temperature. Moreover, with increasing TPL-NS content, the phase transition temperature of the P407 gel increased. The phase transition temperatures of 1%, 5% and 10% TPL-NS-Gel were 24–26 °C, 28–30 °C and 30–32 °C, respectively, which were consistent with the gelling temperatures measured by the magnetic stirring method (24.1 °C, 28.8 °C and 32.3 °C).

DSC diagram of poloxamer gel showed that there was an obvious endothermic peak in the formation process of gel. When no PLGA nanoparticles were added, the endothermic peak appeared at 27.57 °C (Figure 3A), and when 5% PLGA nanoparticles were added, the endothermic peak appeared at 29.84 °C (Figure 3B). PLGA nanoparticles increased the endothermic peak temperature, which was close to the gelation temperature measured by the magnetic stirring method (28.8 °C).

2.3. Changes of Joint Status before and after Administration in Rat Model of RA

Redness and swelling began to appear in SD rats 3–5 days after injection of chicken type II collagen and Friedrin’s complete adjuvant, and the swelling became increasingly obvious after that. Generally, the swelling and joint stiffness reached a very obvious degree in approximately two weeks, accompanied by a certain reduction in diet and activity, indicating that the rat RA model was successfully constructed. After 24 days of administration, the photos of diseased joint sites of experimental animals in the TPL-NS-Gel group, TPL-NS group and control group were observed (Figure 4). The results showed that the degree of joint swelling, bending and stiffness of rats in the control group were aggravated with the extension of time, and the symptoms of incomplete feeding and reduced activity appeared. In the TPL-NS-Gel group, the degree of joint swelling and bending stiffness were significantly improved, and there was no significant decrease in the amount of eating and activity and no weight loss. In the TPL-NS group, the redness and swelling of the joints were alleviated to a certain extent, but the stiffness was not significantly improved. In the control group, the degree of swelling and stiffness was further developed, especially the degree of swelling, which was significantly aggravated.

2.4. Changes of Inflammatory Cytokines in Rat Model of RA before and after Administration

Figure 5 shows the standard curves and regression equations of ELISA methods for the determination of four inflammatory factors, IL-1, IL-6, TNF-α and hs-CRP, with R² values above 0.994, meeting the methodological requirements. Approximately 4–5 weeks after the establishment of the RA model, the levels of four inflammatory factors in the articular fluid and serum of rats were significantly increased compared with those of healthy rats (p < 0.01). Table 1 shows the content determination results of four inflammatory factors in serum and joint fluid of the TPL-NS-Gel group, TPL-NS group and control group before and after administration. The data showed that the contents of hs-CRP, IL-1, IL-6 and TNF-α in serum and articular fluid of the rat RA model significantly decreased in the TPL-NS-Gel group and TPL-NS group compared with the control group after 12 days of administration (p < 0.01). There was no significant difference between the TPL-NS-Gel group and TPL-NS group in other inflammatory factors except TNF-α. After 24 days of administration, the contents of hs-CRP, IL-1, IL-6 and TNF-α in serum and joint fluid of the rat RA model were still significantly decreased in the TPL-NS-Gel group and TPL-NS group compared with the control group (p < 0.01). The four inflammatory factors were also significantly different between the TPL-NS-Gel group and the TPL-NS group (p < 0.05) which indicated that the long-term therapeutic effect of the TPL-NS-Gel group was better than that of the TPL-NS group. On Day 24, compared with the control group, the four inflammatory factors of the TPL-NS-Gel group in serum decreased by 73.79%, 42.00%, 66.32% and 67.99%, and the factors in joint fluid decreased by 74.29%, 42.27%, 73.55% and 82.46%, respectively. Compared with the control group, the percentage of factors in serum decreased by 60.78%, 27.48%, 59.51% and 58.55%, and the percentage of factors in joint fluid decreased by 61.97%, 29.33%, 69.72% and 69.57%, respectively. However, the levels of four inflammatory factors in the control group were continuously increased. At 24 days after administration, there were significant differences in the other three indicators (p < 0.05), except hs-CRP in serum was not significantly increased (p = 0.19), indicating that the degree of inflammatory factors in the RA model without treatment increased. For the same indicators in the same group, the decrease percentage in serum was smaller than that in joint fluid, which may be due to the higher expression of these indicators in RA joints than in serum, while TPL exerts its anti-RA effect mainly by reducing the levels of inflammatory factors such as hs-CRP, IL-1, IL-6, and TNF-α.

2.5. Results of UPLC-MS/MS Methodology Investigation

The secondary mass spectra of tissues and plasma of rats showed that the endogenous substances did not interfere with the determination. The degree of separation between TPL (retention time was approximately 3.1 min) and internal standard (retention time was approximately 2.4 min) was above 1.5. The detection limit was 0.2 ng/mL, and the quantification limit was 2 ng/mL. The standard curve and correlation coefficient of the heart, liver, spleen, lung, kidney, plasma and joint tissue are shown below. y = 0.0306x + 0.1622 (R² = 0.9977), y = 0.0303x + 0.1284 (R² = 0.9958), y = 0.0301x + 0.1452 (R² = 0.9974), y = 0.0316x + 0.0773 (R² = 0.9936), y = 0.0323x + 0.1217 (R² = 0.9962), y = 0.0316x + 0.0825 (R² = 0.9938), y = 0.0254x + 0.1231 (R² = 0.9979). The linear ranges were 2.47–247.25 ng/g (ng/mL), and the relative recoveries were ≥88.3%. The RSDs of intra-day and inter-day precision were ≤4.37% and 6.71%, respectively. The RSDs of stability were ≤4.46% at room temperature for 8h and ≤3.85% after three repeated freeze-thaw cycles. The matrix effect was ≥92.8%.

2.6. The Distribution of Tissues and Plasma Pharmacokinetic Behavior in RA Rats

The tissue distribution data after the injection of TPL-NS-Gel and TPL-NS into the articular cavity are shown in Table 2. The results showed that the peak concentration in the articular tissue of the TPL-NS-Gel group was 102.64 ± 9.47 ng/g, the peak time was the 8th day, and the articular cavity retention time was more than 24 days. The peak concentration in the joint tissue of the TPL-NS group was 197.18 ± 9.54 ng/g, the peak time was the third day, and the retention time of the joint cavity was approximately 2 weeks. Compared with the TPL-NS group, the peak concentration of TPL-NS-Gel in the joint was lower, but the retention time was longer, indicating that the thermosensitive gel could further delay drug release, reduce the rapid elimination of solution form in the joint cavity, and further reduce the number of drug administration in the joint cavity. After administration in the two groups, the drug concentration in other tissues (heart, liver, spleen, lung, kidney, and plasma) except joints was relatively low, suggesting that local drug administration in the joint cavity itself had a certain local targeting effect. However, the peak concentration in the TPL-NS-Gel group was significantly lower than that in the TPL-NS group, indicating that drug accumulation in the joint cavity was beneficial to reduce drug concentration in all tissues, reducing systemic toxicity and side effects, and facilitating the advantages of local drug treatment. The pharmacokinetic parameters were calculated using a non-compartment model, and the main pharmacokinetic parameters are listed in Table 3. The AUC_(0–-t) and MRT_(0–t) of the TPL-NS-Gel group were 1.7 and 2.3 times that of the TPL-NS group, respectively. Based on the concentration–time curves (Figure 6), the in vivo pharmacokinetic data of TPL-NS-Gel and TPL-NS on RA rats were also fitted to a two-compartment model, and the main pharmacokinetic parameters are listed in Table 3. The AUC_(0–t) of the TPL-NS-Gel group was 1.7 times that of the TPL-NS group, which did not show an obvious difference from that of the non-compartment model. The t_1/2Ka, t_1/2α and t_1/2β of the TPL-NS-Gel group were 8.1, 2.4 and 1.6 times that of the TPL-NS group, respectively. It suggested that the poloxamer gel formed an obvious drug reservoir in the joint cavity, prolonging the absorption, distribution and elimination of the drug in vivo.

2.7. Pathological Changes in the RA Rat Model

The heart, liver, spleen, lung, kidney and joint tissues of rats in the TPL-NS-Gel group, TPL-NS group and control group were taken for HE staining 24 days after administration. The pathological morphology was observed as shown in Figure 7. HE staining of joint tissue showed that the inflammatory cell infiltration, the degree of tissue hyperplasia, and the degree of cell swelling and deformation were significantly reduced compared with the control group, and the number and dilation of blood vessels were alleviated in the TPL-NS-Gel Group. In the TPL-NS group, other phenomena were not significantly improved except for the decrease in inflammatory cell infiltration. HE staining of heart, liver, spleen, lung and kidney tissues showed no significant difference in TPL-NS-Gel Group and TPL-NS Group compared with the control group, no obvious histological changes, inflammatory cell infiltration, etc. The HE results showed that local injection of TPL-NS-Gel in the joint cavity had a good effect on the improvement of joint histopathologic morphology, and no systemic organ toxicity was observed.

4.1. Materials and Reagents

Standards of TPL (the purity is above 98.5% determined by HPLC-UV, China National Institutes for Food and Drug Control); PLGA (molecular weight = 15,000 Da, Shandong institute of medical devices); methanol (Merck Co., Gradient Grade), acetonitrile (Merck Co., Gradient Grade), ammonium formate (Merck Co., Gradient Grade); Rat IL-1α ELISA Kit, Rat IL-6 ELISA Kit, Rat TNF alpha ELISA Kit (Abcam^®); Rat CRP (C-Reactive Protein) ELISA Kit (Elabscience^®); anhydrous ethanol, paraformaldehyde, xylene, formaldehyde, hydrochloric acid, neutral balsam, propylene glycol (Sinopharm Chemical Reagent Co., Ltd., analytical grade); Chicken type II collagen (Hefei Bomei biotechnology Co., Ltd.); Phosphate buffer solution (PBS, Life Technologies^®).

4.2. Animals

Male SD rats (weighing 180~220 g) were supplied by the Animal Centre of Chongqing Medical University (Chongqing, China). All the experiments in this study were approved by the Animal Research Ethics Committee of Chongqing Medical and Pharmaceutical College. All the rats were housed with free access to food and water under a standardized light/dark cycle condition (20~22 °C and 45~65% RH).

4.3. Preparation and Properties of TPL-NS-Gel

TPL nanoparticles were prepared by an improved self-emulsifying solvent evaporation method. The organic phase consisting of acetone/anhydrous ethanol dissolved with an appropriate amount of PLGA and TPL was added to poloxamer 188 aqueous phase at a certain concentration under agitation, and all the organic solvent and part of the water were evaporated under pressure to obtain a blue opalescent gel suspension. After centrifugation at high speed (12,000 r/min, 10 °C for 3 h), the precipitates were freeze-dried to obtain white nanoparticle powder. One to two drops of TPL nanoparticle suspension were placed on the copper net, and stained with 2% phosphotungstic acid solution, and the morphology of the nanoparticle was observed under a transmission electron microscope. The average particle size and distribution of the nanoparticle suspensions diluted (1000 times) were measured on the Marvin laser particle size detector. An appropriate amount of nanoparticle was taken and dissolved with dichloromethane, filtered, nitrogen blow-dried, mobile phase dissolved, and filtered through a 0.45 μm filter membrane. The mobile phase volume was adjusted to scale, and mass spectrometry was used to determine the concentration, encapsulation rate and drug-loading ability of the nanoparticles. The thermosensitive gel was prepared by the cold method. P407/P188 (19.5%/5%, w/w) was dissolved in pure water, and sodium hyaluronate (0.5%, w/w) was added and placed in the refrigerator at 4 °C for 24 h. Under the condition of mild agitation, nanoparticles (5% according to TPL, w/w) were added and stored in the refrigerator at 4 °C for later use. The morphology of gel micelles was observed by the ArcturusXT™ Laser Capture Microdissection System at 37 °C.

4.4. The Effect of Nanoparticle Carrier Material PLGA on the Phase Transition Temperature of P407 Gel Was Studied by ¹H Variable Temperature Nuclear Magnetic Resonance and DSC

D₂O was used as the solvent to prepare the D₂O hydrogel. The preparation process was the same as above. Ordinary gel and D₂O hydrogel samples (0.5 mL) were loaded into the nuclear magnetic tube. A 5 mm BBQ probe, Bruker BVT3000 digital temperature controller with the following parameters was used: single pulse, D₂O lock field, a resonance frequency of 500.13 MHz, a pulse delay of 6 s, 16 sampling times, a 90° pulse width of 13.2 μs, D₂O gel as the sample, and D₂O calibration.

The heating range was −40–+50 °C and the heating rate was 5 °C/min. The testing mode was DSC-TG with an empty crucible as the reference. Liquid nitrogen cooling, poloxamer gel without PLGA and poloxamer gel with 5% PLGA nanoparticles as samples.

4.5. The Establishment of Rat RA Model

All the animal experiments approved by the medical ethics committee of Chongqing Medical and Pharmaceutical College were conducted in accordance with the relevant guidelines and regulations. The chicken type II collagen was mixed and emulsified with Complete Freund’s adjuvant. The 0.25 mL emulsifier was injected subcutaneously into the foot plantarum of SD rats. Immunity was further enhanced with an injection of 0.1 mL emulsifier 7 d later. The body weight and paw swelling of the rats were observed every day after the injection. The serum and joint fluid were collected and the expression levels of the inflammatory factors were measured.

4.6. Instrumentation and Conditions of UPLC-MS/MS for the Determination of Biological Samples

Chromatographic analysis was performed using an Agilent 1290 Infinity II ultrahigh-performance liquid chromatography system (UPLC), including a binary pump, an online vacuum degasser, a surveyor auto-sampling system, a column temperature controller and a diode-array detector. The samples were separated on an Agilent ZORBAX SB C₁₈ column (2.1 × 100 mm, 1.8 μm) and eluted with a gradient elution of solvent A (water containing 5 mmol/L ammonium formate) and solvent B (acetonitrile) as follows: 30% B (0–3.7 min), 95% B (3.7–4.0 min), 90% B (4.0–10.0 min), and 30% B (10.1–15 min). The column temperature was set at 40 °C, the flow rate was set at 0.4 mL/min, and the injection volume was set at 2 μL. Mass spectrometric detection was carried out on an Agilent 6460 triple-quadrupole mass spectrometer with positive electrospray ionization (ESI), which was connected to the liquid chromatography system. The MRM mass scan mode was implemented. The precursor ion/product ion were m/z 378.1/361.0 for TPL and m/z 260.0/116.2 for the internal standard (IS, propranolol hydrochloride). The MS/MS conditions were optimized as follows: fragmentor, 100 V; capillary voltage, 4000 V; nebulizer gas pressure (N₂), 35 psi; drying gas flow rate (N₂), 10 L/min and gas temperature, 350 °C.

4.7. Method Validation of UPLC-MS/MS

A series of standard working solutions were obtained by further diluting the stock solution of TPL with methanol for method validation. The calibration standard samples for TPL (actual concentrations were 2.47, 24.72, 49.45, 123.62 and 247.25 ng/mL, respectively) were prepared by mixing TPL standard working solutions and the IS solution of the same volume into blank heart, liver, spleen, lung, kidney, joint and plasma tissues. The calibration curves were constructed as the peak area ratios of TPL to IS against the TPL concentrations. The lower limit of quantification (LLOQ) was determined as the concentration of TPL with a signal-to-noise ratio of 10. To determine intra-day and inter-day precision and accuracy, five replicates of QC samples at low (2.47 ng/mL), medium (49.45 ng/mL) and high (247.25 ng/mL) concentration levels were prepared and analyzed on the same day and on three different days. The extraction recovery was determined by calculating the ratio of QC samples obtained at the three concentrations mentioned above in the blank tissues against those originally spiked in methanol (n = 5). The short-term stability was evaluated by determining QC samples at room temperature for 12 h. The freeze-thaw stability was determined through three freeze-thaw cycles on consecutive days. The matrix effect was determined by calculating the ratio of QC samples obtained at low, medium and high concentration levels against those in the mobile phase (n = 5). The detection of the matrix effect can exclude the influence of endogenous components in those tissues, resulting in ion suppression of the analyte signal. The results of method validation are expressed as the mean ± RSD value.

4.8. Animal Experimental Protocol

A total of 66 RA model rats were weighed and randomly divided into three groups: (A) 30 rats in the TPL-NS-Gel group (TPL-NS-Gel was administered into the joint cavity at 10 mg/kg); (B) 30 rats in the TPL-NS group (TPL-NS was administered into the joint cavity, 10 mg/kg); (C) six rats in the control group (0.9% saline was administered into in the joint cavity). All groups received a single dose. The heart, liver, spleen, lung, kidney, joint and whole blood were taken at 30 min, 8 h, 1 d, 2 d, 3 d, 5 d, 8 d, 12 d, 16 d, and 24 d after administration (three animals were sacrificed at each time point, n = 3). Whole blood was centrifuged at 12,000 r/min for 10 min to separate the plasma. The joints of the dosing side were removed, rinsed with PBS7.4, and wiped dry, and the whole cartilage synovial tissues were removed and weighed. Other tissues were washed with PBS 7.4, dried and weighed. Both plasma and tissues were stored at −80 °C in a freezer. After administration, the animals were observed and recorded daily for signs of status, including joint swelling and stiffness and activity, and feeding condition. Before tissue removal on Days 12 and 24, the joint cavity was injected with 0.5 mL of sterile NaCl solution, and an additional 0.5 mL of joint fluid was withdrawn and stored frozen at −20 °C. Blood was taken, and serum was separated and stored frozen at −20 °C. These joint fluids and serum were used to detect the inflammatory cytokines hs-CRP, IL-1, IL-6, and TNF-α. Part of the tissues taken out on the 12th and 24th days were immersed and fixed in a 10% formaldehyde solution for pathological examination.

4.9. Treatment of Biological Samples

Tissues (0.2 g) or plasma (200 μL) were thawed at room temperature before analysis. After adding 50 μL of IS solution (100 μg/mL), TPL in tissues and plasma was extracted twice with ethyl acetate (total 3 mL) by vortexing for 5 min at a time. The extracted solution was centrifuged for 10 min at 12,000 r/min. The ethyl acetate layers were removed and evaporated at 40 °C under nitrogen. The residues were redissolved in 200 μL methanol and subjected to UPLC-MS/MS analysis. The TPL concentrations in each tissue (ng/g) and plasma (ng/mL) were calculated by the above calibration curves. If necessary, the sample should be diluted and then re-injected to ensure that the concentration is within the linear range.

4.10. Measurement of hs-CRP, IL-1, IL-6 and TNF-α in Serum and Synovial Fluid by ELISA

ELISA was used to measure inflammatory cytokines in the serum and synovial fluid. The assays were performed according to the protocols outlined in the TNF-α, IL-1, IL-6 and hs-CRP ELISA kits. Synovial fluid and serum were incubated at 37 °C for 60 min according to the manufacturer’s protocol: a total of 100 µL synovial fluid or serum was added to the TNF-α, IL-1, IL-6 or hs-CRP antibody-coated plate and incubated at 37 °C for 1 h. After adding the biotin-conjugated detecting TNF-α, IL-1, IL-6 or hs-CRP antibody and incubating for 2 h, streptavidin-HRP was added, and 3,3’-5,5’ tetramethylbenzidine was used for development. The optical density value was measured at 450 nm by Multiskan spectroscopy (Thermo Fisher Scientific, Inc.). Experiments were performed in triplicate.

4.11. Statistical Analysis

Descriptive statistics include the mean and standard deviation (SD) and were determined with Microsoft Excel 2013. The plasma pharmacokinetic data were calculated using DAS2.0 software. The statistical analysis was performed with SPSS 23.0 software (SPSS, IBM Inc.). The statistical significance of differences between the TPL-NS-Gel group, TPL-NS group and control groups in four inflammatory factors were calculated using the independent-sample t-test. Differences were considered statistically significant and very significant when p values calculated were <0.05 and <0.01, respectively.