The Preparation of a Novel Poly(Lactic Acid)-Based Sustained H2S Releasing Microsphere for Rheumatoid Arthritis Alleviation

Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease that mainly erodes joints and surrounding tissues, and if it is not treated in time, it can cause joint deformities and loss of function. S-propargyl-cysteine (SPRC) is an excellent endogenous hydrogen sulfide donor which can relieve the symptoms of RA through the promotion of H2S release via the CSE/H2S pathway in vivo. However, the instant release of H2S in vivo could potentially limit its further clinical use. To solve this problem, in this study, a SPRC-loaded poly(lactic acid) (PLA) microsphere (SPRC@PLA) was prepared, which could release SPRC in vitro in a sustained manner, and further promote sustained in vivo H2S release. Furthermore, its therapeutical effect on RA in rats was also studied. A spherical-like SPRC@PLA was successfully prepared with a diameter of approximately 31.61 μm, yielding rate of 50.66%, loading efficiency of 6.10% and encapsulation efficiency of 52.71%. The SPRC@PLA showed significant prolonged in vitro SPRC release, to 4 days, and additionally, an in vivo H2S release around 3 days could also be observed. In addition, a better therapeutical effect and prolonged administration interval toward RA rats was also observed in the SPRC@PLA group.


Introduction
Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease that mainly erodes joints and surrounding tissues [1][2][3][4] and if it is not treated in time, it can cause joint deformities and loss of function. It is often accompanied by tissue and organ injury, including cardiovascular and lung [5][6][7]. The pathogenesis of RA is still not clear, therefore, an ideal drug has not been found to completely cure this type of disease [8,9]. At present, the treatment of RA is mainly based on non-steroidal anti-inflammatory drugs, glucocorticoids, traditional anti-rheumatic drugs, and biological agents for improving the condition of the disease [10][11][12]. However, the specific treatment of RA is still a dilemma in modern medicine.
Poly(lactic acid) (PLA) is produced by the polymerization of lactic acid. Because of its excellent properties such as good biocompatibility and degradability, it has been widely studied since it was discovered [13][14][15][16]. The initial raw material of PLA is plant starch, which produces extremely low pollution during the production process, and can also be completely decomposed into CO 2 and H 2 O after use and utilized by nature [17,18]. Due to its excellent performance, PLA has been recognized to be a new type of green and environmentally friendly polymer material. Because of its biodegradability, PLA can be mixed with specific drugs to produce microparticles [19]. When these microparticles reach the action site, they slowly decompose into CO 2 and H 2 O, and the drug is gradually released at the corresponding action site, therefore, improving the therapeutic index [13,14,20,21].
Hydrogen sulfide (H 2 S) has always been considered to be a poisonous gas with a smell similar to rotten eggs, and it is produced in large quantities in some polluted environments [22]. With the deepening of research, recent studies have found that H 2 S is also an important physiological gas molecule and is considered to be the third gasotransmitter after nitric oxide (NO) and carbon monoxide (CO) [23,24]. Endogenous H 2 S is produced via the catalyzation of cystathionine pyridoxal-5-phosphate dependent enzymes, including cystathionine-β-synthase (CBS), cystathionine-y-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). In human blood, the concentration of H 2 S at normal physiological levels is about 40 µM, and the local concentration in the brain can reach more than 100 µM. As a gas signal molecule, H 2 S could reduce high glucose-induced myocardial injury [25] or kidney injury [26], however, the half-life of direct administration of H 2 S is too short, and it is also difficult to precisely control the dosage. Therefore, it is of great significance to study a series of H 2 S donor which could be used as a CSE substrate to further release H 2 S in a relatively slow manner [27,28]. S-allyl-cysteine (SAC) is an extract in garlic, which could reduce the area of myocardial infarction by regulating the level of H 2 S in ischemic myocardial tissue [29,30]. According to the structure of SAC, our group synthesized a compound called S-propargyl-cysteine (SPRC, also known as ZYZ-802) [31][32][33], which is a compound with a similar structure to SAC ( Figure 1). Our previous study found that SPRC could be used as a new type of H 2 S donor for ischemia-hypoxic cell models and the treatment of coronary artery ligation rat myocardial infarction models [32,34]. In addition, SPRC could also exert its neuroprotective effect through its anti-inflammatory effect [35,36]. Recent studies have also shown that SPRC could treat rheumatoid arthritis in rats by regulating endogenous H 2 S [31,37,38]. However, the instant release of H 2 S by SPRC might prevent its clinical use, hence, how to achieve a sustained release of H 2 S in vivo through SPRC remains to be a challenging problem. In this study, we aim to solve the problem that SPRC might prompt the H 2 S in an instant manner, a SPRC-loaded PLA-based microsphere was successfully prepared (SPRC@PLA), which showed sustained release of SPRC in vitro, therefore, elevating the plasma H 2 S concentration for almost 3 days. Through this long elevation period, the administration interval for treating RA has also been increased as compared with that determined in a previous study [31].

Materials
Poly(lactic acid) (PLA) polymer, with Mw around 10,000~18,000 Da and viscosity of 0.16~0.24 was purchased from Evonik Industries (AG, Essen, Germany). SPRC was synthesized, as previously reported [39,40]. Poly(vinyl alcohol) (PVA), with MW around 25,000 Da, and 88% mole hydrolyzed, was purchased from Polysciences (Warrington, PA, USA). Elisa kit of TNF-α, IL-1β, IL-6, and IL-10 were purchased from MultiSciences A double emulsion evaporation method (W1/O/W2) was followed with slight modification [41]. First, 50 mg of SPRC was dissolved in 1 mL of distilled water to prepare the inner water phase (W1). Meanwhile, the oil phase (O), which was various amounts of PLA dissolved in 12 mL of DCM, was also prepared. Then, the W1 was dispersed in O, with further emulsification through an Ultraturrax T25 high-speed homogenizer (IKA, Staufen, Germany) at 9000~12,000 rpm for 5 min to prepare the primary water-in-oil emulsion (W1/O). Then, the W1/O was dispersed in 100 mL of 0.5% (w/w) PVA solution with a paddle agitation at 800 rpm for 4 h, until the evaporation of organic solvent. Then, particles were collected through filtration via sieve with 200 mesh, intended to remove the potential bulk shape microspheres, and washed three times with distilled water to remove the excess residual PVA and SPRC on the surface. Finally, the obtained particles were lyophilized overnight to obtain the SPRC@PLA. Table 1 showed the detailed information of the different formulations.

The Production Yeild of SPRC@PLA
The percentage of production yield (PY) was calculated using the following equation: where W is the weight of corresponding component.

The Morphology Study of SPRC@PLA
Samples were firstly dispersed in distilled water, and a Microtrac S3500 (Montgomeryville, PA, USA) was used for the measurement of particle size and size distribution. A Phenom Pro Desktop SEMS-3400 scanning electron microscope (Thermo Fisher Scientific Inc., Waltham, MA, USA) was conducted to observe the morphology of SPRC@PLA. Samples were gold coated before examination.

The Encapsulation Efficiency of SPRC@PLA
The loading efficiency (LE) and encapsulation efficiency (EE) were determined by dissolving 50 mg of SPRC@PLA in 3 mL of DCM with further extract SPRC with 5 mL distilled water and analyzed using a 1290 Infinity II LC System (Agilent Technologies, Inc., Santa Clara, CA, USA). LE and EE were calculated using the following equations: where W SPRCi is the weight of SPRC initially fed, W SPRCs is the weight of SPRC in supernatant, and W SPRC@PLA is the weight of SPRC@PLA.

The SPRC Release In Vitro
The in vitro releasing experiment was conducted, using the method as reported with little modification [43]. First, 50 mg of differently prepared SPRC@PLA was dispersed in vials filled with 3 mL of PBS buffer (pH = 7.4, 37 • C) and placed in a shaker bath with a constant shaking speed of 100 rpm and temperature at 37 • C (Clifton Shaking Bath NE5, Nikel Electro Ltd., Weston-super-Mare, UK). Then, 0.5 mL of the samples were taken out, and then the same volume of PBS was refilled at predetermined intervals, and samples were analyzed using a 1290 Infinity II LC System (Agilent Technologies Inc., Santa Clara, CA, USA).

The Measurement of H 2 S Release In Vivo
The concentration of H 2 S was measured, as reported with little modification [44], and the schematic for detection of H 2 S in vivo is shown in Figure 2  The mass spectrometer was operated in positive ion mode. The scan type chosen was MRM with gas temperature at 325 • C and gas flow at 10 L·min −1 . Scan time was 500 ms and start-stop mass was 100~1000. The sample injection volume was 5 µL. Samples of SPRC powder and SPRC@PLA were dissolved or dispersed in saline for subcutaneous injection, each sample contained the same amount of SPRC, and the amounts used were calculated through the weight of the rats (100 mg·kg −1 ). The rats' serum was collected at predetermined times (0, 0.5, 1, 1.5, 2, 3, 6, 12, 24, 48, and 72 h) into heparin sodium tubes and analyzed. Each group contained 3 rats.

SPRC@PLA Showed Anti-Inflmmation Effect towards Rheumatoid Arthritis
The AIA rat model was established via the injection of CFA (10 mg·mL −1 ), according to the manufacturer's instructions. In total, 30 rats were randomly divided into four groups as follows: Control group (n = 5), no intervention; AIA group (n = 5), injection of 100 µL of CFA; SPRC group (n = 5), after injection of 100 µL of CFA, further subcutaneous injected with 2 mL of SPRC solution every 3 days for 30 days; SPRC@PLA group (n = 5), after injection of 100 µL of CFA, further subcutaneous injected with 2 mL of SPRC@PLA suspension every 3 days for 30 days (the amounts of SPRC used were all equivalent to 100 mg·kg −1 of SPRC.) The paw volume was measured using a UGO Basile 7140 plethysmometer (Ugo Basile, Gemonio VA, Italy) and body weight was measured at the 0, 5th, 15th, 20th, 25th, and 30th day post the injection of CFA. The arthritis index was scored ( Table 2) from 0 to 4 per limb, with 0 = no sign of inflammation and 1~4 = increasing degrees of inflammation, and a maximum score of 16 per rat. Erythema and mild swelling confined to the tarsals or ankle joint 2 Erythema and mild swelling extending from the ankle to the tarsals 3 Erythema and moderate swelling extending from the ankle to metatarsal joints 4 Erythema and severe swelling encompassing the ankle, foot, and digits; ankylosis of the limb might be present At day 30, a blood sample was collected from rats in each group, the pro-inflammatory cytokine levels (TNF-α, IL-1β, and IL-6) and anti-inflammatory cytokine (IL-10) level in serum were measured using ELISA kits, according to the manufacturer's instructions.

Statistical Analysis
Statistical analyses of samples were performed using IBM SPSS Statistics Base (V22, IBM, Armonk, NY, USA.) and GraphPad Prism (V8, GraphPad Software, San Diego, CA, USA). Each experiment was performed at least three times. The data are expressed as the mean ± SD. Statistical significance was determined using a one-way analysis of variance (ANOVA) test, unless otherwise stated, p < 0.05 was considered to be significant.

The Characterization of SPRC@PLA
First, the influence of PLA was investigated. As shown in Table 3, with an increase in the amount of PLA used, a decreasing trend of LE and an increasing trend of EE could be observed. The influence of the amount of PLA used on particle size was also investigated. The particle size showed an increasing trend with an increase in the amount of PLA used. While interestingly, there is no significant influence on PY. The influence of homogenization speed was also investigated. As shown in Table 4, with an increase in homogenization speed, the LE and EE both showed an increasing trend while conversely, the particle size showed a decreasing trend. Interestingly, the PY still showed no significant change with an increase in homogenization speed. The in vitro release profiles from F-1 to F-5 were also investigated for the selection of the optimized formulations, and the results are shown in Figure 3. SPRC might dissolve extremely fast in PBS due to its high hydrophilicity. However, a significantly prolonged in vitro release period could be observed in F-1 to F-5 as compared with the SPRC group, which was up to almost 4 days. F-3 and F-4 both showed an incomplete cumulative release potential due to the larger size usually accompanied with a relatively sustained release manner. In addition, F-1, F-2, and F-5 all showed a sustained and complete release within 96 h. By combining the in vitro release results with the particle size, it could be deduced that particle size might play a vital role in the property of in vitro release. F-1 to F-5 showed a similar PY, which indicated that neither the amount of PLA used, nor the homogenization speed could influence the PY of the prepared SPRC@PLA. Normally, for subcutaneous injection, micro-sized particles with a range between 20 to 100 µm [45] are generally used, since, on the one hand, it usually has sufficient size to incorporate enough active pharmaceutical ingredient, and, on the other hand, sizes between this range are normally suitable, and therefore do not induce inflammation in the injection area. Hence, although F-1 and F-5 both showed a sustained and complete release of SPRC, for further in vivo study, F-1 and F-5 were not selected.
Above all, F-2 was chosen as the optimized formulation for further study due to its high EE, stable PY, relative monodispersed particle size, as well as its sustained and complete release in vitro. The SEM of F-2 is shown in Figure 4. For a convenient expression, F-2 is denoted as SPRC@PLA for the remainder of this paper.

The Elevation of Plasma H 2 S Concentration by Supplementations
Generally, H 2 S is unstable, and can exist as the mixed state of hydrogen sulfide (H 2 S), hydrogen sulfide anion (HS − ), and sulfide anion (S 2− ) under physiological conditions ( Figure 1A). It has been reported [46,47] that H 2 S, HS − , or S 2− could react quickly with MBB to produce a relatively stable SDB in a Tris-HCl buffer (pH 9.5) under 1% oxygen ( Figure 1B). It would be much easier to detect the SDB rather than the H 2 S in vivo. Herein, the LC-MS was adopted for the measurement of SDB in vivo, which could indirectly reflect the equal amount of H 2 S in vivo. Two peaks, as shown in Figure 5A, indicated effective separation of SDB (Peak 1) and internal standard (Peak 2). Then, the calibration curve was calculated with a concentration range from 0.625 to 20 µM, ( Figure 5B) which indicated a good linear correlation of this method. The SPR promoted H 2 S release in vivo in a fast manner, while SPRC@PLA sustained the elevated plasma H 2 S concentration, as shown in Figure 5C. Instantaneous H 2 S production and metabolism could be observed within 6 h after a single injection of SPRC solution. Differently, SPRC@PLA slowly elevated the plasma H 2 S concentration, which was potentially induced by the sustained SPRC release from SPRC@PLA.

Supplementations Increased the Expression of CSE
The SPRC has been reported to be able to promote the H 2 S release in vivo through the CSE/H 2 S signaling pathway, according to our previous studies [6,7], and the CSE mainly distributed in heart and liver [48][49][50][51]. The expression of CSE in the heart and liver were investigated and the increased expression could be found in both SPRC and SPRC@PLA groups. The SPRC@PLA group showed a higher expression of CSE than the SPRC group both in heart and liver, mainly because of the sustained release of SPRC from SPRC@PLA ( Figure 6).

Supplementations Inhibited the Paw Swollen in AIA Rats
As shown in Figure 7, the paw swollen was calculated through the paw volume and arthritis index, while before the 10th day, no significant increase of these parameters could be observed. However, after 10 days, an instant and dramatic increase of paw volume and arthritis index could be observed in the AIA model group, while the SPRC group showed the same trend, which indicated a low therapeutical effect of the SPRC group. Conversely, the SPRC@PLA group showed significant inhibition of both paw volume and arthritis index. Figure 7. The (A) paw volume was measured by plethysmometer, and the (B) arthritis index were assessed using the arthritis scoring system to evaluate the severity of swollen symptoms (n = 5, mean ± SD).
As illustrated in Figure 8, SPRC showed a negligible anti-inflammatory effect in AIA rats as compared with the model group, while a dramatic decrease in pro-inflammatory cytokines and an increase in anti-inflammatory cytokines could be observed in the SPRC@PLA group. Figure 8. The pro-inflammatory cytokine levels of IL-1β, TNF-α, and IL-6, and anti-inflammatory cytokine level of IL-10 in rats were measured. * indicated a significant different as compared with the AIA model group (n = 5, mean ± SD).

Conclusions
In summary, in this study, SPRC@PLA, a spherical-like microsphere, with a diameter of approximately 30 µm was successfully prepared via the W1/O/W2 emulsification method. In addition, SPRC@PLA showed sustained in vitro SPRC release up to 4 days, and this prolonged in vitro release also promoted in vivo H 2 S release in a sustained manner for 3 days with a single injection. In addition, a once per three-day injection of SPRC@PLA showed good therapeutical effect towards AIA, which increased the administration intervals as compared with those in our previous study [31].  Data Availability Statement: Data can be received from the authors upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.