Rheumatoid arthritis (RA) is a chronic destructive inflammatory synovitis that is associated with a wide range of clinical consequences, including comorbidities affecting systemic bone, vasculature, metabolic function, and cognition. Although Resveratrol (Res) has been shown to have pharmacological benefits in the treatment of RA, its limited bioavailability and poor water solubility have prevented clinical usage of this substance. Therefore, changing the crystalline form of Res to boost its solubility and bioavailability can thus significantly improve its therapeutic effect for RA. Based on this, in this study, the crystalline form of Res was milled into nanocrystals by media milling and loaded into soluble microneedles (MNs) prepared from high molecular weight hyaluronic acid by vacuum casting for the treatment of RA. This study addressed the shortcomings of poor water solubility and low bioavailability of Res, and meanwhile, the transdermal drug delivery system mediated by soluble MNs improved patient compliance and extended the duration of drug action in vivo, which provided a novel research idea for the treatment of RA.
Research Article
Resveratrol nanocrystals-based dissolving microneedles for rheumatoid arthritis
https://doi.org/10.21203/rs.3.rs-3787286/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 31 Mar, 2024
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Dissolvable microneedles
Nanocrystals
Resveratrol
Rheumatoid arthritis
Rheumatoid arthritis (RA) is still an incurable illness [1]. Unmet demands include many patients' partial or non-response to treatment, failure to establish immunological homeostasis or drug-free remission, and inability to repair damaged tissues[2, 3]. Inflammation and immunological reactivity are predominantly restricted to the synovium, causing pain and articular injury, but are also linked to a slew of other complications[4, 5]. RA is now understood to represent the culmination of a multi-year prodromal phase in which systemic immune dysregulation, most likely commencing on mucosal surfaces, is followed by a symptomatic clinical phase. Therefore, there is an urgent need to develop a new mode of drug delivery for the treatment of RA, and the emergence of transdermal drug delivery systems has brought new hope to researchers[6–8].
Nanocrystals (NC), also known as nanosuspensions are nanostates (1 ~ 1000 nm) in which the drug is stably dispersed with the help of a small amount of surfactant or in the presence of polymeric materials, and contain close to 100% of the drug[9, 10]. Nanocrystalline drugs can increase the solubility of insoluble drugs and improve their bioavailability by reducing the particle size and increasing the specific surface area of drug particles[11–13]. Therefore, the small particle size, high drug loading and large specific surface area of the drug after the application of nanotechnology can effectively improve the solubility and dissolution of insoluble drugs, with better safety and feasibility of large-scale commercial production, which is considered to be the ideal dosage form for insoluble drugs[14, 15].
Microneedles (MNs) are micrometer-sized needle-like devices with a pointer length of 20 ~ 2000 µm, assembled in the form of arrays on a patch or support substrate[16, 17]. MNs drug delivery is a form of transdermal drug delivery in which the drug is encapsulated in the tip of the microneedle and is absorbed into the blood circulation by capillaries through channels formed by the tip of the needle piercing the cuticle of the skin, thus realizing localized or systemic drug delivery[18–20]. Microneedle-mediated transdermal drug delivery can avoid the digestive and absorptive effects of the gastrointestinal tract, while avoiding the first-pass effect of the liver, thus improving bioavailability[21–23]. MNs have the advantages of flexibility in termination of drug delivery, reduction of drug toxicity and side effects, and improved safety[24, 25]. Also, because of the fine tip of the MNs, it can reduce pain and achieve the effect of painless drug delivery, which greatly improves the patient's compliance.
Natural products have been extensively studied in the treatment and prevention of various chronic diseases[26]. Resveratrol (Res) is a naturally occurring polyphenol that can be found in mulberries, berries, grapes, and peanuts. It is especially abundant in Polygonum cuspidatum and red grape skin. Res is notable for its positive benefits on health, which are linked to a variety of modes of action, such as its ability to scavenge reactive oxygen species (ROS) and exhibit antioxidative and anti-inflammatory properties[27, 28]. However, its drawbacks such as poor water solubility and low bioavailability have severely limited its clinical application[29]. In recent years, scientific studies have demonstrated that Res has the ability to promote the proliferation of osteoblasts and has a non-negligible role in the treatment of rheumatoid arthritis[30, 31].
In this work, we employed the media milling approach to successfully convert Res into NC, thereby enhancing its solubility and bioavailability while also altering its crystalline structure. Additionally, soluble MNs made by vacuum casting technique were filled with resveratrol nanocrystals (Res NC) for the purpose of treating RA. It was found that the transdermal drug delivery system mediated by soluble MNs improved patient compliance and prolonged the duration of drug action in vivo, as well as inhibited the progression of RA by alleviating the inflammatory effects through inhibiting excessive ROS during the development of RA.
2.1 Materials
Resveratrol: Shanghai McLean Biochemical Technology Co., Ltd; Sodium Hyaluronate (HA-TLM 20–40): Huaxi Bio-technology Co., Ltd; Carboxymethylcellulose Sodium (CMC-Na): Shanghai Aladdin Biochemical Technology Co. Ltd; Tween 80: Shanghai Aladdin Biochemical Technology Co., Ltd; Poroxam 188 (P188): Jiangsu Green Leaf Biotechnology Co., Ltd; Tepan Blue test solution: Beijing Solebao Technology Co: Self-made in the laboratory.
2.2 Methods
2.2.1 Preparation of resveratrol nanocrystals (Res NC)
In this study, nanocrystals were prepared by micro-media milling method, using a syringing bottle as the milling chamber, a magnetic stirrer as the power unit, and 0.5 mm ZrO2 beads as the milling medium. The magnetic rotor was added to a clean cilindrical flask, appropriate amount of stabilizer was weighed into the cilindrical flask, 2 mL of deionized water was added to dissolve it ultrasonically, and then appropriate amount of Res was weighed into the cilindrical flask and dispersed ultrasonically, and then 0.5 mm ZrO2 beads were added, sealed, and then driven by a magnetic stirrer to grind the ZrO2 beads for several hours. At the end of grinding, the nanosuspension was sucked into a plastic centrifuge tube with a syringe, and then 3 mL of deionized water was taken in five times to wash in the vial, and the washing solution was added into the plastic centrifuge tube to obtain the resveratrol nanocrystalline suspension. The optimal preparation process was obtained by examining the type of stabilizer, the ratio of stabilizer to drug, the amount of ZrO2 beads, the grinding time and other factors.
2.2.1.1 Screening of stabilizers
After the drug is milled, its particle size reaches the nanometer scale, the specific surface area and surface energy increase, in order to prevent the particles from particle aggregation due to the change of surface energy, a stabilizer is needed to wet the surface of the particles, so the selection of suitable stabilizers has become the key to the preparation of nanocrystals by micro-media milling method.
In this experiment, five stabilizers, carboxymethylcellulose sodium (CMC-Na), Poroxam 188 (P188), Tween 80, polyvinylpyrrolidone (PVP), and sodium dodecyl sulfate (SDS), were screened (Experiment 1), and the experimental procedure was as follows: five washed 10 mL xylitol bottles were taken and labeled 1, 2, 3, 4, and 5, and five clean 10 mL plastic centrifuge tubes with attached lids were taken and labeled 1 ', 2', 3', 4', 5'. The magnetic rotor was added into clean vials, and 20 mg of the five stabilizers were weighed into the above labeled vials, and each vial was processed in the same way: 2 mL of deionized water was added, and ultrasonication was performed to dissolve the vials for 1 ~ 2 min, 20 mg of resveratrol was added, ultrasonication was performed to disperse the vials for 1 min, 5 mL of ZrO2 beads were added, and the vials were sealed and placed in the thermostatic magnetic stirrer at 600 r/min for 12 h. After the end of stirring, the vial was removed, and the vial was stirred for 12 h. After stirring, remove the vial, draw out the liquid with a syringe, put it into a 10 mL plastic centrifuge tube with a lid, take 3 mL of deionized water to wash the ZrO2 beads in the vial five times, and draw the washing liquid into the centrifuge tube. The liquid in the centrifuge tube was taken to measure its particle size in a laser particle sizer, and the components with uniform dispersion and smaller particle size were selected.
2.2.1.2 Screening of stabilizer to drug ratio
The amount of stabilizer determines whether or not the surface of the particles can be completely wetted, thus determining the extent to which secondary aggregation of particles can be avoided. Therefore, the ratio of drug to stabilizer is equally important in influencing the particle size of the drug after milling.
In this experiment, SDS was used as the stabilizer to complete the screening of the ratio of stabilizer to drug (Experiment 2), and the experimental steps were as follows: five washed 10 mL syringes, labeled 1, 2, 3, 4 and 5, and five clean 10 mL plastic centrifuge tubes with attached lids, labeled 1', 2', 3' and 4', and 5'. 5', 2', 3', 4', 5'. Weigh 4 mg, 10 mg, 20 mg, 40 mg, 80 mg of stabilizer in the above vials respectively, the remaining steps were processed according to the operation of experiment 1, and the liquid in the centrifugal tubes was taken to measure its particle size in a laser particle sizer.
2.2.1.3 Screening of ZrO2 beads dosage
ZrO2 is the grinding medium of the experiment, the crushing of drug particles is realized through the collision between ZrO2 and drug particles, therefore, the dosage of ZrO2 beads has a direct relationship with the particle size of the drug particles, theoretically speaking, under the same rotational speed, the more ZrO2 beads, the greater the possibility of collision of drug particles with them, the smaller the particle size of the drug after grinding, but when the dosage of ZrO2 beads is too much, in the limited space, the However, if too many ZrO2 beads are used, in the limited space, the rotation of the magnetic rotor is not enough to drive all ZrO2 beads, resulting in insufficient grinding.
In this experiment, SDS was used as the stabilizer to complete the screening of the amount of ZrO2 beads, and the experimental steps were as follows: five washed 10 mL syringing bottles labeled 1, 2, 3, 4, 5, and five clean 10 mL plastic centrifugal tubes with lids labeled 1', 2', 3', 4', 5'. ', 5'. Add 1 mL, 2 mL, 3 mL, 5 mL, and 7 mL of ZrO2 beads to the five syringes sequentially, and the remaining steps were handled as in Experiment 1. The liquid in the centrifuge tubes was taken and its particle size was measured in a laser particle sizer.
2.2.1.4 Screening of grinding time
Grinding time is likewise an important variable in determining the size of the drug particles, and different grinding times have an important effect on the nanocrystalline particle size.
In this experiment, SDS was used as a stabilizer to complete the screening of the grinding time, and the experimental procedure was as follows: five washed 10 mL syringes, labeled 1, 2, 3, 4 and 5, and five clean 10 mL plastic centrifuge tubes with attached lids, labeled 1', 2', 3' and 4', were taken. 5', 2', 3', 4', 5'. The ratio of sample addition was the same as that in experiment 1, and then placed on a constant temperature magnetic stirrer at 600 r/min for 4 h, 6 h, 8 h, 12 h and 24 h. The sampling steps were as described in the above experiment, and the liquid in the centrifugal tubes was taken to measure the particle size in a laser particle sizer.
2.2.2 Preparation of Res NC lyophilized powder
Take a clean and dry glass dish, pour Res NC into the glass dish, seal the glass dish with plastic wrap and fix it with a leather band, after fixing it, use a needle tip to tie holes in the plastic wrap, requiring fine and uniform, put it into the freezing layer of the refrigerator at -80 ℃ to carry out pre-freeze-drying, and then transfer it to the lyophilizer to carry out lyophilizing after pre-freezing for 12 h. The lyophilized products were put into plastic centrifugal tubes to be preserved.
2.2.3 Determination of Res NC solubility
Prepare three dry and clean 50 mL plastic centrifuge tubes, add 35 mL of PBS solution containing 0.5% SDS, and label the active pharmaceutical ingredient (API), nanocrystals and mixed samples respectively. Weigh 40 mg of resveratrol API in a dry and clean 10 mL plastic centrifuge tube, add 8 mL of the above PBS solution, and dissolve by ultrasonication. According to the optimal preparation process, two Res NC suspensions were prepared, the suspensions were aspirated using a disposable syringe, and the grinding beads were washed with 2 mL of deionized water, resulting in a diluted Res NC suspension of 4 mL. The two Res NC suspensions were transferred into a 10 mL plastic centrifugal tube for mixing, and shaken well.
Use a pipette gun to aspirate 4 mL of the above-prepared resveratrol API solution into a 14,000 Da dialysis bag, squeeze out the air and tie it tightly with a cotton thread, and place it on a clean petri dish for spare. Prepare a dialysis bag containing 4 mL of resveratrol nanocrystal suspension in the same way.
Use a pipette gun to suck up 2 mL of the above preparation of Res API solution in a 14000 Da dialysis bag, and then use a pipette gun to suck up 2 mL of the above preparation of Res NC suspension in the same dialysis bag, squeeze out the air, and then gently squeeze several times after tying with a cotton thread, so as to make the internal solution of the dialysis bag mixed, and then place it on a clean petri dish for spare.
Put the three dialysis bags into the centrifuge tubes with 35 mL PBS solution, put them into a 37°C constant temperature water bath oscillator, and then take 1 mL of the release medium at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h, respectively, and then replenish the solution with 1 ml PBS to do the in vitro release experiments.
Calculation formula :
Vn: sampling volume; Ci: concentration of Res at the sampling (µg/mL); Cn: concentration of resveratrol at the nth sampling (µg/mL); V0: volume of PBS release medium (mL); mdrug: mass of resveratrol weighed (µg).
2.3 Resveratrol nanocrystal cytotoxicity assay
2.3.1 Cell culture
Murine RAW264.7 macrophages were gifted by the School of Pharmacy, Yantai University. The cells were culture d in RPMI 1640 medium containing 10% FBS, penicillin (50 U/mL), and streptomycin (50 U/mL) at 37°C in a humidified incubator with 5% CO2.
RAW264.7 cells in logarithmic growth phase were taken and inoculated in 96-well plates and placed in a 37°C, 5% CO2 sterile cell culture incubator for 24h, and the cells were allowed to attach to the wall completely. The supernatant was discarded, and different concentrations of resveratrol nanocrystal solutions were given, and after incubated in the cell culture incubator for 24 h and 48 h, 20µL of 5 mg/mL MTT solution was added to each well, and the incubation was continued for 4 h. The medium was discarded, and 200µL of DMSO solution was added to each well, and the crystalline filth produced was fully solubilized by shaking on a shaking table at 37℃ for 10 min, followed by detection of absorbance with absorbance was detected by enzyme labeling instrument.
2.4 Free radical scavenging capacity of Res NC by DPPH method
Decolorization reaction occurs when antioxidants are added to the DPPH solution, so the change in absorbance can be used to quantify the antioxidant capacity of the antioxidant substances. Different concentrations of Res NC were added to the DPPH solution, and after the antioxidant reacted with the assay working solution, the absorbance was measured at 515 nm using an enzyme meter. As a control, DPPH solution with the same concentration but without analyzed samples was used.
2.5 Preparation of microneedles
2.5.1 Preparation of blank microneedles
Weigh a certain amount of low molecular sodium hyaluronate in a 50 mL plastic centrifuge tube, add an appropriate amount of deionized water, stir so that the sodium hyaluronate is completely dissolved into a sol-gel, and then leave it to make the air bubbles disappear.
Inject the bubble-free sodium hyaluronate solution evenly into the clean microneedle mold, and gently shake to ensure that the thickness of the liquid in the mold is uniform, and use a pipette gun to suck up the bubbles in the liquid. Put the mold injected with sodium hyaluronate solution into the vacuum drying oven for vacuuming, remove it and dry it at room temperature until it is completely dry, and demold it.
2.5.2 Preparation of Res NC microneedle
Weigh a certain amount of low molecular sodium hyaluronate in a 50 mL plastic centrifuge tube, add an appropriate amount of Res NC solution, stir so that the sodium hyaluronate is completely dissolved into a sol-gel, and then leave it to make the air bubbles disappear.
Inject the bubble-free sodium hyaluronate solution evenly into a clean microneedle mold, and gently shake to ensure that the thickness of the liquid in the mold is uniform, and use a pipette gun to suck up the bubbles that exist in the liquid. Put the mold injected with sodium hyaluronate solution into the vacuum drying oven for vacuuming, remove it and dry it at room temperature until it is completely dry, and demold it.
2.5.3 Res NC MNs performance testing
2.5.3.1 Skin puncture performance testing
Using a scalpel, the two prepared MNs were cut into 11×11 arrays and placed in reserve for skin-piercing experiments. The MNs should be placed with the tip of the needle pointing upwards, and the MNs should be kept dry.
The hair on the back of anesthetized SD rats was removed using a shaving machine, and the area was covered with depilatory agent for 5–10 min to remove small downy hairs, and the treated skin was wiped with a 0.9% saline-soaked cotton ball after washing off the depilatory agent with water, and the treated back skin was clipped off using scissors. The prepared skin was unfolded and fixed on the operating table with a needle, and the unused skin could be placed in 0.9% saline-soaked gauze and placed in a 4 ℃ refrigerator for backup.
The MNs was inserted vertically into the mouse skin, pressed for 1 min, and removed with forceps. After the staining was completed, a cotton ball was used to absorb the excess staining solution, and the stained portion was rinsed with 0.9% saline. The number of stained pinholes was observed and recorded, and the rate of microneedle puncture was calculated by microneedle puncture rate=(number of blue pinholes/total number of microneedles)×100%.
2.5.3.2 Skin recovery performance test
The prepared Res NC MNs were cut into 38 × 38 arrays using a scalpel, taking care not to destroy the MNs tip during the process. Use tweezers to clip the backing layer of the MNs and stick the MNs backing layer to the pre-prepared tape. Holding the adhesive tape with Res NC MNs, the tape was placed on the back of the experimenter's left hand and pressed for 1 min to allow it to pierce the skin of the back of the hand. The microneedle was pricked into the skin of the dorsum of the hand for 3 min, and the MNs was removed after 3 min, and the skin condition was recorded when the MNs was removed at the time of 0 min, 10 min, 30 min, 1 h, 3 h and 6 h.
2.5.3.3 Res NC MNs solubility determination
The prepared MNs were cut into 1×11 arrays using a scalpel, and the edges of the backing layer were clamped using tweezers, and pierced into the treated mouse skin, and pressed for 15 s, 30 s, 1 min, 2 min, and 3 min, respectively. MNs at different times were uncovered from the skin, pasted on slides, and the intradermal dissolution of the MNs tips at different times was observed under an inverted microscope.
2.6 In vivo pharmacodynamic studies
Male SD rats (SPF grade, 180 g ~ 200 g). The experimental procedures were performed in strict accordance with the relevant regulations of the Committee for the Management and Use of Laboratory Animals of Yantai University.
2.6.1 AIA Arthritis Modeling and Grouping
The AIA model was established by subcutaneous injection of complete Freund's adjuvant (FCA) into the right hind foot of SD rats at 0.20 mL each. Saline was injected subcutaneously into the right hind foot of the control group as a blank control. The SD rats were equally divided into five groups, the control group, the AIA model group, the oral Res administration group, and the Res NC MNs administration group, with three rats in each group, The Res dose for each formulation group was 10 mg/kg, and the dosing cycle was once every two days, starting on the 11th day after modeling. The therapeutic effects of different preparations in rats for the treatment of rheumatoid arthritis were evaluated by evaluating the degree of swelling of the hind feet of the rats in the primary and secondary tests, as well as by staining H&E pathology sections of the hind feet of the rats, and by immunohistochemistry to observe anti-inflammatory effects. Finally, the safety of the different preparations in vivo was judged by body weight changes and organ histopathological evaluation.
2.6.2 Measurement of joint swelling rate
In this experiment, the trend in the rate of swelling of the primary measured joints after the start of modeling and the rate of swelling of the secondary measured joints, which differed in the time of the start of measurement, were counted. Significant swelling of the primary joints was seen on the third day after modeling, and the degree of swelling of the primary joints was measured by toe volumetry on days 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30, respectively. The success of the modeling was demonstrated by swelling of the secondary joint on day 11, and the swelling of the secondary joint was measured by toe volumetry on days 12, 15, 18, 21, 24, 27, and 30 after modeling. The weight of each group was weighed at the same time as each toe volume measurement and the change in weight was recorded.
2.6.3 Histopathological studies of joints
After the last administration, the rats were anesthetized with 10% chloral hydrate, photographs of the hindfoot joint area were taken in each group, followed by cardiac perfusion, and the joint tissues were excised 2 cm out from the top and bottom of the ankle joints of the rats and immersed in paraformaldehyde solution for fixation, followed by decalcification, dehydration, paraffin embedding, filming and H&E staining. At the same time, the synovial tissue was removed and labeled with immunoproteins, and the brown area was the positive expression area. Pathologic sections were observed with a light microscope and photographed.
2.6.4 In vivo safety evaluation
In this experiment, the in vivo safety was assessed by monitoring the changes in body weight and H&E staining pathology of major organs (heart, liver, spleen, lungs and kidneys) of rats at the end of drug administration. The skin of the rats was also observed for redness and swelling after microneedle administration.
3.1 Screening of stabilizer type
From the experimental data, it can be seen that in other experimental conditions are the same, when the stabilizer is SDS, the resveratrol drug particle size is the smallest, 229.9 ± 0.15 nm and the particle size of the particles dispersed uniformly, so SDS is selected as the stabilizer for the preparation of Res NC.
|
Stabilizer type |
Nanocrystal grain size/nm |
PDI |
|---|---|---|
|
CMC-Na |
424.3 ± 2.59 |
0.322 ± 0.001 |
|
Poloxamer188 |
342.8 ± 3.94 |
0.203 ± 0.003 |
|
Tween 80 |
298.9 ± 1.01 |
0.298 ± 0.003 |
|
PVP |
266.5 ± 1.13 |
0.224 ± 0.003 |
|
SDS |
229.9 ± 0.15 |
0.221 ± 0.002 |
3.2 Screening of the ratio of stabilizer to drug
From the experimental data, it can be seen that when the ratio of drug to stabilizer is 1:1, 2:1 and 4:1 under the same other experimental conditions, the particle size of the resulting resveratrol nanocrystalline particles is within the range of 200–300 nm, which is in line with the expected requirements of the experimental results. In order to prepare resveratrol nanocrystals with high drug loading capacity, the experimental condition of 1:1 ratio of drug to stabilizer was chosen for this experiment.
|
Stabilizer-drug ratio |
Nanocrystal grain size/nm |
PDI |
|---|---|---|
|
1: 5 |
668.8 ± 1.32 |
0.375 ± 0.002 |
|
1: 2 |
325.9 ± 2.65 |
0.133 ± 0.004 |
|
1: 1 |
275.9 ± 1.06 |
0.194 ± 0.002 |
|
2: 1 |
229.9 ± 0.35 |
0.154 ± 0.003 |
|
4: 1 |
234.0 ± 2.80 |
0.250 ± 0.001 |
3.3 Screening of ZrO2 bead dosage
From the experimental data, it can be seen that the Res NC particle size decreases with the increase of grinding bead dosage within a certain range when other experimental conditions are the same in the limited space, but too large a dosage of grinding beads will lead to insufficient grinding. In this experiment, when the dosage of ZrO2 beads was 5 mL, the prepared Res NC had smaller particle size, therefore, 5 mL of ZrO2 beads were required to be added for the preparation of Res NC.
|
ZrO2 beads dosage/mL |
Nanocrystal grain size/nm |
PDI |
|---|---|---|
|
1 |
705.2 ± 6.79 |
0.262 ± 0.001 |
|
2 |
466.0 ± 1.10 |
0.255 ± 0.002 |
|
3 |
319.4 ± 0.67 |
0.114 ± 0.002 |
|
5 |
228.6 ± 1.35 |
0.122 ± 0.003 |
|
7 |
571.9 ± 4.69 |
0.273 ± 0.003 |
3.4 Screening of grinding time
The experimental results showed that the longer the grinding time, the smaller the grain size of the resulting resveratrol nanocrystals under the same conditions of other experimental conditions. Therefore, a grinding time of 24 h was used for the preparation of resveratrol nanocrystals.
|
Grinding time/h |
Nanocrystal grain size/nm |
PDI |
|---|---|---|
|
4 |
662.5 ± 5.33 |
0.204 ± 0.006 |
|
6 |
439.6 ± 4.92 |
0.164 ± 0.002 |
|
8 |
331.4 ± 5.99 |
0.177 ± 0.002 |
|
12 |
187.6 ± 4.55 |
0.143 ± 0.002 |
|
24 |
100.0 ± 4.29 |
0.124 ± 0.004 |
In summary, this experiment screened out the best preparation process for the preparation of resveratrol nanocrystals as follows: add the magnetic rotor to a clean cilindrical bottle, weigh 20 mg of sodium dodecyl sulfate (SDS) in a 10 mL cilindrical bottle, add 2 mL of deionized water, dissolve with ultrasonication for 1 ~ 2 min, add 20 mg of resveratrol API, dispersed with ultrasonication for 1 min, add 5 mL of ZrO2 beads, add a magnetic stirrer The nanocrystalline suspension of resveratrol was prepared with a particle size of about 100 nm and a dispersion degree of 0.30 or less, which indicated that the screened experimental method was feasible and the results were good.
3.5 Examination of resveratrol nanocrystal morphology
The prepared resveratrol nanocrystal suspension showed light blue milky light (Fig. 2. A), and the light path could be found by irradiating the nanocrystal suspension solution with a laser pointer, i.e. the Tyndall effect could be produced (Fig. 2. B), which could be initially judged that the prepared nanocrystals had appropriate granularity. The lyophilized powder of resveratrol nanocrystals obtained from the preparation was in the form of white compact lumps, which did not disperse when shaken and was soluble in water (Fig. 2. C), i.e., resveratrol nanocrystals could be lyophilized to prolong the preservation time. The resveratrol nanocrystals were observed to be smaller under the scanning electron microscope.
3.6 Res NC particle size and potential distribution
The particle size of Res NC produced by the optimal preparation process was determined to be around 100 nm with PDI below 0.30 using a laser particle size meter. The prepared nanocrystals had uniform particle size and were well dispersed. By analyzing the potential and distribution diagram of the Res NC suspension, it can be learned that the potential distribution of Res NC is around − 37 mV, and the stability is good. From the experimental data, it can be seen that the drug release of Res API reaches about 10% in 1 h, and 20% in 5 h. Until 25 h, the drug release reaches a maximum of 38%. The mixture of Res NC and Res API could reach 20% in 1 h and up to 40% by 25 h. Res NC can release up to 20% in 1 h, 70% in 10 h, and up to about 80% in 15 h. The drug release of Res NC can reach up to 40% in 25 h.
3.7 Examination of Res NC in vitro
Res NC were put in a certain concentration range at 24h and 48h cell survival rate still reached more than 70%, and in vitro antioxidant capacity reached more than 80%.
3.8 Examination of the morphology of Res NC MNs
After demolding the MNs, the MNs as well as the backing layer were transparent, the needle body was full and conical without needle breakage, the backing layer was complete, the thickness was uniform, and there were no air bubbles, so it was initially judged that the prepared MNs was successful. The Res NC MNs under the optical microscope was full conical, consistent with the visual morphology, the needle body and the backing layer were tightly linked, the thickness of the backing layer was uniform, and there were no air bubbles and hollows. The Res NC MNs have a certain mechanical strength to puncture the skin stratum corneum.
3.9 Examination of Res NC MNs performance
After stabbing into the skin, the skin state can be restored in a relatively short period of time, and it has a fast dissolution speed, which can release the drug in time to achieve a certain therapeutic effect.
3.10 In vivo pharmacodynamic evaluation
As shown in Fig. 7, compared with the control group, the joint swelling of the rats in the AIA model group was significantly increased. H&E staining pathology showed that the AIA model group showed a large number of neutrophils and lymphocytes infiltration and a severe inflammatory reaction compared with the control group. In contrast, compared with the AIA model group, the free Res administration group and the Res NC MNs administration group all reduced the inflammatory response to varying degrees, with the Res NC MNs having the best effect in inhibiting the inflammatory response (Fig. 7B). Compared with the control group, the expression content of TNF-α and IL-6 was significantly higher in the Model group. Compared with the Model group, the TNF-α and IL-6 protein contents of synovial and cartilage tissues in each treatment group decreased to different degrees, with the most significant decrease in protein contents in the Res NC MNs group, indicating that the Res NC MNs treatment was able to effectively alleviate inflammation at the joint site (Fig. 7C and D). When the administration of Res NC MNs began on the 12th day, there was a trend of decreasing joint swelling in all administration groups, among which the decreasing trend was more obvious in the Res NC MNs administration group, which indicated that the Res NC MNs administration group was able to significantly inhibit the joint swelling in rats with AIA-type arthritis (Fig. 7E and F).The body weight of the rats in all groups increased to varying degrees, with the most pronounced increase in the control group and a slow increase in the AIA model group and the other administered groups, which may be related to the inflammatory response (Fig. 7G). Subsequently, we evaluated the hemolysis of the formulation and Res NC did not show hemolysis compared to the negative control group, indicating a good safety profile (Fig. 7H).
3.11 In vivo safety evaluation
As shown in the figure, there was no significant histological damage in the H&E-stained pathograms of the major organs (heart, liver, spleen, lungs and kidneys) of the different dosing groups in SD rats compared to the control group; therefore, Res NC MNs had no significant systemic toxicity in SD rats. At the end of the administration, no significant erythema was observed in the Res NC MNs group compared to the other groups.
In this study, a transdermal drug delivery system based on soluble microneedles prepared from hyaluronic acid encapsulating Res NC was constructed. The optimal preparation process of resveratrol nanocrystals was determined by screening the type of stabilizer, the ratio of stabilizer to drug, the amount of grinding beads, and the grinding time. The prepared Res NC were also examined for morphology, particle size, potential and drug release, and the experimental data showed that the nanocrystals prepared by the optimal process had good stability, solubility properties and no obvious cytotoxicity. The optimal sodium hyaluronate concentration for the preparation of Res NC MNs was determined by screening the sodium hyaluronate concentration. The prepared Res NC MNs was examined for appearance, skin puncture, skin recovery, and skin dissolution, and the experimental results showed that the prepared Res NC MNs had good morphology, good skin puncture rate and did not affect the skin recovery. It is feasible to use hyaluronic acid to prepare MNs carrying Res NC to improve their bioavailability. It was found that a transdermal drug delivery system mediated by soluble MNs improved patient compliance and prolonged the duration of drug action in vivo, while mitigating the inflammatory effects and inhibiting the progression of RA by suppressing excess ROS during RA development. Provides a new research idea for the treatment of RA.
Ethics approval and consent to participate
All experiments were conducted by the ARRIVE Guidelines and were approved by the Animal Ethics Committee of Yantai University (Ethical Annotation License Number: YTU20220923). Male Sprague-Dawley rats (180~200g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Shandong, China). All animals have free access to food and water and are housed at a temperature of 22±1℃, relative humidity of 50±1%, and a light/dark cycle of 12/12 h. Before administration, the rats were anesthetized with intra-peritoneal injection of anesthetics (80 mg/kg ketamine and 4.5 mg/kg xylazine) while monitoring body temperature with a feedback-conditioned heating pad. Rats were euthanized by intraperitoneal injection of excessive pentobarbital sodium. The study does not involve human subjects.
Consent to publication
All authors have read and agreed to the published version of the manuscript.
Declaration of Competing Interest
The authors declare no competing interests.
Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Data availability Statement
Neither the entire paper nor any part of its content has been published or has been accepted elsewhere. It is not being submitted to any other journal. Hope this study can be suitable for the journal.
Authors’ contributions
Ningning Diao: Contributed to the conception of the study, experimented. Wenxin Wang and Min Cao: Visualization, Data curation. Xiaowei Liu and Weili Yang: Visualization, Data curation. Tianying Sun and Huijie Pei: Performed the data analyses and wrote the manuscript. Chunjing Guo: Helped perform the analysis with constructive discussions. Daquan Chen: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing-review & editing.
Author information
Authors and Affiliations
School of Pharmacy, Yantai University, 30 Qingquan Road, Yantai, 264005, China
Ningning Diao, Wenxin Wang, Min Cao, Xiaowei Liu, Weili Yang, Tianying Sun, Huijie Pei, Daquan Chen
College of Marine Life Science, Ocean University of China, 5Yushan 10 Road, Qingdao, 266003, China
Chunjing Guo
Funding
All authors acknowledge financial support from Natural Science Foundation of Shandong Province (No. ZR2019ZD24, ZR2019YQ30), Graduate Innovation Foundation of Yantai University, GIFYTU. We would also like to thank Editage (www.editage.cn) for English language editing.
Corresponding author
Correspondence to Daquan Chen, Chunjing Guo
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