| Objective: Capsaicin, a sort of vanillyl amide alkaloids which was extracted from natural peppers, and its chemical name is: N-[(4- hydroxy-3-methoxyphenyl) methyl]-8-methyl-(E)-6-nonenamide. Capsaicin has also shown a wide variety of physiological pharmacology properties, including eliminating inflammation, analgesic effect, bacteriostasis, relieving itching, prevention and cure of angiocardiopathy, promoting lipometabolism, regulating tumor cell apoptosis, and so on, and the most special among them are its long-acting analgesic effect and new analgesic mechanism: relieving pain and itching mainly through act on the release,synthesis and storage of neuropeptide substance P, thus capsaicin has been widely used and posses huge potential on application in medicine, with stronger analgesic effect than opioid analgesics, while without the side effects usually generated by non-steroid anti-inflammatory agents. Clinically, capsaicin has been used to treat severe post-herpetic neuralgia, sciatica, diabetic neuralgia, rheumatoid arthritis, psoriasis vulgaris and so on.However, since capsaicin possesses significant first pass effect of hepar, excessive short half life, low oral bioavailability, now, capsaicin is mostly used in topical drug administration, such as cream, patch, ointments, membrane, cataplasm and so on. But all the above-mentioned routes of administration show various degree of irritant effects on skin which limited its clinical application, such as curtaneous thermalgia, local congestion and neurogenic inflammation. In view of its disadvantages existed in the process of clinical application, the aim of our study is to prepare biodegradable capsaicin-loaded microspheres with polymers, in order to avoid hepatic first pass effect, reduce its stimulation, maintain drug concentration for a long time, so as to reduce the frequency of administration, decrease its side effects, improve patients'compliance, thereby, establish the basis for further development of new form of capsaicin.Method:UV spectroscopy was established in the determination of the content of capsaicin in PLGA microspheres. Capsaicin-loaded PLGA microspheres had been prepared using the solvent evaporation method based on O/W emulsion. The single factor tests were practiced for the identification of main factors affecting the preparation of capsaicin-loaded PLGA microspheres, including the addition method of oil phase, the rotation speed, the reaction temperature, the kind of dispersing agent of aqueous phase, the overall volume of reaction system, the concentration of PLGA, the concentration of gelatin, the mass ratio (CAP/PLGA), the composition of organic solvent and the water/oil phase ratio,and then uniform design was employed to select the optimal prescription and preparation technology, taking particle size, drug loading, entrapment efficiency and score of microspheres as indexes, and then 6 batches of capsaicin-loaded PLGA microspheres were prepared according to the optimal formulation to inspect its reproducibility. We adopt microscopic count to assay the mean diameter and particle size distribution, use the scanning electron microscope to observe the shape and surface of the microspheres, utilize UV spectroscopy to determine the drug loading and entrapment efficiency.High performance liquid chromatography was established in the determination of in vitro release of capsaicin-loaded PLGA microspheres, the in vivo release profile of capsaicin-loaded PLGA microspheres was investigated at 55℃and 37℃, and the accumulative release percentage-time curve was obtained. And then the observed data were fitted to several release models (zero order, first order, Higuchi, Ritger-peppas) ,to clarify the drug releasing mechanism.66 female Kunming mice were injected subcutaneous with capsaicin suspension or capsaicin-loaded PLGA microspheres at dose of 24mg·kg-1. The mice were sacrificed at predetermined intervals and the residual microspheres were removed with surrounding tissue, the residual drug content in tissue was determined via HPLC, so as to obtain the in vivo accumulative release percentage, and the accumulative release percentage-time curve was obtained.The capsaicin-loaded PLGA microspheres were stored under high temperature, huge humidity or highlight condition to perform the influential factor tests, and the permanent stability was investigated by determination of its appearance and drug loading at different times under environmental conditions: sealed up, room temperature, and protected from light.Result:UV-Vis spectroscopy was established in the determination of the content of capsaicin in PLGA microspheres, and the method is reliable ,simple and easy.The initiatory preparative parameters of the capsaicin microspheres was determined by single factor tests: injecting the oil phase into the aqueous phase with glass syringe, gelatin as the dispersing agent for the aqueous phase, the overall volume of reaction system was 150 mL, the concentration of dispersing agent was 0.5%, selecting dichloromethane as the organic solvent, the temperature of water bath was 25℃, and the water/oil phase ratio was 29∶1.The uniform design was employed to refine the prescription and preparation technology,taking particle size,drug loading rate, the entrapment efficiency and score of microspheres as indexes. The quantitative equation was obtained through multiple linear regression,the quantitative effect of the formulation parameters at different levels on the above indexes could be predicted using quantitative equation. The optimal prescription and preparation technology was subsequently obtained:the concentration of PLGA is 2%,the rotation speed is 1000 rpm,the mass ratio (CAP/PLGA) is 1∶2. According to the optimum formulation, the mean particle size,drug-loading rate,entrapment rate and score of 6 batches microspheres prepared from the formulations were: 4.73μm, 27.60%, 82.82% and 7.92, respectively. All the actual indexes were in the extent of predictive value, except for the higher actual mean particle size. Using the scanning electron microscope, we found that the microspheres resulted in spherical shapes and possessed a smooth surface.The dissolubility of capsaicin in PBS (pH 7.4) containing 0.05% Tween 80 and 0.02% sodium azide is 129.47±4.85μg·ml-1,The dissolubility of capsaicin in PBS (pH7.4) containing only 0.05% Tween 80 is 65.80±1.31μg·ml-1。The in vitro release profiles of capsaicin from PLGA microspheres at the previously defined time at 55℃and 37℃demonstrated: after an initial burst (27.16% in 24 hours) , a continuous drug release was observed for up to 164 hours (55.35% in all) at 55℃, whereas, under the condition of 37℃, after an initial burst (21.06% in 24 hours), a continuous drug release was observed for up to 12 days (89.34% in all). By fitting the observed data to several release models (zero order, first order, Higuchi, Ritger-peppas), the drug release from capsaicin-loaded PLGA microspheres can be better agreed with Higuchi equation because of the higher r value, 0.982 and 0.993 respectively. From the Ritger-peppas equation Q=ktn, the releasing indexes (n) were 0.560 and 0.638,respectively, indicated that drug release was non-Fick diffusion controlled, the mechanism may be involved: the dissolution/diffusion of drug from the matrices, and the matrix erosion resulting from degradation/dissolution of PLGA.In vivo drug release was studied by determining the residual drug from mice after subcutaneous injection of capsaicin-loaded microspheres and capsaicin suspension, suggested that the drug suspension released rapidly after subcutaneous injection, the accumulate drug release was more than 97% after 12 hours, while the drug loaded microspheres release profiles showed a large initial burst effect (59%), and then released slowly, nearly 100% of capsaicin released at the end of 20 days.Influential factor test results showed that capsaicin PLGA microspheres are sensitive to temperature, they should be storage under 4℃or 25℃to avoid high temperature, and they are also sensitive to humidity, the morphology of the microspheres would be changed and microspheres were going to aggregate, so they must be sealed up to preserve, they are, however, stable under 4500lx of the light condition. Long-term test suggested that microspheres could be storaged for at least 5 months under environmental conditions: sealed up, room temperature, and protected from light.Conclusions: Capsaicin-loaded PLGA microspheres have been developed using the solvent evaporation method based on O/W emulsion and an optimization strategy, the preparation process is stable, this formulation showed good drug loading rate and encapsulation efficiency, and the microspheres resulted in spherical shapes, smooth surface, with good dispersion. The microspheres showed a prolonged duration of release with regard to suspension when administrated via subcutaneous injection, however, with a large initial burst. The microspheres could be storaged for at least 5 months sealed up under room temperature condition, keeping away from light. |
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