| Objectives: Capsaicin(CAP) is a kind of vanilla amides alkaloid, which is the active ingredients of spicy chili. A beneficial role of capsaicin has been reported in obesity, cardiovascular and gastrointestinal conditions, various cancers, neurogenic, bladder, and dermatologic conditions and so on. Among them, the long-acting analgesic action is unique and the new mechanism of analgesic action, namely by influencing the neuropeptide substance P release, synthesis and storage, which is different from traditional analgesic drugs and antipyretic analgesics drugs. It has a very good development prospects because of no allergic, no gastrointestinal reaction and no addiction. However, Capsaicin possesses a very high degree of hepatic first-pass effect and a excessively short half-life, at present capsaicin mostly used as topical drug administration, but there will be many serious side effects, such as burning sensation, itching, local hyperemia, some even may cause nerve inflammation. In recent years, many new dosage forms has been developed, trying to improve its topical drug side effects. In this process, the researchers found that the half-life of capsaicin in the body is too short, which limits its application to some extent. So we urgently need to find a new dosage form to improve the status.Liposomes is composed of phospholipids and cholesterol, which has similar biological bilayer membrane structure. But not modified liposome used the effect not beautiful.In recent years, the researchers continue to study the modification of liposome surface, hoping to achieve the goal including sustained release effect, extending biologic half life of drug, reducing irritation, activing targeting characteristics, and increasing biological compatibility, etc. At present the common modifiers include: chitosan and its derivatives, polyethylene glycol, galactose, polyvinyl alcohol, transferrin, peptide, etc. In these modified agent, polyethylene glycol(PEG) can extend the cycle time of drug in the body and prolong the half life of drug obviously. So we will preparate the capsaicin liposome with PEG modificated on it, to prolong the half-life of capsaicin, for further development of capsaicin.Methods: The PEGylated capsaicin liposome was prepared by the thin film dispersion method with ultrasound, the mini-column centrifuge method was established to separate the liposomes and the free drug by investigating the height of mini-column and speed; The RP-HPLC method for encapsulation efficiency of PEGylated capsaicin liposome test was established. The single-factor tests were applied to study the formulation and technological factors, which can influence the EE of PEGylated capsaicin liposome. Big impact factors were screened out and the optimal prescription and preparation technology was obtained by the Central Composite Design-Response Surface Methodology. The optimized PEGylated capsaicin liposome were prepared to verification the efficiency of regression prediction and evaluation of the quality.Dissolution apparatus was taken to investigate the vitro release behaviors of PEGylated capsaicin and capsaicin solution. Establishing a RP-HPLC method to detect the concentration of capsaicin in the vitro release media. The cumulative release rate versus time curve was obtained and the data were fitted to some release models, to clarify the drug releasing mechanism of PEGylated capsaicin.The single dose intravenous injection was taken to examine the pharmacokinetic behaviors and compare the half-time of PEGylated capsaicin with capsaicin solution in rats. RP-HPLC-fluorescence detection method was established to analysis the concentration of capsaicin in rat plasma, the drug concentration in plasma versus time curve was obtained and Kinetica 4.4.1 was used to calculate the relevant pharmacokinetic parameters.PEGylated capsaicin was placed at different light conditions and temperature to investigate the leakage rate and observe the changes in the appearance and characters. The long-term stability of PEGylated capsaicin was examined to provide a theoretical basis for its preservation.Results: The elution conditions of micro column centrifugation to separate the free drug from liposome was determined, which include that: column height is 1.1 m L, the volume of sample is 0.2 m L, the eluent is 0.2 m L PBS solution(p H=7.4), two centrifugal rotational speed are both 2500 rpm, the centrifugal time is 3 min, and centrifugal without eluent for the first time. The column recovery rates of Capsaicin solution, blank liposome and PEGylated capsaicin liposome are 98.07%, 97.74% and 100.2%, respectively, which are able to meet the measurement requirements. A RP- HPLC method for the determination of PEGylated capsaicin liposome’s EE% was established, which was strong specificity, and good stability.The PEGylated capsaicin liposome was prepared by the thin film dispersion with ultrasonic method. The single factor test was used to select the prescription and process conditions, which could influence on EE. The results displayed that the main effects of conditions are Drug/SPC(m/m, X1),Chol/SPC(mo L/mo L, X2), PEG-2000/SPC(mo L/mo L, X3), The Central Composite Design-Response Surface Methodology was used to optimizate the optimal formulation, the result was: the X1 =0.05, X2 =0.25, X3 =0.08; Other conditions were that:PH=7.4 PBS solution as hydration medium, 120 rpm as rotary evaporation speed,50℃ as temperature, 3 h as the hydration time, 4 min as ultrasonic time, 200 W as ultrasonic power. According to the optimal prescription preparation for PEGylated capsaicin liposome’s envelopment rate and drug loadings, respectively 76.45%, 2.88%, Average particle size was120.2 nm.According to the results of the in vitro release, the release rate of capsaicin solution is the fastest, which released 87.17% at 4 h; The release of Capsaicin liposome and PEGylated capsaicin liposome were also slow, capsaicin liposome release reached 80.09% at 19 h, however the PEGylated capsaicin liposome release reached 76.49% at 25 h; The results showed that PEGylated capsaicin liposome in vitro release rate was slower than the capsaicin liposome.Model fitting results showed that capsaicin liposome and capsaicin long circulation liposomes are accord with First-order released model. Determination of capsaicin in rats plasma concentration by RP-HPLC fluorescence method, which was strong specificity and good stability. The Plasma concentration versus time curve was obtained after single dose tail vein injection of capsaicin liposome and PEGylated capsaicin liposome. The dates was analyzd by Kinetica 4.4.1 software, the results were that: the half-time of capsaicin liposome and PEGylated capsaicin liposome were respectively 21.63±2.61 min,48.14±3.79 min, which showed that PEGylated capsaicin liposome has more significant slow-release effect.Temperature and illumination have a great influence on the storage of PEGylated capsaicin liposome. The leakage rates and the appearance were changed significantly, when the temperature or the light was became higher. Long-term tests showed that the leakage rate of PEGylated capsaicin liposome was 22.19% after 3 month under the condition of 4 without light. The result ℃should be kept at 4 avoid light conditions.℃Conclusions: RP-HPLC method was used to determine the envelopment rate of PEGylated capsaicin liposome, which is strong specificity and good stability; The Micro column centrifugation method which was used to separate the liposome and free drugs, is reliable and satisfy the measurement requirements; Star design-response surface optimization method can accurately extract the optimal prescription; Experiments in vivo and in vitro showed that PEGylated capsaicin liposome has the remarkable sustained-release effects; Temperature and illumination has a great influence on the storage of PEGylated capsaicin liposome, so the preparation should be storage in the environment of 4 ℃ without illumination. |
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