Prepare Of Sufentanil-loaded Solid Lipid Nanoparticles And Study On Its Targeting Performance To Peripheral Inflamed Tissue

Background In eighteenth century, Serturner reported the isolation of a pure substance in opium that he named morphine. By the middle of the nineteenth century, the use of pure alkaloids rather than crude opium preparations began to spread throughout the medical world. In addition to the remarkable beneficial effects of opioids, the toxic side effects and addictive potential of these drugs also have been known for centuries. Immunohistochemical studies and in situ hybridization analysis have demonstrated that opioid receptors are expressed in various areas in the central nervous system (CNS). These include the amygdala, the mesencephalic reticular formation, the periaqueductal gray matter (PAG), and the rostral ventral medulla. These opioid receptors expressed in various areas are closely related to all kind of side effect of these drugs. The respiratory-depressant actions of opioids represent their most serious adverse effect. Allμreceptor-stimulating opioids cause dose-dependent depression of respiration in humans primarily through a direct action on brainstem respiratory centers. In the locus caeruleus, the major noradrenergic nucleus in the brain, long-term opioid exposure results in inhibition of adenyl cyclase, reduced activity of protein kinase A, and upregulation of the cAMP pathway. Possible mechanisms involve protein kinase signal transduction cascades that link extracellular signals to cellular changes by regulating target gene expression. Opioids may also produce analgesia through peripheral mechanisms. Immune cells infiltrating the inflammation site may release endogenous opioid-like substances, which act on the opioid receptors located on the primary sensory neuron. These studies suggest that opioid analgesics can produce peripheral opioid receptor-specific antinociception, and its external peripheral effects could avoid the side effects of the central nervous system, such as vomiting, respiratory depression, addiction and so on. Peripheral opioid activity is particularly evident in the inflammatory tissue, given that many pain conditions are associated with inflammation (trauma, postoperative pain, cancer pain, arthritis), inspired by the fact that we can develop an ideal drug, to reduce the central role of opioids and increase the peripheral effects of opioids.Solid lipid nanoparticles(SLN) is thought as the next generation on the targeted durg delivery nanosystems with solid lipid as durg carriers, diameters ranging between 50 and 1000 nanometers, which has the advantages of high physical stability, slow speed of durg leakage and low toxicity etc. When a drug is suitably encapsulated within the carriers or deposited in subsurface, in nanoparticulate form, it can be specifically delivered to the selected target site, released in a controlled way and protected from undergoing premature degradation. This results in higher therapeutic efficacy and dramatically minimizes systemic side effects and toxicity. Thus, SLN is a pormising durg delivery system due to its good physiology compatibility, controlled release, easy, and reasonably cheap to prepare; and specifically accumulate in required sites in the body. Due to their unique size-dependent properties, solid lipid nanoparticles are expected to offer the possibility to improve new dosage form of drug by more and more scholars.As a result, under the financial assistance provided by Guangdong provinceal science and technology programme, sufentanil-loaded solid lipid nanoparticles were prepared by a modified solvent evaporation-low temperature curing technology and the physical and chemical properties of SUF-SLN were studied such as particle size, morphology, Zeta potentials, entrapment eficiency and stability. Next, we established a sensitive method for the determination of sufentanil concentration in mice brain tissue by using enzyme-linked immunosorbent assay (ELISA). Finally, we studied the different concentration of sufentanil and SUF-SLN in brain, liver and inflammatory tissue (skin) of rat model, to evaluate the drug targeting on the inflammatory tissue.Purpose 1. To develop the formulation and characterize the physicochemical properties of sufentanil-loaded solid lipid nanoparticles (SUF-SLN).2. To establish a sensitive method for the determination of sufentanil concentration in mice brain by using enzyme-linked immunosorbent assay (ELISA).3. To evaluate the concentration of SUF-SLN on brain, liver, skin of inflammatory rat model.Method 1. Sufentanil-loaded SLNs preparation and characterization:Based on the optimized results of single-factor and orthogonal design, sufentanil-loaded SLNs were prepared by a modified solvent evaporation method and freeze-drying procedure. The morphology was examined by scanning electron microscope, particle size and zeta potential, drug entrapment efficiency of SUF were determined by Particle diameter analysator and enzyme-linked immunosorbent assay respectively, and long-term physical stability of the SLNs were investigated in detail.2. Method for the determination of sufentanil concentration in mice brain by using enzyme-linked immunosorbent assay (ELISA):The assay was developed by establishing the standard curve and testing limit of detection, recovery rate, precision using sufentanil ELISA kits.3. Determination of sufentanil and SUF-SLN concentration in mice brain, liver and foot skin of inflammatory rat model.Rats were randomly divided into rat formalin inflammatory group (A group) and control group (B group), A group was divided into Sufentanil-loaded SLN group (A1) and sufentanil groups (A2 group), B group is also divided into Sufentanil-loaded SLN group (B1 group) and sufentanil group (B2 group).The inflammatory rat model was established by subcutaneous injection 5% formalin in the left rear foot of rat. Rat brain, liver tissue, inflammatory tissue and blood were obtained according to the different times after injecting of 5μg/kg of sufentanil or Sufentanil-loaded SLN from the tail vein.4. Statistics and analysis: Statistic software SPSS 17.0 was used for statistic analysis. Measurement data were expressed with X±S. The test of normality using Shapiro-Wilk test. Homogeneity of variance, the groups were compared using ANOVA (LSD method), Heterogeneity of variance When using the rank sum test, chi-square test line count data (Fisher exact test), analysis of the main effect of each treatment and interactions using factorial analysis; when P<0.05, the difference was statistically significant.Result 1. The prepared suspension of SUF-SLN exhibited translucent, even quality and the light blue external appearance. The formulated SLNs were found to be relatively uniform in size 115.4±1.6nm, with zeta potential-27.3±1.4mv. The average drug entrapment efficiency of SUF in the nanoparticles was 80.21±2.44%. SUF-SLN displayed spherical or elliptical in shape and was stable.2. The assay was linear over the range of 0.0625-2μg/L for Sufentanil (R2= 0.9937), and the limit of detection was 0.06μg/L. The method afforded recoveries was(81.8±11.9)%-(86.9±7.1)%,and the intra-day and inter-day RSD were (5.0-6.6)% and (4.8-9.6)%,respectively.3. After treated by 5μg/kg of sufentanil or Sufentanil-loaded SLN for 15,30and 60min respectively, there were obvious changes between the different groups in rat brain. After 15minutes of the injection, the sufentanil concentration in A1 groups were significantly lower than that in A2 and B2 groups(P<0.01), the sufentanil concentration in B1 groups were significantly lower than that in A2 and B2 groups(P<0.01). After 30minutes of the injection, sufentanil concentration in brain homogenate, each group showed no significant difference(P> 0.05) and in 60min time point, the sufentanil concentration in A1 groups were significantly lower than that in A2 and B2 groups(P<0.01), B1 groups was significantly lower than A2 and B2 groups (P<0.05) and (P< 0.01).4. After treated by 5μg/kg of sufentanil or Sufentanil-loaded SLN for 15,30and 60min respectively, there were obvious changes between the different groups in rat liver. After 15minutes of the injection, the sufentanil concentration in liver homogenate, A1 groups were significantly lower than A2,B1 and B2 groups(P<0.01), A2 groups were significantly higher than B1 and B2 groups(P<0.01). B1 groups were significantly lower than B2 groups (P<0.05). After 30minutes of the injection, the sufentanil concentration in A1 groups were significantly lower than that in A2, B1 and B2 groups (P<0.05)or P<0.01)and in 60min time point, the sufentanil concentration in A1 groups were significantly lower than that in B2 groups(P<0.01), A2 groups were lower than B2 groups (P<0.05), Bl groups were lower than B2 groups (P<0.05).5. After treated by 5μg/kg of sufentanil or Sufentanil-loaded SLN for 15,30and 60min respectively, each group showed no significant difference in rat plasma (P> 0.05).6. After treated by 5p.g/kg of sufentanil or Sufentanil-loaded SLN for 15,30and 60min respectively, each group showed no significant difference in rat inflammatory tissues or subcutaneous tissue (P> 0.05).Conclusion 1. The SUF-SLN has high entrapment efficiency, uniform particle size, good stability, which initially indicates that this formulation and technology are stable and practical.2. Th method to detect the concentrations of sufentanil by using ELISA is convenient and shortcut, and can be applied to study clinical pharmacokinetic.3. After injecting of sufentanil or Sufentanil-loaded SLN from the rat tail vein, the concentration of SUF-SLN were lower than that of sufentanil in brain and liver tissue, but there was no significant difference in rat plasma and inflammed tissues or subcutaneous tissue...