Of: plum, Chentong Kai, Lin Huaqing, Zhang Shu, Deng Hong
[Abstract] Objective To establish Huperzine A solid lipid Nanoparticles (Hup A SLN) Determination of entrapment efficiency. Methods Using dialysis, ultrafiltration separation of solid lipid nanoparticles and free drug, by high performance liquid chromatography Determination of huperzine A content. The results of ultrafiltration can effectively Huperzine A loaded solid lipid nanoparticles and free separation of Huperzine A recovery of 98.09%, measured 3 batches of samples, the average encapsulation efficiency to 57.63%. Conclusion ultrafiltration convenient, accurate and suitable for Huperzine A for determination of solid lipid nanoparticles encapsulation.
[Keywords:] huperzine A; solid lipid nanoparticles; entrapment efficiency; dialysis; ultrafiltration
Abstract: Objective To establish a method for the determination of the entrapment efficiency of solid lipid nanoparticles loading huperzine A (Hup A SLN). Methods The solid lipid nanoparticles and free drugs were separated by ultrafiltration and dialysis with HPLC for the assay of Hup A. Results Free drugs could be effectively separated from the Hup A SLN by ultrafiltration. The average entrapment efficiency of three batches of Hup A SLN was 57.63%, with the recovery of 98.09%. Conclusion It convenient, sensitive and accurate with ultrafiltration, and can be used to determine the entrapment efficiency of Hup A SLN.
Keywords:: huperzine A; solid lipid nanoparticles; entrapment efficiency; dialysis; ultrafiltration
Huperzine A (huperzine A, Hup A) is from the Chinese herbal Medicine Huperzia serrata (Melaleuca tower) in the isolated natural product, is an efficient and reversible cholinesterase inhibitor, can promote and enhance the memory of the memory representation maintained. According to the literature, Huperzine A treatment of Alzheimer's disease there is a genuine clinical effect, is the international treatment of Alzheimer's ideal drug candidate . but the biological half-life of Huperzine A short oral or adverse reactions after injection affect its efficacy . In recent years, solid lipid nanoparticles (solid lipid nanoparticles, SLN) as a new drug carrier, in a different route of administration has a great potential for development and market prospects. the Hup A encapsulated in SLN, again made of transdermal drug delivery agents, can increase the accumulation of drugs in local tissue and play a continuing role in drug release, reduced systemic absorption to avoid adverse reactions.
Entrapment efficiency of SLN is a prescription drug screening and quality assessment processes important indicators, is also the key to its efficacy to play. In this paper, prepared by high pressure homogenization of Huperzine A loaded solid lipid nanoparticles (Hup A SLN), respectively dialysis method, ultrafiltration method for the determination of its encapsulation efficiency, respectively, to compare these two methods and methodological study, preferably a high accuracy method for determination of Hup A SLN encapsulation.
An instrument and reagent
Ultimate 3000 for High Performance Liquid Chromatography (Dionex Corporation USA); NS1001L high-pressure homogenizer (Italy Niro Soavi SPA. Company); Zetasizer 3000HS Laser particle size analyzer (Malvern UK company).
Huperzine A pharmaceutical ingredients (IMC Zhejiang Pharmaceutical Co., Ltd., batch number: 20090402); dialysis bag (MWCO 8000 to 13 000, per Bo Shanghai Co., Ltd.); monostearin (Tianjin Chemical Co., BES company); ultrafiltration centrifuge tube (MWCO 50 000, Sartorius); poloxamer 188 (BASF Corporation, Germany); oleic acid (Tianjin Chemical Reagent Factory-mao); methanol, acetonitrile for chromatography pure; other reagents for the analysis pure.
2 Methods and Results
2.1 Hup A SLN preparation [3,4]
Weigh 1.5 g poloxamer 188, add appropriate amount of distilled water and ultrasonic dispersion to completely dissolve, placing it in 70 �� water bath, as the water phase; that take prescription drugs and mixed primary lipid material (Monostearate glycerol and oleic acid), the main proportion of drug and lipid materials 1:30 (mass ratio), magnetic stirrer at a constant temperature at 70 �� to fully melt, as the oil phase. in magnetic stirring (1 000 r �� min- 1), the phase drops of water added to the oil phase, maintaining constant stirring to stop heating after a certain time, and continue to stir a certain time, get colostrum. the colostrum in the homogenization pressure 70 000 ~ 90 000 kPa, through the high pressure are uniform quality machine milk 4 times, that was Hup A SLN. 3 batches of the same preparation, at 4 �� refrigerator for later use. with the preparation of blank SLN.
2.2 Chromatographic conditions
Column: Agilent C8 column (250 mm �� 4.6 mm, 5 ��m); mobile phase: 0.02 mol �� L-1 potassium dihydrogen phosphate (pH value of 3.90) acetonitrile (volume ratio 86:14); injection volume: 20 ��L ; detection wavelength: 310 nm; flow rate: 0.8 mL �� min-1.
2.3 Specificity Test
Precise amount of empty medicine solid lipid nanoparticles and solid lipid nanoparticles containing the 1 mL, with the volume fraction of 80% methanol solution after ultrasonic emulsion breaking volume to 50 mL volumetric flask, centrifuged and the supernatant filtered by The chromatographic conditions are sampling, the results shown in Figure 1. Figure 1 shows that the peak of huperzine A good separation of the adjacent peaks, retention time is about 6.3 min, the determination of drug materials without interference.
2.4 Linear Relation
Accurately weighed Huperzine A reference standard, with the volume fraction of 20% methanol solution, the concentration of dissolved dubbed 500.0 ��g �� mL-1 stock solution. Precision Pipette stock solution were appropriate, with the volume fraction of 20% methanol diluted concentration of 5.0,10.0,25.0,40.0,50.0 ��g �� mL-1 of the series of standard solutions, according to "2.2" injection under the chromatographic conditions. the peak area (A) of the mass concentration (��) as a linear regression , the regression equation is A = 0.794��-0.1225 (r = 0.999 9), indicate that Huperzine A in the 5.0 ~ 50.0 ��g �� mL-1 a good linear relationship.
2.5 Precision tests
Take "2.4" under 5.0,25.0,50.0 ��g �� mL-1 solution, according to "2.2" injection under the chromatographic conditions, repeated at 1 d of each sample 5 times, calculating day precision, the result of low, medium, High 3 concentrations were 0.28% RSD, 0.03%, 0.07%; consecutive 5 d repeated injection 5 times, calculate inter-day precision, RSD 0.43%, 0.11%, 0.19%. show that the precision of consistent methodology requirements.
2.6 Stability Test
Precision learned from the "specificity test" under the drug-containing test solution 20 ��L, respectively, 0,2,4,6,8,24 h sampling, press the "2.2" was determined under the chromatographic conditions, record the peak area . Results RSD was 0.56%, indicating that the samples within 24 h in the stable.
2.7 recovery test
Precision preparation of low, medium and high concentrations of 3 (equivalent to the concentration of Hup A 400.0,500.0,600.0 ��g �� mL-1) of the Hup A 20% methanol solution, respectively, and the proportion of blank SLN obtained by mixing a 1:1 mixture . Precision Pipette the mixed solution of 1 mL, the concentration of 5 copies of each were set to 10 mL flask, add appropriate amount of volume fraction of 80% methanol solution after 20 min ultrasonic constant volume, and then by 3 000 r �� min- 1 centrifuge centrifuge 10 min, the supernatant was filtered by "2.2" injection under the chromatographic conditions to calculate the recovery rate, the results in Table 1.
2.8 Hup A SLN in the determination of Hup A
Precision Pipette Hup A SLN 1 mL, 50 mL flask set, add appropriate amount of volume fraction of 80% methanol solution after 20 min ultrasonic constant volume, and then by 3 000 r �� min-1 centrifuges centrifuge 10 min, the supernatant filtration and click "2.2" injection under the chromatographic conditions, using external standard content of the sample. measured levels 3 batches of samples were 99.53%, 99.78%, 99.81%. Table 1 recovery test
2.9 Hup A SLN Determination of entrapment efficiencyLinks to free paper download http://eng.hi138.com
188.8.131.52 Choice of dialysis medium
Dialysis in the dialysis water as the main media generally, because of Hup A is insoluble in water, soluble in methanol, so the choice of dialysis media consider using different concentrations of methanol and water. Because of the structure of methanol on the SLN has damaging effects, so avoid the use of high concentration of methanol. pre-trial found that 20% of the methanol-water solution on the dissolution of Hup A has some ability to come out to meet the conditions of free drugs. and that the volume fraction of methanol on the appearance and structure of SLN had no significant effect, will not damage the particles emulsion layer to affect the accuracy of the results. Therefore, select the 20% methanol solution as dialysis medium.
184.108.40.206 determine the conditions of dialysis 
Precision Pipette Hup A SLN 1 mL, home dialysis bag has been handled well, both ends of the truss was used as internal phase, and then moved to take a 20% aqueous solution of 10 mL of methanol as a dialysis medium, the same method to do 8 samples. 25 ��, 120 r �� min-1 under the conditions, the samples were placed in shaking, 0.5,1,1.5,2,3,4,5,6 h time points were removed one of the samples, taken after the dialysis filter media , according to "2.2" injection under the chromatographic conditions to calculate free drug concentration which showed that reached after 2 h of dialysis in the balance.
220.127.116.11 The recovery test
Preparation of low, medium and high concentrations of 3 (respectively equivalent to the concentration of Hup A 400.0,500.0,600.0 ��g �� mL-1) of the Hup A 20% methanol solution, and blank SLN were obtained by mixing 1:1 mixture . Precision Pipette the mixed solution of 1 mL, at 25 ��, 120 r �� min-1 under the conditions of balance will be placed in shaking 2 h, the Foreign Secretary to take dialysis filter, the determination of free drug content, calculated recovery. The results in Table 2.
18.104.22.168 Determination of entrapment efficiency
Precision Pipette Hup A SLN 1 mL, home dialysis bag has been handled well, both ends of the truss was used as internal phase, and then moved to take a 20% aqueous solution of 10 mL of methanol as a dialysis medium .25 ��, 120 r �� min-1 conditions, placed in shaker, the dialysis 2 h, and then take dialysis filter media, according to "2.2" determined under the chromatographic conditions to calculate the amount of free drug which (Wf); while Precision Pipette Hup A SLN 1 mL, with "2.8" Determination of Hup A SLN under the total drug content, calculate the total amount of the drug (Wo), and calculate the encapsulation efficiency [encapsulation efficiency = (Wo-Wf) / Wo �� 100%]. parallel with the above method 3 batches of samples measured encapsulation efficiency was 54.24%, 52.08%, 53.83%, an average of 53.38%.
22.214.171.124 ultrafiltration centrifuge tube through the ability of the SLN study
Hup A SLN is set to take appropriate ultrafiltration centrifuge tube centrifuge 30 min, speed of 4 000 r �� min-1, collecting the filtrate and the determination of particle size. Filtrate was not detected in the SLN, SLN that there is no filtrate, ultrafiltration centrifuge tube SLN can achieve the full effect of retention.
126.96.36.199 The recovery test 
Preparation of low, medium and high concentrations of 3 (equivalent to the concentration of Hup A 400.0,500.0,600.0 ��g �� mL-1) of the Hup A 20% methanol solution, and blank SLN were obtained by mixing 1:1 mixture. Precision Pipette the mixed solution of 2 mL centrifuge tube set ultrafiltration 30 min, speed of 4 000 r �� min-1, taking home the filtrate 1 mL 10 mL volumetric flask, add the volume fraction of 20% methanol diluted to volume through 0.22 ��m filter membrane, press the "2.2" injection under the chromatographic conditions were measured to calculate the recovery. The results in Table 2. Table 2 The recovery results
188.8.131.52 Determination of entrapment efficiency
Take Hup A SLN amount, set ultrafiltration centrifuge tube 30 min, speed of 4 000 r �� min-1, taking home the filtrate 1 mL 10 mL volumetric flask, add the volume fraction of 20% methanol diluted to volume by 0.22 ��m filter membrane, press the "2.2" injection under the chromatographic conditions to calculate the amount of free drug which (Wf); also moved to take precise Hup A SLN 1 mL, with the "2.8" under the determination of Hup A SLN total drug content, calculate the total amount of the drug (Wo), and calculate the encapsulation efficiency [encapsulation efficiency = (Wo-Wf) / Wo �� 100%]. 3 batches of samples measured encapsulation efficiency was 58.42%, 56.36 %, 58.11%, an average of 57.63%.
3.1 Determination of entrapment efficiency of SLN There are several ways, such as Sephadex column method, protamine precipitation, freezing ultracentrifugation, micro-column method, dialysis and ultrafiltration, with its principles are based on certain The approach allows encapsulation of drugs and free drug separation, determination of the concentration of free drug or encapsulated, calculate the encapsulation efficiency [7,8]. As the experiment smaller particle size of the prepared SLN (30 ~ 100 nm), frozen ultracentrifugation (20 000 �� g) 2 h still not stratified, so the method is not suitable for Hup A SLN entrapment efficiency determination. This paper also tried to dextran gel column separation Hup A SLN in the free drug , experiments using distilled water as eluent, the free drug is only about 70% recovery rate may be Hup A easy absorption in the dextran gel surface of distilled water solubility of Hup A small, hard eluted; solubility of Hup A switch to a larger elution of methanol aqueous solution, although the recovery of Hup A close to 95%, but damage to the structure of dextran gel, Sephadex elution process of the gradual collapse.
3.2 The experiment results show that the ultrafiltration method for determination of encapsulation efficiency measured higher encapsulation rate than dialysis because dialysis may be used in the determination process takes longer, in the dialysis process is encapsulated drugs may have some leaks. Ultrafiltration is the role of the centrifugal force, the use of screening principles of macromolecules and small molecules were isolated: some of the molecular weight of solvent and solute through the membrane of small pore membrane to reach the other side, while the molecular the quality of a large solute or nanoparticles are retained, because the operating time is short, but do not dilute the sample, there is no leakage problem is encapsulated drugs. In addition, test results can be seen from the recovery, dialysis recovery of free drug remains the low, its accuracy is low. ultrafiltration method is simple, rapid, reproducible, and applicable to Huperzine A for determination of solid lipid nanoparticles encapsulation.
 Zhang Haiyan, Tang Xican. Huperzine A: a future treatment of neurodegenerative diseases, drugs [J]. Shanghai Pharmaceutical, 2003,24 (3): 112-120.
 Ren Xiaohui, Wang Wei. Cholinesterase inhibition Healing Stone HUP pharmacological effects and clinical applications [J]. China Pharmacy, 2005,16 (16) :1266-1267.
 MULLER R H, RADTKE M, WISSING S A. Nanostructured lipid matrices for improved microencapsulation of drugs [J]. Int Pharm, 2002,242 (5) :121-128.
 KECK C M, MULLER R H. Drug nanocrystals of poorly soluble drugs produced by high pressure homogenisation [J]. Eur J Pharm Biopharm, 2006,62 (1) :3-16.
 Xiong Fei, Jia-Bi Zhu, Tian Xu, et al. RP HPLC method for the determination of nano liposome breviscapine content and entrapment efficiency [J]. Journal of Pharmaceutical Analysis, 2004,24 (4) :364-366.
 Zhang Ziqiang, Xu Mei, Yao Jing, et al. Ultrafiltration method for the determination of irinotecan encapsulation efficiency of liposomes [J]. Journal of Pharmaceutical Analysis, 2008,28 (12) :2081-2084.
 Chen Yun, Chen Tao, Luting Li. HPLC method for the determination of the content of ethinyl estradiol and the liposomal encapsulation efficiency [J]. Guangdong College of Pharmacy, 2008,24 (3) :217-219.
 Ju Ching Hung, REMEDIES, Han Jing, et al. Chuanhuning Determination of liposomal encapsulation efficiency [J]. Chinese Journal of Pharmaceuticals, 2008,39 (7) :514-516.