| Research BackgroundAlopecia (baldness) is a common disease in clinical practice. Many factors can cause alopecia and affect the life of a huge group of people. Alopecia can generally be classified into cicatricial alopecia and non-cicatricial alopecia, and non-cicatricial alopecia is more common and includes androgenetic alopecia and alopecia areata. Take the androgenetic alopecia as the example. Somebody has conducted a survey on 5,779 men in five cities, including Beijing, Shanghai, Guangzhou and Hangzhou, and found that the incidence of androgenetic alopecia reached up to 25%.Alopecia has many therapies. In terms of external therapy by drugs, Minoxidil (MDX) is the preferred drug for most alopecia patients, especially for patients with androgenetic alopecia. This drug was originally used as antihypertensive drug in Vasculocardiology Department. But it was found later that MDX had the side effect of triggering hair growth, so it was then used to treat depilation. According to reports in the literature, MDX can promote hair growth in the following six aspects:1. dilating the capillaries, and increasing local blood supply; 2. directly stimulating the proliferation and differentiation of hair follicle epithelium by mitogen effect; 3. Minoxidil Sulfate is the active metabolite to stimulate hair growth, and it can enhance anabolism of hair matrix, outer root sheath and fibrocyte surrounding the hair, and extend hair cycles; 4. This drug is potassium channel activator, and it can prevent calcium ions from entering the cells, since calcium ions and epidermic growth factor can inhibit the growth of hair follicle; 5. MDX can trigger Dermal Papilla Cells to express VEGF and stimulate hair growth. Lachger has reported that MDX could promote the expression of VEGFmRNA in hair follicle cells; 6. MDX can activate Peroxidase PGHS and protect hair follicle cells. For a long time, Minoxidil Tincture has been the main dosage form of this drug, and had many shortcomings, e.g.1. Tincture is highly volatile. Once it is rubbed on the skin, it is likely to crystallize and affect the absorption of the drug; 2. Propylene glycol and alcohol in Minoxidil Tincture can cause skin allergy; 3. This drug can only stay a short period of time within the skin, so it is hard to produce continuous curative effect. Moreover, it can easily pass through the skin and cause side effects in other parts of body, such as hairiness and drop of blood pressure; 4. Alcohol has degreasing effect and cause the dry skin in certain areas. So it is quite urgent for us to develop a new dosage form which can not only prolong drug efficacy, but also overcome the above side effects.Due to the various shortages of Minoxidil tincture, the study on the new dosage form of Minoxidil has never stopped, from tincture containing propylene glycol to tincture excluding propylene glycol, to gel and foam. Although these dosage forms did overcome some shortcomings of the original tincture, they could not fundamentally improve the drug retention within the skin or target at hair follicle, so they did not the replace the traditional tincture. In recent years, the study on liposome has become a new hotspot. More than ten kinds of liposome drugs have been launched in the market, but most of them are oral or intravenous preparations, and few are applied on the skin. Mura S, Pirot F, et al prepared Minoxidil Liposome, and proposed that liposome without alcohol is a very promising treatment for alopecia. Hasanovic A, Hollick C once studied Acyclovir and Minoxidil cationic liposome, and pointed out that cationic liposome has better stability and better diffusion rate on the skin. Jain B, Singh B believed that neutral liposome can deliver the drug to hair-follicle sebaceous glands more efficiently. The above studies are mainly based upon the dosage form of SLN (solid lipid nanoparticles). This dosage form has weak stability and low entrapment efficiency, and it has been gradually replaced by new liposome. Nanostructured lipid carrier (NLC) is a new drug-loading system originated from solid lipid nanoparticles (SLN). It replaces some solid lipid in SLN by liquid lipid, forms lipid skeleton with certain nano-compartments, makes nanoparticles exist in crystal-defect or amorphous structure, avoids the decrease of entrapment efficiency caused by drug efflux during storing process, and thereby slows and controls the drug release. Meanwhile, since it is mainly absorbed by hair follicle, it is particularly suitable for transdermal drug delivery systems (TDDS).We took Minoxidil as main drug, took Stearic acid, Oleic acid and Lecithin as ancillary drugs, determined the optimal proportion of drugs through Orthogonal Design, prepared Minoxidil nanostructured lipid carriers by solvent diffusion & low-temperature curing method, and then observed its property, optimized the formula and conducted transdermal permeation test in vitro, and planned to develop a new dosage form for Minoxidil that has good and lasting curative effect and few side effect, and can replace the traditional Minoxidil tincture.Objective:prepare Minoxidil nanostructured lipid carriers (Minoxidil-NLC), optimize its formula, and conduct comparative transdermal experiment in vitro with Minoxidil tincture, with an aim to develop a new dosage form that can overcome the shortcomings of Minoxidil tincture, enhance and prolong curative effect, reduce side effects and has a wide range of applications.Method:preparation of Minoxidil-NLC:(1) dissolve Minoxidil, Stearic acid, Oleic acid and Octadecane amide in methylene chloride; (2) dissolve Lecithin in alcohol, and mix the solutions obtained by these two steps to form oil phase; (3) dissolve Brij in water to form water phase, slowly add the oil phase to water phase under the same temperature, mix up and vaporize, and add the same volume of ice water, go through ultrasonic treatment for 10 minutes, and pass through 0.45-micron filter membrane, and finally obtain Minoxidil-NLC.Drawing of standard curve:preparation of standard solution:accurately weigh 20 mg Minoxidil, put in 100 mL volumetric flask, add methanol to dissolve it, reach the constant volume, and obtain the standard stock solution at 200μg·L-1. Dilute the standard stock solution by methanol into standard series with different concentrations at 100,50,25,10,5,1,0.5 and 0.1μg·mL-1. According to the pre-experimental results, chromatographic condition:Luna C18 column (150.0mm×4.6 mm,5μm); detection wavelength:281 nm; mobile phase:methanol-water (30:70), flow rate:1.0ml·min-1; column temperature:30℃, sample size:20μl. Take concentration (X) as horizontal coordinate, peak area (Y) as vertical coordinate, and obtain regression equation.Observation on the shape of nanoparticles by transmission electron microscope (TEM):test the size and potential of nanoparticles by Malvern Particle Size Analyzer; measure PH value of nanoparticles by PH meter, and test the organic residue in the drug, observe the stability of the drug.Test on Minoxidil Entrapment Efficiency:separate liposome and free drug by high-speed centrifugation, accurately extract 1.5 mL Minoxidil-NLC into centrifuge tube, centrifuge, take all the supernatant for test, and obtain the content of free drug Wf. Accurately extract 1.5 mL Minoxidil-NLC into 10 mL volumetric flask, add methanol to reach the constant volume, take sample for test, and obtain the total drug content of 1.5mL Minoxidil-NLC, Wt. And then, Entrapment Efficiency (EE%)= (Wt-Wf)/Wt×100%. Orthogonal experiment:based on the result of pre-experiment and entrapment efficiency test, analyze 4 factors that affect Entrapment Efficiency of the drug, i.e. A:mass ratio between Stearic acid and Lecithin, B:ratio between solid and liquid lipid, C:ratio between drug and lipid, D: concentration of emulsifier. Select three levels of each factor to test the entrapment efficiency of each formula, in an effort to optimize the formula.In vitro transdermal permeation test in Franz diffusion cell:Transdermal permeation test:select abdominal skin of SD rat, use puncher to make skin samples with diameter at 22mm (effective internal diameter:18 mm), select 2 samples for each rat, put the samples in diffusion cell, with epidermis facing the supply pool. The receiving solution is PBS (PH=7.4). Add 1mL Minoxidil-NLC, Minoxidil tincture and NLC to different supply pools successively. During the experiment, keep the temperature constant at 37℃, stir up the receiving pool by magnetic stirring apparatus at 200r·min-1. Extract 400μL samples from the receiving pool after 0.5,1,2,3,5,7, 9 and 12h respectively, and meanwhile immediately add the same volume of fresh receiving solution at 37℃. Filter the sample, and measure the peak area by HPLC. Rectify by Cr:Cr=Cn+V1∑Ci-1/V(i=1～n), thereinto, Cn is measured mass concentration, V1 is sample volume, and V is capacity of receiving pool. Transdermal quantity per unit area Q= CrV/A. Conduct linear regression analysis on cumulative transdermal quantity per unit area (Q) and time (t), and obtain the slope, i.e. Permeation Rate Constant "Kp".Test on Minoxidil residue in skin:12 hours after transdermal permeation, take out the skin, wash off the remaining drug and receiving solution by normal saline, sip up by filter paper, scissor the skin in diffusion area, weigh and cut into pieces, add 1 mL normal saline, homogenate, extract Minoxidil from the skin by absolute alcohol for several times (3 times in total, and 1mL for each time), centrifuge at 10,000 r·min-1 at low temperature for 30min, add them together to constant volume of 3mL, filter by millipore filter of 0.45μm, inject 10μL into liquid chromatograph, measure peak area, substitute it in standard curve, and calculate the drug residue in skin.Skin irritation test:complete skin irritation test:select 21 Guinea pigs, each weighing (230.0±55.5)g, male and female equal in number, divide them randomly into three groups, i.e.0.5% Minoxidil tincture group,0.5% Minoxidil NLC group and empty lipid carrier group. Cut off the hairs on both sides of the back of Guinea pigs, and apply the drug on the next day, covering an area of (3 X 3) cm.24 hours later, wash off the drug; 48 hours later, visually inspect the area, and check the erythema in local skin. Non-complete skin irritation test:select another 21 Guinea pigs (ditto), mark the shape of "#" on the back skin by needle head to the limit of just pricking the skin, apply the drug, and observe 48 hours later. After the observation, kill the Guinea pigs for anatomy, and observe the pathological changes of vital organs.Skin sensitization test:select 21 healthy Guinea pigs (ditto), divide them randomly into three groups according to literature method, i.e. Minoxidil nanostructured liposome group, Minoxidil tincture group and 2,4- dinitrochlorobenzene positive control group. Unhair the skin on both sides of spinal column of Guinea pigs, with an area of 3cm×3cm on each side, apply the drug twice a day, and sensitize 1 day,7 days and 14 days afterwards, and observe the anaphylactic reaction of the skin, grade according to the standards in literature and calculate the sensitization strength. Calculate the reaction score according to the formula, i.e. reaction score= (total score of erythema+total score of edema)/total number of animals.·Statistic analysis:statistic analysis was conducted on data by SPSS 13.0 statistical software. Measured data was indicated in the form of X±S. Orthogonal design adopted descriptive analysis. Significance test between two groups adopted single factor analysis of variance. P<0.05 indicated statistical difference.ResultThe Minoxidil-NLC prepared in this research looked clear without any sediment, and showed light blue opalescence. Entrapment efficiency was (86.89±6.26)%, average particle size was (58.20±16.50) nm, Zeta Potential was (23.15±2.60) mv, and the surface of this liposome was positively charged. PH was 4.03±0.23. Under the transmission electron microscope, nanoparticles was in oval or round shape, and evenly distributed. After storing in the refrigerator for two months, the property, particle size and entrapment efficiency of the sample had no significant change; while at room temperature, the sample would show turbid with little sediment. According to the results of Orthogonal experiment, when mass ratio between Stearic acid and Lecithin is 7:5, ratio between solid and liquid lipid is 1:3, ratio between drug and lipid is 1:5, and concentration of emulsifier is 0.045g/ml, the highest entrapment efficiency can be obtained at 86.89%. According to the test on methylene chloride, the average value of 5 batches of methylene chloride residue was 0.0035%.Transdermal quantity of MDX tincture gradually increased as time went by, while the transdermal quantity of MDX-NLC was relatively smaller, and the transdermal concentration rose at a slow pace, lower than MDX tincture at every time point. The permeation rate Kp of MDX tincture (26.82±6.17μg·cm-2h-1) was significantly higher than Kp of MDX-NLC (4.30±1.27μg-cm-2-h-1). After tested by the independent sample "t",t=3.572, P<0.01, showing significant statistical difference between two groups.12 hours after transdermal permeation, drug retention of MDX-NLC in skin was (99.73±9.85)μg·g-1, and drug retention of MDX tincture was (57.00±5.89)μg·g-1, showing significant statistical difference between two groups (F=69.539, P<0.01).Conclusion1. Using a method of emulsion evaporation at a high temperature and solidification at a low temperature was used to prepare Minoxidil nanostructured lipid carriers,the average particle diameter is small,the entrapment efficiency, Physical Properties is stable in low temperature and Organic solvent residue is low.2. The characteristics of MDX-NLC transdermal speed is lower and the drug retention of MDX-NLC in skin is higher than that of MDX-tincture.3. MDX-NLC is no irritation and no allergic to the guinea pig's skin. No pathological changes in guinea pig's skin, heart, kidney and lung... |
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