Preparation Of Matrine Lipid-based Cubic Liquid Crystalline Nanoparticle Gel And Its Pharmacological Evaluation Study

Matrine(MAT)is a quinolizidine derivative of lupine alkaloids,which is widely exists in many Chinese herbal medicines,such as Sophora flavescens,Sophora alopecuroides and Tonkin Sophora root.It has the characteristics of weak alkalinity,easy to pass through biofilm barrier,and easy to be absorbed by oral and percutaneous administration.In clinical,it was mainly used for the treatment of inflammatory diseases,such as hepatitis,gynecological inflammation,respiratory disease,and dermatitis.In recent years,modern pharmacological reports on MAT in the treatment of skin diseases are increasing,such as the treatment of hypertrophic scar,skin cancer and various types of skin inflammation,indicating that MAT has a broad prospect in the field of external drug use.Through searching the drug database of National Medical Products Administration,none external skin application MAT preparations were on sale at present.Researches on MAT new preparation mainly includes liposomes,ethosomes,microspheres and phospholipid complex,which were mainly used in oral preparation or injection application.In terms of transdermal delivery,there were sporadic reports about microemulsion gels and transporters,but these two preparations both contain lots of surfactants,which may cause certain irritantion.Therefore,it is of urgent practical significance and good application prospect to explore suitable MAT preparation for external administration.To expand the scope of application of MAT in topical preparations,we supposed to prepare a topical preparation with high drug loading and good stability.Based on summarized large number of literatures,lipid-based cubic liquid crystalline was chosen as the preferred drug delivery carrier,since it has many advantages,such as high drug loading and good biological affinity.Compared with MAT liposomes and MAT aqueous solutions,MAT cubic liquid crystal nanoparticles have better transdermal effect,which may attributed to its unique structure.However,the study was not deep enough and more systematical study was also need to investigate its suitability on drug dilivery.In this experiment,drug loading,encapsulation efficiency(EE)and particle size were chosen as the main indexes,and its formula and process were also optimized.Finally,we successfully prepared the MAT cubic liquid crystal nanoparticle gel and its quality were also evaluated,which may enrich the application experience of cubic liquid crystal in drug delivery systems.1.Pre-prescribing study of MATThe solubility of MAT in phosphate buffer with different pH and the solubilizing effect of different surfactants were determined.The stability of MAT in high temperature and intense light condition were investigated.For all the solvent we detected,ethanol shows the highest solubility,with 729.46mg·mL-1 and its solubility in water(25℃)was(79.4789±1.1147)mg·mL-1.With the increase of solvent pH value,the solubility of MAT decreased(pH=3,168.97mg·mL-1 and 39.74mg·mL-1 at pH=8).The oil-water partition coefficient of MAT in octanol/water system was logP=0.864.2.Preparation and characterization of lipid-based cubic liquid crystalline nanoparticles(MAT-LLCN)The ultrafiltration centrifugal method was used to determine the encapsulation efficiency(EE)of MAT-LLCN,and its detection parameters were optimized.Systematic methodological investigation was also completed.The determination method of the EE was as follows:the sample were diluted 5 times with purified water and place it in ultrafiltration tube(10 kDa),centrifuge at 8000 r·min-1 for 20 min,collecting the lower filtrate as the free MAT.Through detecting the content of free MAT with HPLC,the EE can be calculated.The particle size determination method were also established:samples diluted 100 times with purified water and determined by Malvern particle size analyzer.The preparation method was optimized with the EE as the index,and the mechanical stirring method was chosen as the preparation method of MAT lipid-based cubic liquid crystalline(MAT-LLC).The ratio of GMO to P407 was investigated by a single factor test and GMO/P407 with 9:1 was chosen.Then,the preparation process parameters of MAT-LLC(stirring time,stirring speed and temperature)were optimized with the orthogonal design method.Then high-pressure homogenization method was used to get the nanosized MAT-LLC(lipid-based cubic liquid crystalline nanoparticles,MAT-LLCN).The optimized prescription of MAT-LLCN was GMO-P407(9:1)11%,matrine 1%-12%,adding water to sufficient amount.The optimal process is that the stirring speed is 600r·min-1,with stirring temperature at 60℃,2 h,he high-pressure homogenization parameters were at 1200 bar,with temperature 20℃ circulated 6 times.r·min-1,The obtained MAT-LLCN was characterized,under the polarizing microscope,and a dark field was observed,which indicated that it may formed the cubic liquid crystal,and the lattice parameters were further measured by small-angle X-ray scattering to comfirm its structure.The ratio of each scattering peak was measured as q1:q2:q3=(?),which confirms the body-centric(Im3m)cubic liquid crystal.With the different drug loading,the average particle size of MAT-LLCN was 104.10-463.7nm,and the drug loading can up to 12%.The accelerated stability of 2%、4%、8%MAT-LLCN was investigated,and the content of MAT had no significant changes after 10d storage at 30℃,which indicated that the stability of the samples was good.3.Study on preparation of MAT-LLCN gel and its quality standard researchesOn the basis of obtain the stable MAT-LLCN,gel matrix material was added to prepare the MAT-LLCN gel.The Capom-940 dosage was optimized as 0.6%.Meanwhile,the hydrochloric acid was used to adjust the pH to 6.0～7.0.According to the guideline of Chinese Pharmacoypoeia(2020 edition),the quality of MAT-LLCN gel was studied,and the appearance,pH value,particle size and MAT content of three batches of MAT-LLCN gel were determined respectively.The results of quality showed that MAT-LLCN gel was a white gelatinous semi-solid preparation with good spreadability,pH6.0～7.0,with the MAT content 10.1976～41.7259 mg·g-1,the particle size was about 97.27～348 nm,and the viscosity of 920.3～5332 mPa·s.The influencing factor experiment of MAT-LLCN gel under high temperature and light conditions were investigated to determine the storage conditions.At high temperature of 60℃,the MAT-LLCN gel appeared slightly yellow in appearance,and the particle size increased slightly,but the pH and content has no significantly changes.Under the condition of strong light irradiation,there was no significant change in almost all indexes,except the content of MAT.Therefore,MAT-LLCN gel was suggested to store at low temperature and light avoidance.The appearance,particle size,pH and content of MAT-LLCN gel with different drug loading did not change significantly when the gel was store at 30℃ for 1 month.4.In vitro evaluation of MAT-LLCN gelThe modified Franz diffusion cell method were used to compare the in vitro release and in vitro transdermal behavior of 4%MAT-LLCN gel and 4%MAT ordinary gel.The release curves of the two groups looked exactly like each other,and there was no significant difference in the drug cumulative release(P>0.05).The MAT cumulative release curve from MAT-LLCN gel conformed to the Weibull model,with R2=0.954 0.The drug release mechanism follows Fick’s law of diffusion.In vitro transdermal experiment was performed by modified Franz diffusion cell method,and the difference between MAT-LLCN gel and ordinary gel in transdermal effect was compared.The cumulative release rate and steady-state release rate were calculated,and the retention amount of MAT in cuticle,active epidermis and dermis was determined.The 24h cumulative release of 4%MAT-LLCN gel and ordinary gel was(2229.13±558.52)μg·cm-2,(1671.69±276.62)μg·cm-2,and the steady state release equation was y=97.692x-67.883(R2=0.9924),y=72.002x-17.167(R2=0.9942),steady release rate was 97.692 μg·cm-2·h-1,72.002 μg·cm-2·h-1,cuticle retention was(93.83±57.66)μg·cm-2,(19.19±6.96)μg·cm-2,respectively.The retention of active epidermis and dermis was 345.28±75.33μg·cm-2 and(163.76±56.92)μg·cm-2.In vitro transdermal test showed that the cumulative release rate,steady release rate and skin retention of matrine in cubic liquid crystal gel were significantly higher than those in ordinary gel(P<0.05).These results suggest that with the higher drug loading,it’s easier for LLCN to form the drug storage in deep skin.MAT-LLCN gel also showed better bioaffinity,which was beneficial to maintain the stability of cell structure.Through HE staining of rat skin in vitro at different times,the skin of rats in the normal saline group had a clear layered structure and the stratum comeum was still closely arranged within 8 hours.After 24 h in vitro transdermal penetration,there were obvious gaps between epidermis and dermis in normal saline group and MAT gel group,with disorder arrangement of basal cells,loose arrangement of dermis collagen fibers,and enlargement of intercellular cracks,indicating obvious damage of dermis skin structure.However,in MAT-LLCN gel group,the arrangement of dermal collagen fibers was still regular,and the cell space was less,suggesting that MAT-LLCN gel had a certain biological affinity,which was beneficial to maintain the structural stability of deep skin cells.In conclusion,through this study,a stable,quality-controllable MAT-LLCN gel was prepared,and it was confirmed that cubic liquid crystal has obvious advantages in increasing drug loading,accelerating drug transdermal barrier and increasing drug storage concentration in dermal layer of skin,which enriched the application of cubic liquid crystal in transdermal drug delivery.