Novel Transdermal Photodynamic Drug-hexaminolevulinate Ethosomes Preparation And Experimental Research On Pharmacodynamics

BackgroundPhotodynamic therapy is a kind of technology,it combines light,oxygen molecules in organization and he photosensitizer,using a specific wavelength excitation light gathered in human tissue photosensitizer,photochemical reaction under the participation of tissue oxygen and produce a large number of singlet oxygen and free radicals,and thus to the organization or pathogenic microorganisms in selective damage treatment in order to achieve the purpose.Because has many advantages,such as minimally invasive,curative effect,avoid oral dosing system side effects,PDT is becoming more and more attention and widely used in clinic.In addition to used in the treatment of condyloma acuminatum,PDT in the treatment of diseases in other applications have also made the exact curative effect,such as acnec[1],psoriasis[2],skin tumor[3],and even the skin beauty[4].5-aminolevulinic acid,the second generation of porphyrin photodynamic drugs,which the advantages of good curative effect,high selectivity,small damage,small side effects,no obvious stimulation,tolerance are excellent,not easy to form scar,does not affect the function of important organs,healing,no drug resistance,has become an important means to some skin disease medication[5,6].5-aminolevulinic acid mechanism is that drug after percutaneous penetration into the skin,through a series of enzymatic action produce strong photosensitization protoporphyrin?(Pp?)and play effect.But in practical application,5-aminolevulinic acid exist the following problems:On the one hand,ALA due to poor stability need temporary preparation;On the other hand,the gel dosage forms such as currently used in clinic and hydrophilic properties of ALA itself,the percutaneous permeability is not ideal and need long time the packet,also lead to drugs can't effectively into the skin and seriously affect the clinical treatment effect and cause the waste of most drugs,these problems have severely to ALA in photodynamic therapy on the clinical application of the skin.Transdermal drug delivery system,can avoid the a first effect,liver kidney toxicity and gastrointestinal reaction,thus improve the bioavailability,but the transdermal drug delivery system difficult to absorb or absorb slow problem in the practical application of common drugs,it's much harder the macromolecular drug percutaneous absorption,in recent years,new type quick release percutaneous drug delivery carrier is widely research,including polymer nanoparticles and solid lipid nanoparticlesx:nano emulsion,polylactic acid(PLA),etc.However,these carrier promotes the drug percutaneous penetration,due to its own entity is still unable to solve the insufficient and defect and difficult to obtain practical applications at the same time,such as:The prepared nanoemulsion must be added up to 30%concentration of surfactant,which may cause potential skin irritation,but also some of the multi-polymer components applied to different skin will appear different reactions,that is to say Nano-emulsion can not guarantee the safety;now prepared solid lipid nanoparticles showed poor stability,etc.[7].Ethosomes is a vesicle bilayer structure like liposomes,by Touitou first proposed in 2000[8],ethosomes can be wrapped drug percutaneous penetration,preparation stability and coating rate is high,the more easily through the skin barrier,and has the advantages of non-toxic,no stimulation for transdermal drug delivery system,also provides a new research direction.Therefore,ethosomes is an ideal drug carrier for photodynamic drugs.ALA instability and the polarity is reason for its' percutaneous penetration and the importantthe clinical application and problems,in recent years,research and development of ALA derivatives has become an important breakthrough direction,and has successfully developed some new derivatives,such as hexyla-minolevulinate,methylaminolevulinate,etc.Many studies have shown that ALA derivatives more stable than ALA,lipid solubility also significantly superior to ALA,it is ALA can produce more PpIX in the skin,it also means that at a lower dose can produce equal treatment in the organisation of PpIX,thereby si gnificantly reducing drug usage,the PDT treatment costs and the side effects[9].This topic will combine Hexaminolevulinate which more lipophilic than ALA with ethosomes this high quality carrier to prepare Hexaminolevulinate ethosom es.Research results will provide a promise for clinical work faster and less sid e effects of new photodynamic drugs.ObjectiveResearch and development of a new type of pertacuneous Photodynamic drugs-hexylaminolevulinate ethosomes,increased penetration ability of hexylaminolevulinate ethosome in order to improve the clinical effect of photodynamic therapy.MethodsAnalysis of Hexylaminolevulinate hydrochloride(HLA),5-aminolevulinic acid hydrochloride(ALA)and Methylaminolevulinate hydrochloride(MLA)in the skin by CLSM Produce the efficacy of Pp?;use Box-Behnken effect surface method to screen out the optimal formulation for preparing HLA ethosomes;perform physicochemical properties and quality control methods of HLA ethosomes through appearance,particle size,encapsulation efficiency,drug loading rate,etc.research;Preliminary analysis and evaluation of pharmacodynamics of prepared HLA ethosomes by in vivo analysis and pharmacodynamic experiments in living animals.Results1.Photodynamic drugs-the efficacy of HLA producing PpIXThe mean fluorescence intensity observed by CLSM can be regarded as the amount of PpIX produced after transdermal delivery of the three drugs at different times.HLA was significantly better than MLA and ALA(P<0.01),MLA and ALA at the 2h and 4h administration of the three drugs.No significant difference(P>0.05).After three photodynamic treatments of psoriasis mice with three drugs,histological HE staining revealed that the HLA group was thinner than the MLA and ALA groups,and immunohistochemistry showed that IL-6 was expressed in HLA.Less than MLA,ALA.This shows that the effect of HLA photodynamic therapy on psoriasis mice is significantly greater than that of MLA and ALA.2.The preparation technology of HLA ethosomes2.Preparation and Quality Control of HLA ethosomesDesignExpert 8.0 software used to minimum size of index screening,it is concluded that hexylaminolevulinate ethosomes optimalprescription:ethanol(31.31%,v/v),soybean lecithin(3%,w/v),ultrasonic power(104 w).The optim ization to return a value,the value of 0.994,the predicted value is close to th e target.hexylaminolevulinate ethosomes appearances are transparent light blue,were observed under transmission electron microscopy(SEM)is relatively unif orm round or oval structure form.The average particle size was 80.87±1.82 nm,PDI was 0.31 ±0.032.The envelopment rate was 38.63 ± 0.003%,drug-load ing rate was 82.85 ±0.004%.3.In vivo experiments of HLA ethosomesAfter dosing 1,2,3 and 4h,The average fluorescence intensity of 0.5%HLA ethosomes group compared 0.5%ALA aqueous solution,5%ALA group were statistically significant differences(P<0.05).4.The pharmacodynamic study of hexylaminolevulinate ethosomesHE can be seen in the dyeing test hexylaminolevulinate ethosome group stratum spinosum cell was thinner than 0.5%ALA,5%ALA group,immunohistochemical display hexylaminolevulinate ethosome group expression of IL-6 was much less than 0.5%ALA,5%ALA group(P<0.05).Conlusion1.Photodynamic drug efficacy studies show that:HLA produced more PpIX than MLA and ALA.In the treatment of psoriasis mice,HLA treatment was superior to MLA and ALA.2.The preparation technology of hexylaminolevulinate ethosome show that the preparation of the Box-Behnken design response surface methodology to screen out the optimal formulation consistent with the preset target prescription results,confirmed the reliability and validity of the method.3.The study of physicochemical properties and quality control of HLA ethosomes showed that the prepared HLA ethosomes are small,uniform,light blue transparent liquids with high encapsulation efficiency and good stability.4.In vivo experiments with HLA ethosomes showed that 0.5%HLA ethosomes had better percutaneous penetration than 0.5%ALA and 5%ALA.5.The pharmacodynamic experiments of animal models of HLA ethosomes psoriasis showed that the HLA ethosomes group was superior to 0.5%ALA group and 5%ALA group.