| BackgroundAlzheimer' s disease(AD)is the most common reason of senile dementia,and it is a degenerative disease of central nervous system characterized by progressive cognitive dysfunction,which is greatly harmful to health of the elderly.The exact pathological mechanisms of AD are still under investigation,among which the A? cascade hypothesis is rather recommended.The main pathological alteration is senile plaques(SP)formed by depositions of ?-amyloid protein(A?),and AP depositions could induce intracerebral oxidative stress,activate neuroinflammatory response,promote cell apoptosis,cause disturbances of A? metabolisms and decrease neuronal synaptic plasticity.There is no effective drug for the prevention and treatment of AD at present,thus,it is in urgent need to develop related drugs for the treatment of AD.Lycopene,a pigment belonging to carotenoid family,mostly exists in tomatoes and other fruits with red color.Lycopene could serve as an efficient antioxidant and singlet-oxygen quencher,and has demonstrated diverse and remarkable bioactivities,such as anti-oxidative stress,anti-inflammation,anti-apoptosis and anti-cancer.There has been several evidence indicating that lycopene could exert neuroprotective effects against various neurological disorders.Nevertheless,due to extremely poor solubility,instability and low oral bioavailability,its pharmaceutical applications in treatment of neurological diseases are restricted.Therefore,it is significant to develop novel dosage form for lycopene to elevate solubility and stability,as well as oral bioavailability and further brain efficacy.Microemulsion(ME)is a thermodynamically stable system mixed by oil,surfactant,co-surfactant and water,with droplet size less than 100 nm.In addition to manifold advantages of smaller size and monodispersibility,microemulsion is known to have higher solubilization capacity for hydrophobic substances and possesses promising stability to protect bioactive components from undesired damage,so this system has been extensively used to deliver drugs.Some studies have suggested that microemulsion with specific formations could be successfully utilized to enhance oral bioavailability of poorly water soluble compounds and promote targeted drug delivery to the brain.Hence,the microemulsion system becomes an attractive choice for lycopene oral delivery,and the development of lycopene-loaded microemulsion(LME)with specific formations could facilitate targeted drug delivery to the brain and exert its neuroprotective effects.ObjectiveThis study aimed to develop LME dosage form via formulation selections,and various characteristics of LME were observed and investigated;with the conventional dosage form of lycopene dissolved in olive oil(LOO)as the control,the in vivo metabolic characteristics of LME was analyzed via pharmacokinetic study and tissue distribution study,and the oral bioavailability and brain-targeting of LME were further evaluated;the A? injury moue model was constructed by intracerebroventricular(ICV)stereotaxtic injection,and the neuroprotective effects of LME on A? injury were explored via intracerebral oxidative stress,neuroinflammatory response,cell apoptosis,A? associated metabolisms and synaptic plasticity.By means of the abovementioned serial investigations,we aimed to develop an oral dosage form of LME with high bioavailability,superior physicochemical properties and brain-targeting characteristics,which provided experimental evidence for AD treatment investigations and offered novel strategies and protocols for brain-targeting treatments of other drugs.Methods1.Preparation and characterization of LME1.1 HPLC analysis of lycopeneIn this study,we conducted serial investigations for lycopene determinations,including selection of determination wavelength,chromatographic conditions,specificity,construction of lycopene standard curve,precisions,methodological recovery and stability.The high-performance liquid chromatography(HPLC)analytical method of lycopene was established,and lycopene solubility in oils as well as lycopene content incorporated into LME formulations were determined.1.2 Initial screening of microemulsion compositionsAccording to demands of this study,microemulsion compositions including oil,surfactant and co-surfactant were initialially screened.The lycopene solubility in six different kinds of oils((R)-(+)-limonene,ethyl oleate,oleic acid,olive oil,soybean oil and corn oil)was determined,and three kinds of oils were initialially selected based on their lycopene solubility;Tween 80 was selected as the surfactant for its reported property of superior emulsifying capacity,appropriate hydrophilic-lipophilic balance value(HLB=15),accepted safety for pharmaceutical applications and enhanced brain-targeting for coated nanoparticles;Transcutol HP,PEG 400 and glycerol were selected as co-surfactant candidates due to high biocompatibility and safety,so they were chosen for further investigations.1.3 Orthogonal optimization and construction of pseudo-ternary phase diagramsOn the basis of initial screening of microemulsion compositions,the oil,co-surfactant and surfactant to co-surfactant ratio(Km)were selected as three main factors affecting microemulsion preparation and formation,each containing three levels,and the standard L9(34)orthogonal design was used for further optimization.The surfactant and different co-surfactants were mixed at various ratios(Km=2:1,3:2 and 3:1)according to orthogonal design to make the surfactant and co-surfactant mixture(Smix).Afterwards,selected oil phases and Smix were mixed homogeneously,where the ratios of oil to Smix were set at 9:1,8:2,7:3,6:4,5:5,4:6,3:7,2:8,1:9(w/w),and the blank microemulsion was prepared by the water titration method.In accordance with the weight ratios of oil,Smix and water at the titration critical point(occurrence of transparency-to-turbidity transition),the pseudo-ternary phase diagrams of blank microemulsion were constructed using OriginPro 8.5 software.The optimum combinations and formulations of blank microemulsion were determined based on areas of microemulsion region.1.4 Preparation and formulation optimization of LMEWe prepared blank microemulsion based on its optimum combinations and formulations,and the blank microemulsion possessed superior stability when the ratios of oil to Smix were set at 1:9 and 2:8(w/w).However,when the ratios varied from 3:7 to 9:1(w/w),a little precipitation was observed when samples were subjected to centrifugation after storage.Therefore,several samples of LME with oil to Smix ratios of 1:9 or 2:8(w/w)were prepared by the water titration method,and they were characterized in terms of various parameters for formulation optimization,including physical stability,droplet size,polydispersity index(PDI),zeta potential and lycopene content to Smix ratio.1.5 Characterization of LMEThe characteristics of LME formulation were evaluated in terms of physical stability,droplet size,PDI,zeta potential,lycopene content,ultrastructural morphology and storage stability.The ultrastructural morphology of LME was observed by transmission electron microscopy.As for the stability study during storage,LME and LOO samples were prepared and stored in light-protected and tightly sealed containers flushed with nitrogen gas at 4? and room temperature(25?)for a totol of 8w,respectively,and the storage stability of LME was assessed and compared with LOO.2.Pharmacokinetic study of LME in vivo2.1 HPLC analysis of lycopene in plasmaWe conducted serial investigations for lycopene determinations in plasma,including chromatographic conditions,analysis of plasma samples,specificity,construction of plasma standard curve,precisions,accuracy,extraction recovery,stability of plasma samples,and the HPLC analytical method of lycopene in rat plasma was established.2.2 Animal study of pharmacokineticsA total of 12 male Wistar rats were randomly divided into the test group(LME group)and control group(LOO group)(n=6 in each group).These two groups of rats were orally administered LME and LOO dosage forms separately at a dose of 8 mg/kg based on lycopene concentration,and at different time points after oral administration,blood sample was collected from the jugular vein.The plasma was isolated from the obtained blood sample,and the lycopene concentration in rat plasma was determined by HPLC analytical method.2.3 Statistical analysis of experimental dataThe lycopene concentration in rat plasma at different time points after oral administration was analyzed,and the plasma concentration-time profiles of lycopene were plotted.The pharmacokinetic parameters of plasma were calculated by non-compartmental analysis using DAS software 2.0,including area under the concentration-time curve(AUC),peak concentration(Cmax),time to reach peak concentration(Tmax),half-life(ti/2),mean residence time(MRT)and plasma clearance(CL),among which the measured values of Cmax and Tmax were employed,and the relative bioavailability of lycopene was calculated as well.3.Tissue distribution study and brain-targeting evaluation of LME in vivo3.1 HPLC analysis of lycopene in tissuesWe conducted serial investigations for lycopene determinations in different tissues,including chromatographic conditions,analysis of tissue samples,specificities,construction of tissue standard curves,precisions,accuracies,extraction recovery,stability of tissue samples,and the HPLC analytical methods of lycopene in mouse plasma,brain,liver,kidney,heart,lung and spleen tissues were established.3.2 Animal studyA total of 96 male C57BL/6 mice were randomly divided into the test group(LME group)and control group(LOO group)(n=48 in each group).These two groups of mice were orally administered LME and LOO dosage forms separately at a dose of 8 mg/kg based on lycopene concentration.At different time points after administration,blood was collected from six mice at every time point in each group,respectively,and the plasma was isolated from the obtained blood sample.Thereafter,mice were humanely sacrificed,then samples of brain,liver,kidney,heart,lung and spleen tissues were promptly harvested,and the lycopene concentrations in different tissues were determined by HPLC analytical method.3.3 Tissue distribution of lycopene in miceThe lycopene concentrations in mouse plasma,brain,liver,kidney,heart,lung and spleen tissues at different time points after oral administration were analyzed,and the concentration-time profiles of lycopene in different tissues were plotted,respectively.The pharmacokinetic parameters of corresponding tissues were calculated by non-compartmental analysis using DAS software 2.0,including AUC,Cmax,Tmam,ti/2 and MRT,among which the measured values of Cmax and Tmax were employed,and distributions of lycopene in mouse plasma,brain,liver,kidney,heart,lung and spleen tissues were analyzed.3.4 Drug brain-targeting evaluationAccording to distributions of lycopene in mouse plasma,brain,liver,kidney,heart,lung and spleen tissues,we used targeting parameters,including the relative rates of uptake(Re),the ratio of peak concentration(Ce)and drug targeting index(DTI),to evaluate the targeting efficiency of LME towards brain and other tissues.4.Neuroprotective effects of LME against A? injury4.1 Animal studyA total of 48 male C57BL/6 mice were randomly divided into the peudosurgery group(NC group),A? model group(A? group),LME treatment group(A?+LME group)and LOO treatment group(A?+LOO group)(n=12 in each group).The ICV stereotaxtic injection was performed for all groups of mice,among which mice in the NC group were injected ICV with 2?L PBS(pH7.4),while mice in the A?,A?+LME and A?+LOO groups were injected ICV with 2?L A? solution ?mg/mL).After surgery for 1w,mice in the A?+LME and A?+LOO groups were orally administered LME and LOO dosage forms separately at a dose of 4 mg/kg daily based on lycopene concentration,while mice in the NC and A? groups were orally administered equal aliquot of normal saline.The oral administration lasted for 3w for all groups of mice.4.2 Handling of brain tissue sampleWhen oral administration finished,three mice were randomly selected from each group.Under anesthesia condition,cardiac perfusions of normal saline,followed by 4%paraformaldehyde,were performed for mice,and then mouse brain was removed and fixed in 4%paraformaldehyde.Afterwards,mouse brain tissue was dehydrated,transparentized and embedded by paraffin,and coronal sections were prepared for immunohistochemistry determinations.Moreover,cardiac perfusion of normal saline was performed for other mice in all groups,then mouse brain was removed and stored at-80? for oxidative stress and western blotting analysis.4.3 Determination of oxidative stress level in brain tissueThe malondialdehyde(MDA)content in brain tissue was determined by thiobarbituric acid(TBA)method,and the activities of superoxide dismutase(SOD)and catalase(CAT)were determined by hydroxylamine method and ultraviolet colorimetric method,respectively.The effects of LME on oxidative stress in brain tissue were observed.4.4 Determination of neuroinflammatory response markersThe glial fibrillary acidic protein(GFAP)and ionized calcium binding adapter molecule I(Iba1)are specific markers of astrocytes and microglia,respectively,which are main inflammatory cells in brain tissue and reflects neuroinflammatory response.The expression levels of GFAP and Iba1 in hippocampus and cortex were determined by immunohistochemistry and western blotting respectively to investigate the effects of LME on neuroinflammatory response.4.5 Determination of cell apoptosis markers in brain tissueB cell lymphoma-2(Bcl-2)could suppress cell apoptosis,while Bcl-2 associated X protein(Bax)could induce cell apoptosis,thus,the ratio of Bax/Bcl-2 is a significant factor in determining cell apoptosis;in addition,cysteinyl aspartate specific proteinase-3(Caspase-3)serves as the crucial apoptotic protease in the downstream of various apoptotic pathways,and it is activited to trigger final apoptosis.The expression levels of Bcl-2 and Bax in brain tissue were determined by western blotting,and the ratio of Bax/Bcl-2 was calculated;furthermore,the Caspase-3 activity in brain tissue was determined by microscale spectrophotometry method to explore the effects of LME on cell apoptosis.4.6 Determination of A? associated metabolisms in brain tissueA? is the metabolite of ?-amyloid precursor protein(APP).The cleavage of APP in brain tissue is involved in A? metabolic pathway and non-Ap metabolic pathway,and?-site amyloid precursor protein cleaving enzyme 1(BACE1)and a disintegrin and metalloprotease 110(ADAM 10)are key enzymes of these two pathways,respectively.The expression levels of APP,BACE1 and ADAM10 in brain tissue were determined by western blotting to evaluate the effects of LME on A? associated metabolisms.4.7 Determination of neuronal synaptic plasticity related proteinsSynaptophysin(Syn)is a calcium binding glycoprotein existed on presynaptic vesicular membrane,and postsynaptic density material 95(PSD95)is a scaffolding protein existed in postsynaptic density region,both of which are important neuronal synaptic plasticity related proteins.The expression levels of Syn and PSD95 in brain tissue were determined by western blotting respectively to assess the effects of LME on neuronal synaptic plasticity.Results1.Preparation and characterization of LME1.1 Preparation and formulation optimization of blank microemulsion and LMEOn the basis of initial screening of microemulsion compositions,the orthogonal optimization was designed and pseudo-ternary phase diagrams were constructed.Using areas of microemulsion region as the evaluative index,the optimized formulation and compositions of blank microemulsion were finalized,i.e.,the microemulsion compositions contained(R)-(+)-limonene,Tween 80,Transcutol HP and water,and Tween 80:Transcutol HP=2:1(w/w).The optimized LME formulation was as follows:8%w/w of lycopene and(R)-(+)-limonene(oil),32%w/w of Tween 80 and Transcutol HP(Km=2:1,w/w)and 60%w/w of water,respectively.1.2 Characterization of LMEThe prepared LME based on optimized formulation showed an average droplet size of 12.61 ± 0.46 nm,with PDI value of 0.086 ± 0.028.The zeta potential was determined to be-0.49 ± 0.12 mV,and 463.03 ± 8.96 mg/mL lycopene was found to be soluble and could be incorporated into final microemulsion system.LME possessed superior physical stability,and the spherical and homogeneous morphology was captured by transmission electron microscopy imaging.During storage for a total of 8w,LME demonstrated superior thermodynamic stability.At both 4? and 25 ?,the average percentages of lycopene remaining(PLR)were significantly higher for LME compared with LOO after 2,4,6 and 8 w of storage(Ps<0.05 for 2 w,Ps<0.01 for 4,6 and 8 w),indicating that LME could better protect lycopene from degradation during storage,and this protective effect was more conspicuous when LME was stored at 4?.2.Pharmacokinetic study of LME in vivoAfter oral administration of LME and LOO to rats respectively,the AUC of lycopene in the LME group(4775.93 ± 634.00 h.ng/mL)was significantly increased compared with that in the LOO group(2270.96 ±455.46 h·ng/mL,P<0.01),and the Cmax value for LME was 220.48±30.84 ng/mL,which was 1.82 times greater than that for LOO(121.32±13.47 ng/mL).In addition,the relative bioavailability of LME was elevated to 210.30%,indicating a dramatic enhancement in absorption and oral bioavailability of lycopene.Furthermore,significantly enhanced t1/2 and longer MRT(Ps<0.05)and significantly lower CL(P<0.01)were observed in LME-treated rats,suggesting a slower in vivo elimination rate and prolonged residence time in blood circulation for LME compared with LOO.3.Tissue distribution study and brain-targeting evaluation of LME in vivo3.1 Tissue distribution of lycopene in miceThe AUC values of mouse plasma,brain,liver,kidney,heart,lung and spleen tissues were all enhanced in the LME group in comparison to those in the LOO group,among which the enhancement of brain tissue was most conspicuous,and it was increased from 416.81 h.ng/g(LOO group)to 3549.52 h·ng/g(LME group);As for Cmax,its values of plasma,brain,heart,liver and spleen were markedly enhanced in the LME group compared to those in the LOO group(Ps<0.01),among which the enhancement of brain tissue was most significant,and it was increased from 20.62 ±3.39 ng/g(LOO group)to 143.86 ± 15.27 ng/g(LME group).Neverthelss,for lung and kidney tissues,the differences did not reach significant levels(Ps>0.05);In terms of t1/2 and MRT,in comparison with LOO,longer ti/2 and MRT were observed for LME,among which the magnitude of MRT prolongation was largest for brain tissue.The aforementioned results indicated that,compared with LOO,the biodistributions of lycopene were greatly altered for LME,especially the phenomenon that LME distinctly facilitated distribution of lycopene in brain tissue.3.2 Brain-targeting evaluation of LMEThe differences of drug concentration in blood circulation could be observed due to distinct absorption of oral dosage forms.Although the parameters of Re and Ce could not be employed to directly evaluate drug targeting to tissues just like intravenous administration,they could reflect alterations of tissue distribution after oral administration of drugs.Among all tissues,the maximum values of Re and Ce were obtained for brain tissue,with 8.52 and 6.98 respectively,proving a dramatic increase of lycopene distribution in the brain for LME compared with LOO.More importantly,the targeting parameter of DTI was implemented for the purpose of better evaluating blood-to-tissue direct transport,and DTI>1 was considered as the targeting distribution.The DTI values were determined to be less than 1 for the kidney,heart,lung and spleen tissues,thus,the targeting of LME towards the abovementioned tissues was not shown;the DTI value was 1.09 for liver,indicating a slightly preferential targeting distribution;with regard to the brain tissue,this value was 3.45,implying that LME possessed good brain-targeting efficiency.4.Neuroprotective effects of LME against A? injury4.1 Oxidative stress level in brain tissueCompared with the NC group,the MDA content in mouse brain tissues of the A?group was significantly enhanced(P<0.01).The MDA contents of the A?+LME and A?+LOO groups were both remarkably decreased in comparison with the A? group(Ps<0.01),while the content was also markedly lowered in the A?+LME group than that in the LOO group(P<0.01).In terms of SOD and CAT activities in brain tissues,the values were significantly decreased in the A? group compared with the NC group(Ps<0.01),while in comparison with both of the A? and AP+LOO groups,SOD and CAT activities were remarkably increased in the A?+LME group(Ps<0.01).These results indicated that LME could significantly decrease oxidative stress level in brain tissue and possessed protective effects against A?-induced brain injury.4 2 Neuroinflammatory responseThe immunohistochemistry results of GFAP and Ibal showed that,compared with mice in the NC group,the GFAP+and Iba1+cell numbers and positive area fractions were all significantly increased in mouse hippocampus and cortex of the A? group(Ps<0.01).In comparison with both the A? and AP+LOO groups,the oral administration of LME markedly decreased GFAP+and Iba1+cell numbers and positive area fractions in hippocampus and cortex(Ps<0.01).Similarly,western blotting analysis also suggested that the expressions of GFAP and Iba1 in mouse brains of the A?group were remarkably enhanced than those in the NC group(Ps<0.01).In comparison with the A? group,the expressions of GFAP and Iba1 in the A?+LME group were significantly lowered(Ps<0.01),and these phenomena existed when compared with the AP+LOO group as well(P<0.05 and P<0.01,respectively).The aforementioned results implied that LME effectively attenuated neuroinflammatory response in brain tissues.4.3 Cell apoptosis in brain tissueWestern blotting analysis demonstrated that,compared with the NC group,the expression level of Bcl-2 was significantly decreased in mouse brain tissues of the A?group(P<0.01),while the expression of Bax was remarkably increased(P<0.01),and the ratio of Bax/Bcl-2 was markedly enhanced(P<0.01).In comparison with both the A? and A?+LOO groups,the expression of Bcl-2 was significantly enhanced in the A?+LME group(Ps<0.01),while the expression of Bax was remarkably decreased(Ps<0.01),and the ratio of Bax/Bcl-2 was markedly lowered(Ps<0.01).The results of Caspase-3 activity determination implied that Caspase-3 activity in mouse brains of the A? group was significantly increased than that of the NC group(P<0.01).Compared with both the A?and A?+LOO groups,the oral administration of LME markedly decreased Caspase-3 activity in brain tissues(Ps<0.01).The abovementioned results suggested that LME could attenuate A?-induced cell apoptosis.4.4 A? associated metabolisms in brain tissueWestern blotting analysis demonstrated that,compared with the NC group,the expression levels of APP and BACE1 were significantly increased in mouse brain tissues of the A? group(P<0.05,P<0.01),while the expression level of ADAM10 was markedly decreased(P<0.01).In comparison with the A? group,the expressions of APP and BACE1 were remarkably lowered in mouse brain tissues of the A?+LME group(P<0.05,P<0.01),while the expression of ADAM 10 was significantly enhanced(P<0.01).There all existed significant differences between two groups of A?+LME and A?+LOO in expression levels of APP,BACE1 and ADAM10(P<0.05,P<0.01 and P<0.01,respectively).The aforementioned results implied that LME could regulate A? associated metabolisms and reduce productions of A? in brain tissues.4.5 Neuronal synaptic plasticityThe western blotting results showed that expressions of Syn and PSD95 in mouse brains of the A?group were remarkably lowered in the A? group than those in the NC group(Ps<0.01).In comparison with both the A? and A?+LOO groups,the oral administration of LME markedly increased expressions of Syn and PSD95 in mouse brains(Ps<0.01).The abovementioned results indicated that LME could effectively attenuate A? injury towards neuronal synaptic plasticity and exert protective effects on synaptic plasticity.Conclusions1.LME with superior physicochemical properties was successfully prepared via formulation selections,and it possessed good physical stability,owned small droplet size and PDI value,together with high lycopene content.During storage at 4? and 25? for a total of 8w,LME was observed to better protect lycopene and reduce its degradation compared with LOO.2.In vivo pharmacokinetic study indicated that,in camparison with LOO,LME could enhance absorption and oral bioavailability of lycopene dramatically,as well as decreasing elimination rate and prolonging residence time of lycopene in blood circulation.3.In vivo tissue distribution study suggested that,in camparison with LOO,LME could strikingly facilitate distribution of pharmaceuticals in the brain tissue,and the DTI of brain was 3.45,implying that LME possessed good brain-targeting efficiency.4.Compared with the NC group,brain oxidative stress level of the A? group was significantly elevated,together with the enhancement of neuroinflammatory response and cell apoptosis,the increment of A? metabolic pathway,the decrement of non-A(3 metabolic pathway and the reduction of synaptic plasticity.In camparison with the A? group,brain oxidative stress level of the A?+LME group vas significantly decreased,together with the decrement of neuroinflammatory response and cell apoptosis,the decrement of A? metabolic pathway,the increment of non-AP metabolic pathway and the enhancement of synaptic plasticity,and these effects of LME were significantly more effective compared with the A?+LOO group,suggesting that LME owned good neuroprotective efficacy against AP injury.SignificanceThis study successfully developed an oral dosage form of LME,and it possessed superior physicochemical properties,enhanced absorption and oral bioavailability of lycopene dramatically and owned good brain-targeting efficiency.The animal experiments demonstrated that LME had excellent neuroprotective effects against A?injury,thus providing experimental evidence and novel strategies for pharmaceutical brain-targeting treatment of AD and other central nervous system disease,which also showed great value and importance in practical applications. |
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