Preparation Of Fucose Coupled Berberine Hydrochloride Liposomes And Its Inflammatory Targeting Effect

Berberine hydrochloride has good anti-inflammatory activity,but its oral absorption is poor.Injection can cause serious side effects.Nanodelivery systems such as liposomes can target drug delivery.If appropriate targeting ligands are selected to couple with berberine hydrochloride liposomes,targeted delivery can be achieved,reducing toxicity,and improving anti-inflammatory effects.Due to the high expression of P-selectin in vascular endothelial cells at the inflammatory site,low molecular weight fucoidan(LMWF)has a good binding ability to P-selectin.Berberine hydrochloride is encapsulated in the liposome and then modified with LMWF,enabling the liposome to specifically bind to activated vascular endothelial cells expressing P-selectin,thereby achieving targeted delivery of berberine hydrochloride to inflammation.Considering the specific binding of P-selectin to fucoidan,this study intends to prepare LMWF-coupled berberine hydrochloride liposomes(LMWF-Lip/BBR)and investigate their uptake,uptake patterns,systemic distribution,and anti-inflammatory effects,in order to evaluate the anti-inflammatory targeting delivery effect of berberine hydrochloride.The main contents of this study are as follows.1.Preparation of low molecular weight fucoidan coupled berberine hydrochloride liposomesThe preparation of LMWF-Lip/BBR mainly involves establishing a method for determining the content of BBR,establishing a method for determining the entrapment efficiency of berberine hydrochloride liposomes(Lip/BBR),optimizing the formulation of Lip/BBR,and characterizing the liposomes.The results show that:(1)The content of BBR detected by HPLC is within the concentration range of 0.25-50 μg/m L,exhibiting a good linear relationship with a regression curve equation of Y=33.34X-7.939 and R~2 = 0.9994.The stability meets the standard.(2)The Sephadex gel column method was used to separate Lip/BBR from the unencapsulated BBR.The peak interval was 3 minutes,and the recovery rate met the standard.This method was suitable for the separation of Lip/BBR,enabling the calculation of the encapsulation rate.(3)The optimal formulation and process of LMWFLip/BBR are as follows: a phospholipid concentration of 10 mg/m L,a phospholipid cholesterol ratio of 1:1,a phospholipid drug ratio of 35:1,and hydration at p H 6.0.The preparation process involves dissolving the membrane material in 10 m L of chloroform,rotating and evaporating at 40°C to remove the solvent,adding 10 m L of PBS solution with p H 6.0 for hydration for 30 minutes.Then,the washed membrane material is placed in an ice water bath and subjected to ultrasound conditions of 400 W for 2 seconds with intervals of 2 seconds,and ultrasound is applied for 10 minutes.The resulting liposome is filtered using a 0.22 μm microporous filter membrane,followed by extrusion through a 100 nm polycarbonate membrane using a liposome extruder.The aqueous phase p H of the liposome is adjusted to 7 with Na OH,LMWF is added,and the reaction is conducted overnight at room temperature to obtain a yellow translucent LMWF-Lip/BBR.(4)The particle size of the LMWF-Lip/BBR prepared with the optimal formulation was 104.13±1.18 nm,the Zeta potential was-12.2 ± 0.46 m V,the encapsulation efficiency was 79.73 ± 1.06%,and the drug loading was 0.45 ± 0.01%,meeting the requirements of nanopreparations.2.Study on the targeting effect of low molecular weight fucoidan coupled berberine hydrochloride liposomes on human umbilical vein endothelial cells in vitroLPS was utilized to activate human umbilical vein endothelial cells(HUVECs)for evaluating the cytotoxicity,uptake,mode of uptake,and anti-inflammatory effects of LMWF-Lip/BBR.The results are as follows:(1)LMWF-Lip/BBR at concentrations ≤ 80μg/m L did not significantly affect the activity of activated HUVECs(P>0.05).(2)The uptake of LMWF-Lip/BBR by activated HUVECs was 1.5 times higher compared to unmodified Lip/BBR.(3)The uptake of LMWF-Lip/BBR by activated HUVECs is predominantly mediated by caveolin-and reticulin-mediated endocytosis,which is an energy-dependent process.(4)Calculated based on the content of BBR.At a concentration of 10 μg/m L,LMWF-Lip/BBR significantly inhibited the production of IL-1β and IL-6 by activated HUVECs(P<0.01).The above findings indicate that LMWF-Lip/BBR at concentrations below 80 μg/m L does not exhibit significant cytotoxicity toward activated vascular endothelial cells.Moreover,LMWF-Lip/BBR at a concentration of 10 μg/m L demonstrates remarkable anti-inflammatory capabilities,surpassing the effects of Free BBR or Lip/BBR at the same concentration.LMWF-Lip/BBR can bind to P-selectin and be actively internalized by activated HUVECs,exhibiting potential for inflammation targeting.3.Study on the anti-inflammatory targeting effect of low molecular weight fucoidan coupled berberine hydrochloride liposomes in vivoThe distribution of LMWF-Lip/BBR in inflammatory sites and various organs,its in vivo anti-inflammatory effect,and the effects of histopathological changes in a rat model of foot swelling were investigated.The results are as follows.(1)The concentration of LMWFLip/BBR in the swollen right foot was higher compared to unmodified berberine hydrochloride liposomes,and it was also higher than its concentration in the uninflamed left foot.However,there was no significant difference in the distribution of LMWF-Lip/BBR and unmodified liposomes in various organs.(2)LMWF-Lip/BBR significantly alleviated the elevation of white blood cell count and monocyte count in the plasma of rat foot swelling models(P<0.01).It also significantly reduced the increased levels of IL-1β and IL-6 and prevented the increase in toe weight.(3)After 7 days of intravenous administration of 3mg/kg of free berberine hydrochloride or Lip/BBR,a small amount of inflammatory cell infiltration was observed in the hearts of rats,localized necrosis occurred in the liver,and liver cells exhibited steatosis.However,there were no significant pathological changes in the organs of rats injected with the same concentration of LMWF-Lip/BBR.The above results indicate that compared to unmodified liposomes or free berberine hydrochloride,LMWF-Lip/BBR can enhance the anti-inflammatory effect and exhibit targeted effects in rat models of foot swelling inflammation.In summary,this study successfully prepared the LMWF-Lip/BBR.The optimized formulation and preparation conditions were as follows: phospholipid concentration of 10mg/m L,phospholipid cholesterol ratio of 1:1,phospholipid drug ratio of 35:1,and hydration p H of 6.0.The preparation process involved dissolving the membrane material in chloroform,evaporating the solvent,hydrating with PBS solution,and subjecting the liposome to ultrasound and filtration.The resulting liposome was further adjusted to p H 7 and reacted with LMWF overnight to obtain a yellow translucent LMWF-Lip/BBR.The encapsulation efficiency was found to be 79.73 ± 1.06%,and the drug loading was 0.45 ± 0.01%,which met the requirements of nano preparations.In vitro experiments demonstrated that LMWFLip/BBR exhibited specific binding to P-selectin on vascular endothelial cells,leading to increased uptake and protection against lysosomal degradation.In vivo,LMWF-Lip/BBR showed enhanced drug concentration at inflammatory sites while reducing concentration in non-inflammatory organs,achieving targeted delivery to inflamed areas through the interaction with vascular endothelial cells.This ultimately improved the anti-inflammatory effect of berberine hydrochloride and exerted its inflammatory targeting effect.