| BackgroundHemangiomas,also known as infantile hemangioma,are the most common benign vascular tumors.The incidence of hemangiomas worldwide ranges from 4%to 5%,and they are increasing year by year.As the hemangioma will bring psychological harm to children and parents,and about 10%of hemangioma may occur with various complications such as airway obstruction.Therefore,most hemangiomas require aggressive treatments as soon as possible.Surgical treatment,laser treatment,cryotherapy and oral or injection drug treatment of different efficacy,side effects or complications more,some still more serious.Propranolol,a non-selective P-blocker,hav been commonly applied in hypertension and other diseases.Numerous clinical studies have confirmed that propranolol is effective and safe.HemangeolTM oral solution(propranolol hydrochloride),approved by the U.S.Food and Drug Administration(FDA),is the first-line drug in hemangiomas nowadays.However,side effects caused by propranolol still can not be ignored,including symptomatic bradycardia and so on.In addition,propranolol takes twice daily and also reduces patient compliance.Therefore,we need to decrease the side effects and doses of propranolol.Topical injection is a direct way to target the affected area and achieve the desired concentration of drug delivery,fast onset of drugs,other organs in normal distribution,less side effects.Drug delivery system is a slow delivery system to achieve the drug delivery system,can reduce the number of administration,to extend the role of drugs,blood concentration can maintain a relatively stable and lasting effective range,improve drug safety.Therefore,the slow-release drug delivery system for local injection is expected to decrese the frequency of administration and side effects of propranolol.Liposomes and polylactic acid-polyglycolic acid copolymer(PLGA)carriers are two types of typical sustained release drug delivery systems.Liposomes,cmposed of a lipid bilayer or multiple lipid bilayers,possess the advantages of good biocompatibility,easy surface modification and simple technology.However,the liposome membrane stability is poor,and the drug is prone to leakage,which is also an important factor that restricts the clinical application of liposomes.PLGA microspheres have been widely used in sustained release drug delivery,by changing the comonomer ratio,resulting in the drug release time ranging from weeks to months.Therefore,PLGA microspheres can reduce the number of drug administration,prolong drug action time and improve drug safety.The researchers found that the liposomes loaded in PLGA microspheres prepared into lipid microspheres can overcome the shortcomings of the release of liposomes,and better drug delivery and treatment effect.Herein,we envisaged that propranolol could be loaded in liposomes,and then propranolol liposomes were encapsulated in PLGA microspheres to construct a propranolol-loaded liposome PLGA microspheres(propranolol lipid microspheres),to achieve long sustained propranolol release,reduce the number of propranolol administration and side effects,and achieve safe and effective treatment of hemangiomas.Hemangioma stem cells have been shown to be the seed cells for the development of hemangiomas.Hemangioma stem cells are able to differentiate into multiple hemangiomas and form hemangiomas,making them the key cells for hemangioma formation.Targeted killer hemangioma stem cells can fundamentally inhibit the proliferation of hemangiomas.CD 133 is a 5-transmembrane glycoprotein that is a cell surface marker for a variety of stem cells.The study found that CD 133 is also a surface marker of hemangioma and carcinoma stem cells.CD133-positive hemangioma stem cells can be re-differentiated into hemangiomas in immunodeficient nude mice,and CD133-positive hemangioma stem cells that are resorted from hemangiomas formed from CD133-positive hemangioma stem cells can still be re-differentiated into hemangiomas,that is,CD 133 positive hemangioma stem cells have a continuous hemangiomas formation ability.Therefore,CD 133-positive hemangioma stem cells are the seeds of hemangiomas,and targeted killing of CD 133-positive hemangioma stem cells can fundamentally inhibit the proliferation of hemangiomas.Due to the specific expression of CD133 by hemangio-lineage stem cells,according to this feature,we can select aptamers to specifically target CD133.Aptamer is a low-molecular-weight oligonucleotide fragment that specifically targets cell-surface antigen.Because of its ease of synthesis and low immunogenicity,aptamer is widely used in molecular targeted therapy application.Therefore,CD 133 aptamers are able to specifically target CD 133-positive hemangioma stem cells.Therefore,in this study,we envisaged constructing a nanocarrier loaded with propranolol modified CD 133 aptamers(CD 133 targeting propranolol nanocarriers)to achieve specific killing of hemangioma stem cells.In addition,PLGA nanocarriers can also achieve long-term sustained release of propranolol,and reduce the number of administration of propranolol and side effects,to achieve safe and effective treatment of hemangiomas.Research methods(1)Isolation and culture of hemangioma cells:Hemangiomas excised from patients with hemangiomas were stored in cell growth medium.Thereafter,the specimens were cut into 1mm3 tissue sections and then harvested by trypsinization in endothelial cell growth medium to obtain hemangioma endothelial cells.The use of CD 133 beads sorting hemangioma endot:helial cells obtained CD 133 positive hemangioma cells.(2)Preparation and characterization of propranolol lipid microspheres and CD133-targeted propranolol nanocarrier? Preparation of propranolol lipid nicrospheres:Propranolol liposomes were developed by film hydration.Then the liposomes were extruded to be liposomes of uniform particle size was performed by gel filtration.Uncoated propranolol was removed from the liposomes.The modified liposomes wer'e then dispersed in the oil phase of polylactic acid polyglycolic acid-polyethylene glycol-polylactic acid polyglycolic acid(PLGA-PEG-PLGA)in ethyl acetate.Then by severe vortexing,the water/oil emulsion was obtained.The water/oil emulsion was instilled into the aqueous polyvinyl alcohol solution and mechanically stirred.The obtained water/oil/water emulsion is poured into a polyvinyl alcohol aqueous solution,and the organic solvent is further volatilized by mechanical stirring to form solid microspheres,and the propranolol lipid microspheres are obtained after being washed and freeze-dried.? Preparation of CD 133-targeted propranolol nanocarrier:Polylactic acid polyglycolic acid-polyethylene glycol-carboxyl(PLGA-PEG-COOH)was dissolved in methylene chloride,and propranolol was dissolved in deionized water,Thereafter,the propranolol solution was emulsified in the oil phase.Then the emulsion was prepared by the sonication by a probe.This water/oil emulsion is transferred to a polyvinyl alcohol aqueous solution.The resulting water/oil/water emulsion was gently stirred to evaporate organic phase.The nanocarrier is centrifuged and suspended in deionized water twice to purify the nanocarrier.The solution is transferred to aqueous polyvinyl alcohol solution and the mixture is sonicated.The CD 133 aptamer was then linked to the propranolol nanocarrier by the carbodiimide method to prepare CD 133 targeting propranolol nanocarrier.? Characteristics of microspheres and nanocarriers:The microspheres were tested for size distribution by Malvern Mastersizer 2000 particle size analyzer.For the size and potential of the nanocarrier,Zetasizer Nano S was performed.HPLC was used to test the encapsulation efficiency and drug loading of propranolol.? The drug release of microspheres and nanocarriers:The microspheres and nanocarriers were placed in a dialysis membrane.The molecular weight cut-off of the membrane is 1000 Daltons.After then the dialysis membrane was placed in a beaker containing different media,and the beaker was put in a water bath at 37? with gentle agitation.At different time points,a small amount of dialysate was taken out to determine the content of propranolol by high performance liquid chromatography.(3)Targeting of hemangioma stem cells by CD 133 targeting propranolol nanocarriers:Hemangioma stem cells were plated in plates,and the fluorescein loaded CD133 targeting nanocarriers and CD 133 targeting propranolol nanocarriers were treated with hemangioma stem cells for 4 hours.The phosphate buffer solution was used to wash cells for the analysis of fluorescence with flow cytometry.At the same time,the cells were lysed to be analyzed by high performance liquid chromatography.(4)In vitro cytotoxicity of propranolol lipid microspheres and CD133-targeting propranolol nanocarriers and their effect on cytokine secretion:hemangioma stem cells and endothelial cells were cultured in plates.After then,the propranolol lipid microspheres and CD 133 target propranolol nanocarrier were added to treat them.The cell proliferation kit was used to detect cell proliferation,and enzyme-linked immunosorbent assay(ELISA)was adopted to evaluate the concentration of VEGF-A and basic fibroblast growth factor.(5)The mechanism underlying the anti-hemangioma activity of propranolol lipid microspheres and CD 133 targeting propranolol nanocarriers:The hemangiomas excised from the patient were cultured for hemangioma stem cells.Hemangioma stem cells were resuspended in Matrigel and then inoculated into the flank of nude mice.After waiting for the hemangiomas to reach a volume of 25 mm3,propranolol lipid microspheres or CD 133 targeting propranolol nanocarriers were intratumourally injected to the tumors.The change of hemangioma volume at different time points was assessed to evaluate the anti-hemangioma effect in vivo.After treatment,the nude mice were killed to excise the hemangiomas to weigh their weight.The hemangiomas were evaluated by hematoxylin-eosin staining and microvessel density assessment.At the same time,the body weight of nude mice was detected for the analysis of the toxicity of the drugs in vivo.Results(1)Preparation and characterization of propranolol lipid microspheres and CD 133-targeting propranolol nanocarrier:Propranolol lipid microspheres possessed a particle size of 77.8 ?m.The polydispersity index of the microspheres was 0.26.The encapsulation efficiency(EE)of propranolol in the microspheres was 23.9%,which was a little lower than propranolol liposomes(40.8%).The drug release experiments showed that only 8%propranolol was released on day 1,35%on day 4,and 75%on day 20 of propranolol.Propranolol lipid microspheres released drug faster in serum-containing phosphate buffered saline than phosphate buffered saline,suggesting that serum may destabilize propranolol lipid microspheres to some extent.The particle size of CD133-targeted propranolol nanocarriers was 143.7 nm,and the polydispersity coefficient was 0.16.The particle size distribution of C3133-targeted propranolol nanocarriers was uniform and the zeta potential was lower than-20 mv.Transmission electron microscopy showed that the particle size of CD133-targeted propranolol nanocarrier was uniform and relatively smooth.The encapsulation efficiency of CD 133-targeting propranolol nanocarrier was 51.8%,and the drug loading was 6.2%.Drug release experiments showed that CD133-targeted propranolol nanocarriers were able to rapidly release propranolol(40%in phosphate buffer,55%in serum-containing phosphate buffer)within the first 48 hours.Over the next 144 hours,the cumulative release of propranolol from the nanocarrier reached about 70%in phosphate buffered saline and about 85%in serum-containing phosphate buffered saline.(2)The targeting of CD133-targeted propranolol nanocarriers to hemangioma stem cells:The average fluorescence intensity of CD133-targeting nanocarrier containing fluorescein was significantly higher than the nanocarrier containing fluorescein.However,after the pretreatment of the cells with CD133 aptamers,the mean fluorescence intensity of the fluorescein-loaded CD 133-targeting nanocarriers was significantly reduced,indicating that the binding of the fluorescein-loaded CD 133-targeting nanocarriers and hemangioma stem cells was attributable to the CD133 aptamer.In addition,the endocytosis rate of CD133-targeting propranolol nanocarriers in hemangioma stem cells was 67%,significantly higher than propranolol(8%)and propranolol nanocarrier(26%).After the pre-treatment of CD 133 aptamers with hemangioma stem cells,the propranolol endocytotic rate of CD 133 targeted propranolol nanocarriers was significantly reduced,indicating that the targeting of CD133-targeted propranolol nanocarriers and hemangioma stem cells was attributed to the CD 133 aptamers.(3)The effect of propranolol lipid microspheres and CD133-targeted propranolol nanocarriers on the in vitro cytotoxicity and cytokine secretion:The 72-hour IC50 values of propranolol,propranolol liposomes,and propranolol lipid microspheres were 110.5,120.8 and 379.3 ?M,respectively,indicating that propranolol liposomes showed similar cytotoxicity to propranolol.Propranolol lipid microspheres were 3.4-fold less effective compared with propranolol.Furthermore,the 120-hour IC50 values of propranolol,propranolol liposomes and propranolol lipid microspheres were 40.8,56.5 and 120.7 ?M,respectively,indicating that propranolol liposomes and propranolol possess similar cytotoxicity.Propranolol lipid microspheres were 2.95-fold less effective than propranolol.Compared with propranolol lipid microspheres,both propranolol and propranolol liposomes inhibited VEGF-A mRNA expression more effectively,in the range of 30-500 ?M of propranolol.For example,propranolol and propranolol liposomes suppressed the VEGF-A mRNA expression by 80%at 500 ?M,whereas the same concentration of propranolol lipid microspheres only inhibited 40%.Likewise,propranolol and propranolol liposomes were more potent than propranolol lipid microspheres in inhibiting VEGF-A protein expression at propranolol concentrations in the range of 30-500 ?M.Similarly,propranolol and propranolol liposomes suppressed bFGF expression more effectively than propranolol lipid microspheres.The 72 hour IC50 values for propranolol,propranolol nanocarriers and CD133-targeted propranolol nanocarriers in hemangioma stem cells were 83.2,97.6 and 35.8 ?M,respectively,suggesting that propranolol and propranolol nanocarriers had similar cytotoxic effects,but the toxicity of CD 133 targeting propranolol nanocarriers was 2.7 times stronger than that of propranolol.The IC50 values for propranolol,propranolol nanocarriers and CD133-targeted propranolol nanocarriers for 120 hours were 63.5,78.7 and 16.9?M,respectively,indicating that propranolol and propranolol nanocarriers have similar cytotoxicity,however,the toxicity of CD 133 targeting propranolol nanocarrier was 3.8 times stronger than that of propranolol.Propranolol,propranolol nanocarrier and CD133-targeted propranolol nanocarrier all could dose-dependently inhibit VEGF-A and bFGF mRNA and protein in hemangioma stem cells.CD 133 targeting propranolol nanocarriers were more effective than propranolol in inhibiting VEGF-A and bFGF mRNA and protein.For example,propranolol and propranolol nanocarriers suppressed VEGF-A mRNA expression by about 60%at 500 ?M,whereas the inhibitory rate of CD133-targetedpropranolol nanocarriers was 80%.Propranollol and propranolol nanocarriers showed 65%inhibition of VEGF-A protein expression at 500 ?M,whereas the inhibition rate of CD133-targeting propranolol nanocarriers was 90%.(4)In vivo anti-hemangioma effect of propranolol lipid microspheres and CD 133 targeting propranolol nanocarriers:We evaluated the antitumor effect of propranolol,propranolol liposomes and propranolol lipid microspheres on hemangiomas in mice in vivo.Propranolol lipid microsphere treatment induced a 84%reduction in hemangioma volume on day 35.However,propranolol liposomes and propranolol induced in a 44%and 13%reduction.The hemangioma volume of propranolol lipid microsphere treatment group was the smallest among all the groups(saline group:215 mm3,propranolol group:187 mm3,propranolol liposomes group:121 mm3,propranolol lipid nicrosphere group:35 mm3),The mean hemangioma weight of the propranolol lipid microsphere-treated group was the smallest(saline group:0.28 g,propranolol group:0.21 g,propranolol liposomes group:0.13 g,propranolol lipid microspheres:0.03 g).As a result,all treatments had good tolerability in hemangioma bearingmice.All treated mice did not show any significant behavioral changes and no significant changes in body weight,indicating that propranolol,propranolol liposomes and propranolol lipid microspheres did not severe side affects in mice.In the end,H&E staining was adopted to stain hemangiomas,and microvessel density analysis of tissue sections was carried out.The mean microvessel density of propranolol lipid microspheres treatment group was the lowest.We can safely conclude that propranolol lipid microspheres could significantly suppress the angiogenesis of hemangiomas.The therapeutic efficacy of propranolol,propranolol nanocarriers,and CD133 targeting propranolol nanocarriers was evaluted in the hemangiomas in mice.At the end of treatment,CD133 targeting propranolol nanocarriers induced a 76%reduction in the volume of hemangiomas,using the saline-treated group as a blank control,whereas propranolol nanocarriers and propranolol alone resulted in only a reduction of hemangiomas 57%and 34%by volume.The hemangioma volume of CD133 targeting propranolol nanocarrier-treated group was the smallest(saline group:256 mm3,propranolol group:169 mm3,propranolol nanocarrier group:110 mm3,CD133 targeting propranolol nanocarrier group:62 mm3).Moreover,the hemangiomas treated with CD 133 propranolol nanocarriers were significantly lower than the other groups(0.26 g in the saline group,0.18 g in the propranolol group,0.11 g in the propranolol nanocarrier group,0.05 g in CD133 targeting propranolol nanocarrier group).The results also indicated that propranolol nanocarriers and CD 133 targeting propranolol nanocarriers did not cause significant behavioral changes in mice and had no adverse effects on body weight in mice.In the end,propranolol significantly inhibited hemangioma angiogenesis in mice compared to saline injections.The density of CD133 targeting propranolol nanocarriers-treated group was the lowest(saline group:68 vessels/mm,propranolol group:40 vessels/mm,propranolol nanocarrier group:27 vessels/mm 2,CD133 targeting propranolol nanocarrier group:13 vessels/mm2),indicating that CD133 targeting propranolol nanocarriers were the most effective at inhibiting vascularization of hemangiomas.ConclusionPropranolol lipid microspheres achieved long-term sustained release of propranolol,reduced the frequency and side effects of propranolol administration,and achieved safe and effective treatment of hemangiomas.CD133 targeting propranolol nanocarrier achieved specific killing of hemangiomas stem cell,but achieved long-term sustained release of propranolol,reducing the number of propranolol administration and side effects and achieving the safety of hemangiomas treatment.This study,for the first time,used liposomal microspheres and nanocarriers to realize the controlled release of targeting hemangioma stem cells,providing new insights into the treatment of hemangiomas.The research can provide two kinds of new anti-hemangioma drug delivery carriers with high efficiency and low toxicity. |
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