Construction And Evaluation Of Functionalized Calcium Phosphate Nanoparticles Through Caveolae-mediated Endocytosis

ObjectiveCalcium phosphate?CaP?nanoparticles had low transfection efficiency,poor stability and weak capability to achieve transfection in vivo.To solve these problems,two series of functionalized organic-inorganic hybrid CaP nanoparticles via caveolae-mediated uptake pathway were designed.The first kind of nanoparticles employed calcium phosphate nanoparticles as biomaterials to condense DNA and then form the biocorona with bovine serum albumin?BSA?to obtain the BSA-CaP nanoparticles,which could activate the phosphorylation of caveolin-1 to promote the budding and release of caveolae.We hypothesized that those BSA-CaP nanoparticles can retain the inherent endocytosis profiles of BSA and be internalized via caveolae-mediated endocytosis.The second kind of functionalized nanoparticles were constructed by introducing biomimetic moiety of mannitol onto the surface of CaP nanoparticles.Through coordination interaction between the phosphonate groups of the MA-AL and the Ca²⁺of CaP,different degrees of MA-AL were anchored on CaP to self-assembly form the organic-inorganic hybrid CaP-MA nanoparticles.The constructed hybrid nanoparticles with mannitol groups may simulate caveolae-mediated endocytosis,the non-destructive delivery pathway,to reduce the gene degradation in endosomes or lysosomes occurred with clathrin-mediated endocytosis.In addition,we hoped that the introduction of MA-AL could improve the stability and finally obtained the gene delivery system with high transfection efficiency,good stability,which can be further used for gene transfection in vivo.Methods1.In this study,two methods with four different BSA/CaP weight ratios were used to prepare BSA-CaP nanoparticles through the electrostatic interaction between BSA and Ca²⁺,called BSA-CaP-5/15/30/50-M1 and BSA-CaP-5/15/30/50-M2,respectively.2.Through the coordination interaction between the phosphonate groups of mannitol-alendronate?MA-AL?and the Ca²⁺of calcium phosphate,MA-AL was modified onto the surface of the CaP with different degrees,named CaP-MA-5,CaP-MA-20 and CaP-MA-40 nanoparticles,respectively.3.The zeta potential and stability of two series of nanoparticles were evaluated by Delsa?Nano C Particle Analyzer.The DNA protection experiment was carried out to assess the ability of nanoparticles to protect nucleic acid in DNase I condition.Hemolytic experiments and MTT assay were utilized to estimate the biocompatibility of two series of nanoparticles.The gene encoding green fluorescent protein?EGFP?was used as the reporter gene,and the transfection activities of two kinds of nanoparticles were evaluated by flow cytometry and fluorescence microscopy.The specific endocytic inhibitors were employed to investigate the endocytic pathway of two series of nanoparticles.Western blot was performed to evaluate the effect of the nanocarriers on the phosphorylation of caveolin-1.The cellular uptake of the second kind of nanoparticles was studied using fluorescently labeled DNA and the molecular probes were utilized to explore the intracellular trafficking of the second kind of nanoparticles.The green fluorescent protein-encoding gene was used as reporter gene to evaluate the transfection activity of two serious of nanovectors in vivo.ResultsPart one:BSA-CaP nanoparticles1.With four varied weight ratios of BSA and CaP,BSA-CaP-5/15/30/50-M1 and BSA-CaP-5/15/30/50-M2 nanoparticles were prepared through two methods.The particle size of BSA-CaP-M1 nanoparticles was around 181-411 nm and that of BSA-CaP-M2 was about 113-359 nm.With the increase of weight ratio,the particle size declined.The BSA-CaP nanoparticles displayed a homogeneous size distribution and possessed negative zeta potential.The DNase I protection assay showed that all the BSA-CaP nanoparticles prepared by both methods could resist gene degradation upon exposure to DNase I.In addition,the BSA-CaP nanoparticles had better stability comparing with traditional CaP nanoparticles.2.The results of MTT assay showed that the cytotoxicity of BSA-CaP nanoparticles on HEK293T cells was still negligible even at high concentration,and the cell survival rate was significantly improved comparing with the traditional CaP nanoparticles.Hemolysis results revealed that the hemolysis rates of BSA-CaP nanoparticles were less than 5%,which met the safety requirements of biomaterials.3.Transfection experiments in vitro showed that the transfection efficiency of BSA-CaP nanoparticles was significantly increased comparing with CaP nanoparticles.Among them,BSA-CaP-50-M2 nanoparticles had highest percentage of positive transfected cells and mean fluorescence intensity.The fluorescence microscopy obtained the results similar to those of flow cytometry.4.Transfection experiments were carried out in the presence of specific cellular uptake inhibitors and showed that transfection efficiency of CaP nanoparticles and BSA-CaP-50-M2 nanoparticles were not affected in the presence of amiloride?inhibitor of macropinocytosis?.When cells were pre-incubated with chlorpromazine?inhibitor of clathrin-mediated endocytosis?,the transfection efficiency of CaP nanoparticle was significantly decreased,whereas that of BSA-CaP-50-M2 nanoparticles only slightly declined at high concentrations.In the presence of genistein?inhibitor of caveolae-mediated endocytosis?,the transfection efficiency of CaP nanoparticles decreased and the transfection efficiency of BSA-CaP-50-M2 nanoparticles plunged significantly.These results demonstrated that CaP nanoparticles were internalized through the cathrin-mediated and caveolae-mediated endocytosis,but mostly relied on cathrin-mediated pathway.While the constructed BSA-CaP-50-M2 nanoparticles were mainly internalized via the caveolae-mediated endocytosis,which could avoid the acidic and enzymatic environment following clathrin-mediated pathway that largely contributed to the low transfection.5.Western-blot analysis manifested that BSA did have the ability to up-regulate the expression of phosphorylated caveolin-1?P-CAV1?.When treated with the BSA-CaP-50-M2 nanoparticles,biocorona-coated with BSA,cells expressed a much higher level of P-CAV1 than control and CaP nanoparticles group.Moreover,when genistein,the inhibitor of Src tyrosine kinase,was added,the expression of P-CAV1 was significantly down-regulated.According to above results,BSA-CaP-50-M2 nanoparticles,with the BSA corona,could retain the inherent uptake profiles of BSA and stimulate the phosphorylation of caveolin-1,thus activating the caveolae-mediated endocytosis and change the cellular uptake pathway of CaP nanoparticles.6.In vivo transfection experiments revealed that the DNA could be protected and delivered successfully in vivo by BSA-CaP-50-M2 nanoparticles.After a single dose injection,the fluorescence of EGFP was captured one hour after injection and the effects lasted at least 72 hours,achieving a long-term transgene expression.Given the transfection activity both in vitro,ex vivo and in vivo,BSA-CaP-50-M2 nanoparticles were considered to be a promising delivery nanovector for gene therapy,Part two:CaP-MA nanoparticles1.The structure of MA-AL was confirmed by ¹H NMR and IR spectra,which was consistent with that of target product.2.A facile co-precipitation method was employed to prepare CaP-MA-5/20/40nanoparticles with different degrees of MA-AL.Characterization of nanoparticles showed that the stability of CaP-MA-5/20/40 nanoparticles was significantly improved comparing with the traditional CaP nanoparticles,and the particle size of CaP-MA-20/40nanoparticles did not change obviously within 5 days.Results of scanning electron microscopy demonstrated that the nanoparticles were spherical with uniform size.The results of DNA protection experiment indicated that all the three functionalized CaP-MA nanoparticles showed the excellent capability of DNA protection as CaP nanoparticles.3.The results of MTT assay revealed that the biocompatibilities of CaP-MA groups were enhanced to a large extent comparing with the unmodified CaP nanoparticles.The cytotoxicity of MA-AL was negligible even in the case of the concentration of 800?g/mL.The hemolytic effect of CaP-MA nanoparticles was also determined and the hemolytic rates of all modified nanoparticles were less than 5%,which met the safety requirements of biomaterials.4.The results of in vitro transfection experiments indicated that all the nanoparticles modified with MA-AL exhibited distinguished transgene expression and their transfection efficiency were superior to CaP nanoparticles.Among them CaP-MA-40 nanoparticles displayed highest percentage of positive transfected cells and mean fluorescence intensity.Fluorescence microscopy showed the similar results.5.Transfection experiments were carried out in the presence of specific cellular uptake inhibitors and showed that when cells were treated with amiloride,the inhibitor of macropinocytic pathway,transfection efficiency of CaP-MA-40 and CaP were unaffected.When cells were pre-incubated with chlorpromazine?inhibitor of clathrin-mediated endocytosis?,the transfection efficiency of CaP nanoparticles was significantly decreased,whereas the decrease of CaP-MA-40 nanoparticles was much smaller than that of CaP nanoparticles.In the presence of genistein?inhibitor of caveolae-mediated endocytosis?,the decrease of transfection efficiency in CaP-MA-40 nanoparticles was significantly higher than that of CaP group.These results preliminarily revealed that CaP-MA-40nanoparticles had enhanced sensitivity to the inhibitor of genistein associated with caveolae-mediated endocytosis.6.Results of cellular uptake evaluation in the presence of specific cellular uptake inhibitors showed that the addition of amiloride did not impact the cellular uptake both of unmodified CaP nanoparticles and CaP-MA-40 nanoparticles.When unmodified CaP nanoparticles were treated with chlorpromazine or genistein,the uptake was inhibited57.6%or 28.3%,respectively.As for CaP-MA-40 nanoparticles,the uptake reduced 11.5%or 63.8%,respectively.The cellular uptake images observed by laser scanning confocal microscopy were consistent with the results analyzed by FACS.7.Intracellular trafficking showed that when cells were incubated with unmodified CaP nanoparticles and then marked by LysoTracker^?Deep Red,the merged image exhibited extremely high level overlap of subcellular colocalization in lysosome.On the contrary,much lower degree of overlap was observed in the colocalization image with caveosome.However,when cells were treated with CaP-MA-40 nanoparticles,the degree of overlap in lysosome obviously reduced comparing with CaP,and fluorescein-labeled nanoparticles were mainly observed in caveosome.These results indicated that the unmodified CaP nanoparticles were mainly internalized through clathrin-mediated pathway and transferred into lysosome.It was convinced that the introduction of MA-AL stimulated and increased the cellular uptake of CaP-MA-40 nanoparticles through caveolae-mediated pathway avoiding the subsequent gene degradation in lysosome,which was of great importance in enhancing transgene performance.8.The results of Western blot demonstrated that when cells were treated with mannitol and MA-AL,quantitative analysis exhibited accelerated expression of P-CAV1,which testified that the mannitol and MA-AL truly activated the phosphorylation of CAV1,and stimulated the expression of P-CAV1.On the other hand,as for unmodified CaP nanoparticles,P-CAV1 did not change too much compared with the control.When treated with mannitol-modified CaP-MA-40 nanoparticles,P-CAV1 was up-regulated significantly during the cellular uptake of CaP-MA-40 nanoparticles.Furthermore,when cells were pre-incubated with genistein the expression of P-CAV1 plugged into a low level,which signified that GE could inhibit the phosphorylation of caveolin-1 activated by mannitol-modified CaP-MA-40 nanoparticles.Combined the previous results,we were able to address that mannitol moiety on the surface of CaP-MA-40 nanoparticles could induce the phosphorylation of caveolin-1 and activate the caveolae-mediated endocytosis,thus altering the cellular uptake pathway of traditional CaP nanoparticles.9.In vivo transfection experiments showed that CaP-MA-40 nanoparticles exhibited the highest fluorescence imaging among four types of nanoparticles,which was much better than CaP nanoparticles.The fluorescence intensity of CaP-MA groups reached highest value 6 hours after post-administration,and the GFP fluorescence could sustain at least 72 hours in vivo after only single administration,achieving a long-term transgene expression,which could be a promising candidate as delivery vector both in vitro and in vivo for further application in the clinics.ConclusionsWe designed and constructed two series of functionalized gene delivery systems based on calcium phosphate nanoparticles.All of these nanocarriers can promote the caveolae-mediated endocytosis and alter the cellular uptake pathway of traditional calcium phosphate nanoparticles through stimulating the expression of P-CAV1.The constructed delivery systems exhibited efficient transfection performance which offers new opportunities to develop the novel and efficient delivery nanovectors for the gene therapy of inherited or acquired diseases and military-related diseases.