Preparation,Characterization And Evaluation Of Two Kinds Of Carbon-based Nanoparticles For Anti-tumor Drug Delivery

Premature drug release from nano-sized drug delivery systems,such as mesoporous silica nanoparticles?MSN?,nano-gels and polymeric micelles are great concerns for cancer therapy,which might cause reduced therapeutic effects and increased side effects or toxicity.To circumvent this issue,it is necessary to develop novel drug nano-carriers or drug delivery systems which can prevent drug release before drugs reach pharmacological sites and trigger drug release at the sites.Herein,I study two kinds of carbon-based nanoparticles for anti-tumor drug delivery:DOX-PCM@MCN-SLPD and Ce6-PCM@MCN-RBCM.DOX-PCM@MCN-SLPD is the mesoporous carbon nanoparticle?MCN?loaded with chemotherapeutic agent doxorubicin?DOX?and a phase change material?PCM?1-tetradecanol followed by surface modifying with a bio-surfactant sophorolipid?SLPD?for cancer chemotherapy.Ce6-PCM@MCN-RBCM is the mesoporous carbon nanoparticle loaded with a photosensitizer chlorin e6?Ce6?and PCM and coated with the red blood cell membrane?RBCM?for cancer photodynamic therapy.MCN is a kind of photo-thermal mesoporous nanoparticle with good compatibility and a great specific surface area,which can load a great amount of drug,take photo-thermal effects and generate heat under NIR irradiation,being a promising drug nano-carrier.DOX is the first-line chemotherapeutic agent clinically and has a potent antitumor effect.1-tetradecanol is a kind of temperature sensitive phase-change material which is in a solid state at or below the human physiological temperature?37°C?and becomes liquid at a higher temperature?higher than its melting point,38?³9°C?.Once 1-tetradecanol and DOX or Ce6 are co-loaded inside MCN,DOX or Ce6 is trapped in solid PCM matrix,which cannot release below 38 ^oC³9°C and can be triggered to release at higher temperatures due to the occurrence of solid-liquid phase change of 1-tetradecanol at or above its melting point.SLPD is a biosurfactant which has an excellent biocompatibility.The MCN loaded with DOX and PCM is surface modified with SLPD to provide a hydrophilic surface,which can effectively improve the stability and dispersibility of the MCN in aqueous media,prevent drug release to some extent.So,DOX-PCM@MCN-SLPD can keep stability and prevent drug release in aqueous media?such as blood circulation?and be triggered to release drug under near-infrared?NIR?irradiation due to the occurrence of solid-liquid change of 1-tetradecanol when the temperature rises to its melting point.Ce6-PCM@MCN-RBCM is an RBCM coated nanoparticle loaded with photodynamic agent Ce6,which has an excellent biocompatibility and a long elimination half-life time.MCN was synthesized using low-concentration hydrothermal route,and unfortunately,the obtained carbonized carbon spheres contained many impurities and showed poor solubility in any kind of solvent.To make a stable MCN aqueous dispersion,the carbonized MCN was further treated with an acid solution?98%H₂SO₄ and 68%HNO₃,3:1?,and carboxyl groups were introduced.The introduction of carboxyl groups on the surface of MCN causes better dispersibility of carboxylated MCN in aqueous solution.The introduction of carboxyl group to the surface of MCN was verified after acidification.The change in the zeta potential of the MCN from-11.1±0.2 mV to-32±1.1 mV showed successful acidification.There are lots of pores in the carboxylated MCN with an average pore diameter of 2.5 nm and a pore volume of 0.34 cm³/g.The DOX-PCM@MCN is prepared by filling the pores of the carboxylated MCN with DOX and then covering with PCM.The obtained DOX-PCM@MCN is characterized by zeta potential and drug loading measurements,which has a zeta potential of 8.9±0.2 mV and a DOX loading of 24%.Moreover,there is the absence of fluorescence characteristic peak of DOX for the DOX-PCM@MCN,which indicates that the loaded DOX is trapped inside of the MCN,because of the shielding effect of MCN.The DOX-PCM@MCN shows poor dispersibility in aqueous media.In order to address it,The DOX-PCM@MCN is further coated with SLPD to obtain the DOX-PCM@MCN-SLPD.The DOX-PCM@MCN-SLPD has an excellent water dispersibility with a zeta potential of-17.4±0.2mV.It is also worth mentioning that MCN and DOX-PCM@MCN-SLPD exhibit almost the same rise in temperature after NIR laser irradiation at an intensity of7.5 W/cm² for 5 min.This shows that the DOX loading,PCM gating and further surface coating of SLPD do not affect the photo-thermal property of MCN.There is few amount?less than 5%?of DOX released from DOX-PCM@MCN-SLPD at normal body temperature?37 ^oC?.On sharp contrast,once the temperature is increased beyond the melting point of the PCM,most of the DOX?about 98%?is released from the DOX-PCM@MCN-SLPD at 42 ^oC in 60 min.DOX-PCM@MCN-SLPD releases 80%of the DOX under NIR irradiation?laser at808 nm?at 7.5 w/cm² for 50 min.The temperature rises to 43 ^oC in the aqueous system containing DOX-PCM@MCN-SLPD under the NIR irradiation,which triggers drug release due to the phase change from solid to liquid of the PCM caused by the photothermal effects of the nanoparticles.Furthermore,the cellular uptake of free DOX or DOX-PCM@MCN-SLPD by MCF-7/ADR is evaluated both qualitatively and quantitatively.The DOX uptaken by MCF-7/ADR is investigated by observing DOX fluorescence with confocal laser electronic microscope?CLSM?.The fluorescence intensity within cells is very weak for free DOX and very strong for DOX-PCM@MCN-SLPD,which imply that there is very few DOX in MCF-7/ADR for free DOX and there is a great amount of DOX in cells for DOX-PCM@MCN-SLPD.These results show that a very minute amount of DOX is up taken by cells for free DOX,on contrary,a great amount of DOX can be up taken by cells.MCF-7/ADR cells are multidrug-resistant human breast cancer cells which overexpress many drug efflux pumps,such as P-gp and thus prevent the uptake of DOX.DOX-PCM@MCN-SLPD is up taken by cells via endocytosis and the DOX is trapped inside the nanoparticles up taken within cells,which bypass the P-gp's efflux of DOX.The CLSM observations show that the fluorescence intensity of DOX is very weak within the cells with DOX-PCM@MCN-SLPD at 37 ^oC,however,the cells present very strong fluorescence at 42 ^oC or under NIR irradiation.These results demonstrate that the application of NIR laser irradiation can facilitate or even trigger DOX release from the nanoparticles within MCF-7/ADR cancer cells.The cytotoxicity of DOX-PCM@MCN-SLPD against MCF-7/ADR is evaluated.The cytotoxicity of DOX-PCM@MCN-SLPD is related to temperature and NIR irradiation.In comparison with the cytotoxicity at normal body temperature,for the same concentrations of DOX-PCM@MCN-SLPD,the cytotoxicity of the nanoparticles is much higher at 42 ^oC or under NIR laser irradiation.This is because that much more of the DOX is released from DOX-PCM@MCN-SLPD at 42 ^oC or under NIR laser irradiation.Similarly,the as-synthesized carboxylated MCN is loaded with Ce6 and PCM and further coated with RBCM to obtain Ce6-PCM@MCN-RBCM for combined photo-thermal and photodynamic therapy.The Ce6-PCM@MCN is prepared by filling the pores of the carboxylated MCN with Ce6 and then covering with PCM.The obtained Ce6-PCM@MCN is characterized by zeta potential and Ce6 loading measurements,which has a zeta potential of-21.3±0.1 mV and a Ce6 loading of 21%.The stability and biocompatibility of prepared Ce6-PCM@MCN is enhanced by coating its surface with red blood cell membranes?RBCM?by extrusion method,and Ce6-PCM@MCN-RBCM is obtained.Moreover,the confirmation of RBCM coating on the surface of Ce6-PCM@MCN is evaluated with SDS-PAGE protein analysis of Ce6-PCM@MCN-RBCM and natural RBCM in comparison.The results show that,majority of endogenous membrane proteins are well-maintain on Ce6-PCM@MCN-RBCM.The change in the zeta potential from-21.3±0.1 mV to-28.8±0.7 mV also reveal successful coating of the RBCM.The PCM act as a gatekeeper and prevent the leakage of Ce6 at normal body temperature.Upon exposure to NIR laser irradiation,the Ce6-PCM@MCN-RBCM releases Ce6 once the temperature rise above melting point of the PCM.The temperature of Ce6-PCM@MCN-RBCM and MCN is evaluated under NIR laser irradiation at an intensity of 7.5 W/cm² for 5 min.The results show that the PCM and RBCM coating did not affect the photo-thermal property of MCN.Moreover,the Ce6 release is evaluated at different temperatures?37 ^oC,38 ^oC,39 ^oC,40 ^oC,41 ^oC and 42 ^oC?.At 37 ^oC,the release amount of Ce6 is extremely less?almost 1%?,which shows successful entrapment of Ce6 inside the pores by solid the PCM.As the temperature is increased,we notice a gradual increase in the release of Ce6 from Ce6-PCM@MCN-RBCM.The maximum amount of Ce6is released at 40 ^oC,41 ^oC and 42 ^oC?84%,92%and 97%respectively.?This shows that,the solid-liquid conversion of PCM allow the drug to release from Ce6-PCM@MCN-RBCM.To conclude,both NPs?DOX-PCM@MCN-SLPD and Ce6-PCM@MCN-RBCM?successfully block premature drug/PS release at normal body temperature and delivered the payload in a high amount into cancer cells?MCF-7/ADR and MCF-7?as compare to free DOX and Ce6.Moreover,both MCN-based NPs containing DOX and Ce6 demonstrate the synergistic effect of combined chemo-photothermal therapy and photothermal-photodynamic respectively.We believe that,MCN-based NPs have great potential for anti-tumor therapy in breast cancer.