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Objective:To investigate the therapeutic efficacy of the combination therapy with CD47-based nanoparticles and anti-PD-L1 monoclonal antibody (αPD-L1) for preventing tumor recurrence and metastasis in vivo.Methods:BALB/c mice were used to construct 4T1 tumor-bearing mouse models. The mouse model was treated with the combination therapy to analyze the effects on local tumor recurrence, tumor growth volume, survival time and lung metastasis in the 4T1 mammary tumor-bearing mouse model.Results:The combination therapy could effectively inhibit local tumor recurrence and prolong the survival time of tumor-bearing mice ( P<0.001). Compared with the αPD-L1 group, the combination therapy can increase the expression of cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in mouse serum (all P<0.05) and effector memory T cells in mouse spleen ( P<0.001). In addition, the results on the 4T1-Luc mammary tumor-bearing mouse lung metastasis model showed that the combination therapy could effectively inhibit tumor lung metastasis. Conclusions:The results strongly suggested that combination therapy with CD47-based nanoparticles and αPD-L1 can effectively elicit the memory immune response, and prevent tumor recurrence and lung metastasis.
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Objective To study the maturation and activation effects of hyaluronic acid (HA) modified polymer nanoparticles co-delivering adjuvants and antigens on mouse bone marrow dendritic cells (BMDCs). Methods HA-modified polylactic acid-glycolic acid copolymer (PLGA) and cationic lipid DOTAP were used as nanocarriers (DOTAP-PLGA) to co-deliver adjuvant CpG with model antigen ovalbumin (OVA). In the drug-loaded nanocarriers, CpG was covalently bound to the surface of HA, and OVA was physically blended into DOTAP-PLGA nanocarriers. The nanoparticles were characterized by transmission electron microscopy and dynamic light scattering. The in vitro release of CpG and OVA in the nanoparticles was investigated. The uptake and distribution of nanoparticles in mouse BMDCs were studied by flow cytometry and laser scanning confocal microscopy. The maturation and cytokine expression of mouse BMDCs were evaluated by flow cytometry and enzyme-linked immunosorbent assay, respectively. Results The CpG-HA-OVA-PLGA nanoparticles loading CpG and OVA were prepared. The average particle size was (305.1±2.2) nm and the polydispersity index was 0.203. A core-shell structure of the nanoparticles modified by HA was clearly observed by transmission electron microscopy. Cellular experiment results showed that CpG-HA-OVA-PLGA nanoparticles could be efficiently uptaken by mouse BMDCs, and promote lysosomal release of CpG and cytoplasmic delivery of antigen OVA. Compared with free OVA group and free OVA+CpG group, the CpG-HA-OVA-PLGA nanoparticles significantly up-regulated the expression of co-stimulatory molecules CD86 and CD40 (all P<0.01), major histocompatibility complex I (MHC-I) (P<0.01), and cytokine tumor necrosis factor-α (TNF-α) (P<0.01). Conclusions HA-modified CpG and OVA nanoparticle co-delivery vectors can effectively promote the maturation and activation of dendritic cells, which provides a basis for the development of novel vaccine vectors for the co-delivery of antigens and adjuvants.
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Objective To establish a simple and gentle antigen loading method to prepare pH-responsive and biodegradable microcapsules for efficient antigen delivery.Methods Co-precipitation method was used to embed chicken egg albumin (OVA) in CaCO3 particles.Then,TA and Al (Ⅲ) were coated on the surface of CaCO3 particles template by metal-organic coordination bonds.The CaCO3 template was removed from disodium edetate to obtain TA-Al(Ⅲ) microcapsules carrying OVA,i.e.the OVA@TA-Al(Ⅲ) microcapsules.The microcapsules were characterized by field emission scanning electron microscopy,transmission electron microscopy,X-ray energy spectrometry and atomic force microscopy.The distribution of OVA in the microcapsules was observed by laser scanning confocal microscopy.The cumulative release rate of OVA in the microcapsules at different pH phosphate buffers was also investigated.The cytotoxicity of the microcapsules on immortalized mouse dendritic cells DC2.4 was observed by thiazolyl blue assay.The phagocytosis of the microcapsules by DC2.4 cells was observed by laser scanning confocal microscopy.Results The results of field emission scanning electron microscope and transmission electron microscopy showed that the OVA@TA-Al(Ⅲ) microcapsules have a intact structure and a hollow and collapsed appearance with a diameter of about 4 μm.X-ray energy spectrum showed that there are five kinds of elements,i.e.C,O,Al,Si and Na,in the microcapsules,among which C,Al and some O elements belong to the composition of the microcapsules.Atomic force microscopy showed that the microcapsules have an ultra-thin wall,and the walls of the microcapsules are uniform in thickness (about 16 nm).Laser scanning confocal microscopy showed that OVAs were evenly distributed in the CaCO3 particles.Moreover,the pH sensitivity of the coordination bond makes the OVA@TA-Al(Ⅲ) microcapsules have pH responsiveness.In addition,the microcapsules also have good biocompatibility,and the DC2.4 cells also have good phagocytic ability to the microcapsules.Conclusion A simple and gentle antigen-encapsuling method was developed to achieve effective antigen payload and pH responsive delivery.The prepared microcapsules are expected to be used as a novel antigen delivery vector for clinical research.
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Objective To study the antigen-specific immune response induced by the graphene oxide (GO) in mice.Methods OVA-loaded GO nano-immunocomplexes (GO-OVA) were prepared by co-incubation of nano GO with model antigen ovalbumin (OVA).Nano GO was characterized by atomic force microscopy and laser particle sizeanalyzer.The cytotoxicity of GO to mouse bone marrow dendritic cells (BMDCs) was detected by cell counting kit (CCK-8).The GO-OVA uptake of BMDCs were observed by fluorescent staining.C57BL/6 mice were divided into OVA group,aluminum adjuvant OVA (Al-OVA) group and GO-OVA group (6 mice in each group) by body weight for in vivo immunization.The levels of OVA-specific antibody IgG (total IgG,IgG1,and IgG2a) in serum of mice were detected by enzyme-linked immunosorbent assay (ELISA).The T lymphocyte subsets in spleen and inguinal lymph nodes of mice were detected by flow cytometry.Results The average particle size of the prepared nano GO was (294.34±4.68) nm,and the polydispersity coefficient was 0.208.Nano GO has less toxicity to mouse BMDCs.The results of in vitro experiments indicated that GO-OVA nanovaccine can be efficiently internalized by mouse BMDCs.The antigen-specific IgG antibodies induced by the GO-OVA was similar to that of aluminum adjuvant and the difference was not statistically significant (P＞0.05),and the Th1-type response was predominant.The proportions of CD4+ and CD8+ T lymphocytes in the spleen and inguinal lymph nodes in GO-OVA group were significantly higher than those in OVA and Al-OVA groups,and the differences were statistically significant (all P＜0.05).Conclusions GO-OVA nano-immunocomplexes can induce both humoral and cellular immune responses in mice,which provides basis for the development of novel vaccine vectors and adjuvants.
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In recent years,cancer immunotherapy has developed rapidly due to its significant advantages compared with the traditional cancer treatment methods.Tumor immunotherapy aims at mobilizing or stimulating the body's own immune function,thereby inhibiting and killing cancer cells.With the development of nanotechnology,biological nano-carrier materials provide a new insight into the vaccine development.Nano-vaccines are therapeutic or prophylactic vaccines based on nanotechnology including exogenous antigens for inducing immune responses,vectors delivering antigens,and adjuvants for enhancing immunogenicity and accelerating and prolonging the availability of cancer vaccines.Nano-delivery vectors have good biocompatibility as well as unique physical and chemical properties.They can effectively deliver the antigens,and further activated the immune response of antigenspecific cellulars based on the activation of the body's humoral immunity by regulating the presentation pathways in the antigen-presenting cells.In this paper,the applications of nano-delivery systems in cancer vaccine research were summarized.
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In recent years,cancer immunotherapy has developed rapidly due to its significant advantages compared with the traditional cancer treatment methods.Tumor immunotherapy aims at mobilizing or stimulating the body's own immune function,thereby inhibiting and killing cancer cells.With the development of nanotechnology,biological nano-carrier materials provide a new insight into the vaccine development.Nano-vaccines are therapeutic or prophylactic vaccines based on nanotechnology including exogenous antigens for inducing immune responses,vectors delivering antigens,and adjuvants for enhancing immunogenicity and accelerating and prolonging the availability of cancer vaccines.Nano-delivery vectors have good biocompatibility as well as unique physical and chemical properties.They can effectively deliver the antigens,and further activated the immune response of antigenspecific cellulars based on the activation of the body's humoral immunity by regulating the presentation pathways in the antigen-presenting cells.In this paper,the applications of nano-delivery systems in cancer vaccine research were summarized.
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With the development of nanotechnology,nanomaterials have great application potential in drug delivery system.Understanding the cellular uptake mechanism of nanoparticles has important scientific significance and application value in understanding life processes in cellular level,mechanisms of drug action and gene therapy,which provides the basis for developing safer and more effective nanosized drug carrier.This review summarizes the latest advancement about cellular uptake mechanisms of nanopatricles.Based on a brief introduction of the endocytic pathway of nanoparticles,influencing factors of endocytosis pathway are discussed,and the common methods used to study nanoparticle endocytosis pathways are also introduced in detail.
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Objective To develop paclitaxel-loaded polymeric micelles from poly (ε-caprolactone)-poly (ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL),and to evaluate in vitro cytotoxicity as well as in vivo antitumor activity against EMT-6 tumor breast cell.Methods Paclitaxel-loaded polymeric micelles were prepared by thin-film hydration and ultrasonic method.The physical status of paclitaxel inside the polymeric micelles was investigated by differential scanning calorimetry (DSC).In vitro cytotoxicity of paclitaxel-loaded polymeric micelles against EMT-6 cell line was assessed by MTT assay.In vivo anticancer activity was evaluated against EMT-6 tumorbearing mice,with commercially available Taxol injection as control.Results Paclitaxel-loaded polymeric micelles exhibited homogeneous spherical shapes with apparent core-shell morphology.The average diameter of paclitaxelloaded polymeric micelles was 93 nm.DSC study indicated that paclitaxel was in solid amorphous state after being encapsulated in the polymeric micelles.In vitro cytotoxicity demonstrated that the cytotoxic effect of paclitaxelloaded polymeric micelles was lower than that of Taxol injection at the same paclitaxel content.Paclitaxel-loaded polymeric micelles showed greater tumor growth-inhibition effect in vivo on EMT-6 breast tumor in comparison with that of Taxol injection,with tumor growth inhibition of 85.79％ and 63.37％,respectively (P＜0.05).Conculsions The prepared paclitaxel-loaded polymeric micelles showed high anti-tumoral efficacy and low toxicity,and might have the potential to be developed as an effective anticancer drug-delivery system for cancer chemotherapy.
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ObjectiveThe aim of the present study was to investigate the incorporation of plasmid DNA (pDNA) onto a coronary stent by chemo-immuno-conjugation for achieving site-specific gene delivery.MethodsA gene eluting stent was fabricated by reacting with polyallylamine bisphosphonate (PAA-BP) to introduce amine reactive groups on the surface.Then an anti-DNA antibody was chemically coupled and pDNA was immunologically tethered on the stent surface.Radioactive-labeled antibody was used to evaluate binding capacity and stability.ResultsThe presence of amine groups on the modified stent surface was confirmed by XPS and AFM analysis.The isotope label assay indicated that the amount of antibody chemically linked on the stents was 15-fold higher than that of the control stent and its retention time was also significantly longer.ConclusionThe results suggested that a large amount of reactive amine groups were introduced on the PAA-BP modified 316L coronary stent surface.This study provide a potential metal surface modification method that could facilitate coupling and tethering of biological molecules such as anti-DNA antibody and plasmid DNA (pDNA) to achieve sustained and highly localized gene delivery for substrate-mediated gene transfection.
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ObjectiveTo develop doxorubicine-loaded nanomicelles based on a type of novel starshaped 4-arm PLGA-PEG-NH2 amphiphilic block copolymers.Methods 4s-(PLGA-PEG-NH2) synthesized by 4s-PLGA and (H2N-PEG-NH2) according to N,N'-dicyclohexylcarbodiimide(DCC) condensation reaction was demonstrated by 1H NMR spectroscopy and gel permeation chromatography(GPC); DOX-loaded 4s-(PLGA-PEG-NH2) nanomicelles were self-assembled by doxorubicin(DOX) and 4s-(PLGA-PEG-NH2) via emulsion-solvent evaporation method and characterized in terms of morphology,particle size and size distribution,drug loading,encapsulation efficacy,cell uptake and cytotoxicity studies.Results4s-(PLGA-PEG-NH2) were capable of selfassembling intocore-shell nanomicelles structure and encapsulating DOX into their hydrophobic cores.The mean size of DOX-loaded 4s-(PLGA-PEG-NH2) was nanometer size; drug loading and encapsulation efficacy were around 7.5％ and 75.2％,respectively.Mean surface charge of the micelles was around -17.6 mV.In vitro cell uptake and cytotoxicity studies indicated that comparing to the DOX-loaded linear-(PLGA-PEG-PLGA)nanomicelles,DOX-loaded 4s-(PLGA-PEG-NH2) nanomicelles showed better performance in uptaking by HeLa cells and higher cytotoxicity to cancer cells.Conclusion4s-(PLGA-PEG-NH2) amphiphilic block copolymers can be successfully used in encapsulating DOX,self-assemblingcore-shell nanomicelles in aqueous solvent.Therefore,4s-(PLGA-PEG-NH2) copolymers can be considered as a promising drug carrier in effectively carrying hydrophobic drug,improving the efficacy while reducing the side effect.
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Objective To construct an injectable controlled delivery system of paclitaxel based on thermosensitive PCL1250-PEG1500-PCL1250 hydrogels. Methods A thermosensitive PCL1250-PEG1500-PCL1250 triblock copolymer was synthesized by ring-opening polymerization of e-CL using PEG (Mw=l 500) as the initiator and Sn(Oct)2 as the catalyst. The synthesized PCL1250-PEG1500-PCL1250 copolymers were characterized for their composition,structure, and molecular weight via 1H NMR and GPC techniques. A series of Paclitaxel loaded hydrogels with various predesigned hydrogel concentrations and initial drug loadings were prepared to investigate their gelation ability, in vitro drug release behavior and in vivo biodegradability. Results The results calculated from 1H NMR and GPC indicated that EG/CL ratio(1.55) was consistent with the initial feed ratio(1.6), which offered a strong proof to their composition and molecular structure. The thermosensitive PCL1250-PEG1500-PCL1250 hydrogels exhibited a desirable sol-gel transition ability within the concentration range of 15%-30%. The in vitro release rate of paclitaxel from the paclitaxel/PCL1250-PEG1500-PCL1250 hydrogels was controllable by altering the hydrogel concentrations and initial drug loadings. The PCL1250-PEG1500-PCL1250 hydrogels showed a good in situ gelation ability after subcutaneously injected into mouse back. The in situ formed hydrogels gradually degradated with time and almost disappeared after 45 days in vivo. Conclusion Both the controllable drug release behavior and promising biodegradability of this new thermosensitive PCL1250-PEG1500-PCL1250 hydrogels paved a way to develop a novel delivery system for paclitaxel.
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Objective To prepare paclitaxel-loaded poly(ε-caprolactone)(PCL)/pluronic F68(F68)blend microspheres as a controlled release system. Methods Paclitaxel-loaded PCL/F68 blend microspheres were prepared by the oil-in water(O/W)emulsion/solvent evaporation method. Characterization of the microspheres followed to examine the particle size, the drug encapsulation efficiency, the surface morphology, in vitro release behavior and DSC analysis. In vivo antitumor activity of paclitaxel-loaded PCL/F68 blend microspheres was evaluated in mice bearing with hepatoma H22 cells ascites tumor. Results The results showed that the porous structure can be formed in the surface of PCL/F68 blend microspheres. Faster and controlled release of paclitaxel from PCL/F68 blend microspheres was achieved in comparison with the PCL microspheres. In animal tests, paclitaxel-loaded PCL/F68 blend microspheres showed the potent antitumor activity against hepatoma H22 cells in ascites tumor model. Conclusion The paclitaxel loaded PCL/F68 blend microspheres were found to own a faster release rate and a remarkably controlled release behavior.