The Design And Evaluation Of Brain Tumor Targeted Drug Delivery Systems Based On Different Strategies

The treatment of a brain tumor is still one of the most difficult challenges in oncology. The poor treatment outcome may due to the complex microenvironment of the tumors. Although enhanced permeability and retention effect (EPR effect) do exist in brain tumor, the cut-off size of the pore in neovasculature was much smaller than the peripheral tumors. Generally speaking, blood brain barrier (BBB) was integrated in. the early stage of brain tumor and but was damaged later in middle and advanced stage. However, BBB still remained comparatively intact in the metastasis and infiltration area of brain tumor, rendering itself a big hurdle for the treatment of brain tumor.Recently, more and more studies have been focused on brain tumor targeted delivery accompanied with the development of nanotechnology. However, most researches ignored the complex microenvironment of brain tumo. It is very important that the brain tumor targeted delivery strategy was established based on the different characteristics of the brain tumors. In this dissertation, we developed several targeted drug delivery systems based on this concept, which may further improve the brain tumor treatment effect.Based on the EPR effect, in this dissertation we prepared a brain tumor active targeting delivery system (ILNPs) that utilized IL-13p as brain tumor targeting ligand. ILNPs significantly enhanced intracellular drug delivery and tumor spheroid penetration. Assays for cell apoptosis and growth inhibition of tumor spheroids identified that docetaxel (DTX)-loaded ILNPs could significantly induce cell apoptosis and inhibit tumor spheroid growth (decreased to62.5%compared with control). In vivo imaging of brain tumor-bearing mice demonstrated that ILNPs could target brain tumor and accumulate at the tumor site (1.73-fold as that of NPs), which was further verified by fluorescence imaging of brain slices. These studies demonstrated the modification with targeting ligands could enhance the uptake by targeted cells, facilitate the cell internalization. alter the dominant uptake pathways and increase the location in brain tumor. Pharmacodynamic results indicated that docetaxel-loaded ILNPs significantly prolonged the median survival time of brain tumor-bearing mice compared to unmodified nanoparticles (NPs), DTX and control.To address the two barriers faced in treatment of brain tumors-BBB and brain tumor barrier, a dual targeting delivery strategy was developed. In this dissertation, a phage-displayed TGN peptide and an AS1411aptamer, specific targeting ligands of the BBB and tumor cells respectively, were conjugated with NPs to establish the dual brain tumor targeting delivery system (AsTNPs). In vitro cell uptake and cell monolayer penetration studies demonstrated that TGN peptide increased the uptake of particles by1.61times on bEnd.3cells and facilitated their penetration through cell monolayers. Furthermore, AS1411aptamer could not only enhanced the cellular uptake of particles by1.75times on C6, but also improved their penetration capability in the In vitro three-dimensional tumor spheroids The coculture of spheroids with bEnd.3monolayers further demonstrated dual modification could enable NPs to conquer the two barriers constituted by bEnd.3and C6cells. The coculture of spheroids with bEnd.3monolayers demonstrated dual modification could enable NPs to conquer the two barriers constituted by bEnd.3and C6cells. In vivo imaging further demonstrated that the AsTNPs provided the highest tumor distribution and tumor/normal brain ratio (2.4for AsTNPs vs1.6for NPs). As a result, the DTX-loaded AsTNPs presented optimal anti-brain cancer effect with improved brain tumor bearing survival (increased by88%compared with control) at a dose as low as6mg/kg. while DTX and NPs failed to show any treatment effect. In conclusion, the AsTNPs could precisely target to the brain tumor, which was a valuable strategy for brain tumor imaging and therapy.Dual targeting of tumor cells and neovasculature is also an effective strategy for cancer treatment. Herein, we employed RGD for neovasculature targeting and IL-13p for brain tumor cell targeting. In vitro, the dual-ligands modified nanoparticles (IRNPs) can effectively target to C6cells and human umbilical vein endothelial cells (HUVEC). The selective targeting effect of RGD and IL-13p led to remarkable cell apoptosis when encapsulated with DTX. and the half inhibit concentration was as low as6.84ng/mL. which was only27.6%as that of DTX-loaded NPs. In vivo, the neovasculaure targeting effect of RGD and brain tumor targeting effect of IL-13p were further demonstrated by immunofluorescent staining. IRNPs could effectively target to brain tumor with a3.82-fold increase compared with unmodified NPs. which was significantly higher than NPs modified with single ligand. A2.06-fold higher median survival time was observed for brain tumor-bearing mice treated with DTX-loaded IRNPs when compared with control, which further demonstrated the superiority of this targeting delivery strategy.Although these targeting delivery systems significantly enhanced the treatment effect of chemotherapeutics, the delivery systems mentioned above were mostly limited to the fundamental research because of the hindrance posed in factory-scale production and a lack of pharmaceutic adjuvant. By utilizing the high DTX-binding property of albumin, a new formulation-DTX-incorporated albumin-lipid nanoparticles (DNPs), was prepared and evaluated. However, DNPs could not be further developed into a new commercial drug because of the patent protection. Therefore we try to apply this system to other drugs. Lapatinib is a small molecule that possesses high potential in cancer treatment. Herein, we utilized the albumin-lipid nanoparticles system to prepare lapatinib-incorporated albumin-lipid nanoparticles (LTNPs). Because of the high binding efficiency (>99%) of lapatinib to blood albumin, lapatinib was directly bound to albumin in vitro, and subsequently egg yolk lecithin was added to improve dispersion characteristics. Results indicated that the particles contained a lipid corona and a core of lapatinib and albumin with a size of approximately60nm. The relative bioavailability of LTNPs is320%, while the relative bioavailability of Tykerb (the commercial formulation of lapatinib) was only60.9%. In vitro, LTNPs could be taken up by U87cells in concentration-and time-dependent manner. LTNPs could distribute and accumulate in tumor site by EPR effect. At2h and8h post administration, the concentration of LTNPs in brain tumor was2.19-fold and2.76-fold as that of Tykerb respectively, indicating LTNPs possessed better brain tumor targeting effect. Both LTNPs (10mg/kg) and LTNPs (30mg/kg) could significantly expand the median survival time of brain tumor-bearing mice. At similar anti tumor effect, accumulative dose of LTNPs was only5%as that of Tykerb. Acute and long-term toxicity studies demonstrated the half lethal dose of LTNPs was over250mg/kg, while there was no obviously change in organs under therapeutic dose according to HE staining, suggesting LTNPs possessed low toxicity and good tolerance. Mechanism study suggested the high expression of SPARC protein in tumor site may mediate the tumor targeting delivery of LTNPs. which led to improved anti-brain tumor effect. This strategy could effectively improve the solubility of lapatinib and make the preparation of injection possible, presenting excellent developing value. In conclusion, based on the development and physico characteristics of brain tumor, we developed several systems to treat the brain tumor. Results demonstrated these systems effectively improved the outcome of brain tumor treatment.