Targeting Polymeric Micelles For The Small Molecular Therapeutics Brain Delivery

The morbidity and mortality of brain and central nervous system (CNS) major diseases (such as brain tumor, CNS infections and neurodegenerative diseases) are yearly increasing. The presence of the blood-brain barrier (BBB) makes above98%small molecular drugs and100%macromolecular drugs restricted into the brain tissue, which has become a big challenge for the brain disorders therapy.At present, clinical drug in the treatment of infection of the brain or brain tumor, is usually small molecules. However, there are many challenges encountered in the clinical meditation:(1) the nature of the drug itself-many anti-infection or anti-tumor drugs are poor solubility, which makes it difficult for clinical medication, especially the intravenous administration;(2) the poor accumulation capacity of drugs in the disease region-drug distributes into other non-targeted organs along with the blood circulation, causing serious side effects;(3) resistance problem-the pathogens or tumor cells readily get resistance to the therapeutics, especially after continuously repeated dosing, causing the invalid treatment.However, besides the advantages of the traditional targeting polymeric micelles, they also still have many defects in practice:(1) the target molecules are usually selected with peptides or antibodies, adopting the receptor-mediated strategy, therefore the improvement of targeting capacity is limited;(2) the structural stability is poor, the polymer materials are usually selected with traditional amphiphilic copolymers, under the condition of large amounts of blood dilution, the micelles are bound to disassociate and cause the drug leakage, due to lower than the critical micelle concentration (CMC). Caused by these problems, the targeting efficiency is reduced greatly.Therefore, in order to solve these problems, this study has innovatively designed a kind of smart polymeric micelles, the advantages are reflected in:(1) to use the transporter-mediated strategy, developed effective target molecules. This study designed a high affinity of targeting molecule, dehydroascorbic acid (DHA), which could specifically recognize the facilitative glucose transporter1(GLUT1), both highly expressed on the BBB and tumor cells. Modifying the DHA on the micelles surface could achieve the brain and glioma dual targeting delivery;(2) to develop a kind of poly (amino acid)s copolymers with high safety and biocompatibility. The active groups on the side chains could react with the crosslinking agents bearing the cellular microenvironment sensibility, to produce the smart polymeric micelles with the cellular microenvironment triggered drug release. Combination of these two advantages, the designed smart polymeric micelles could:(1) keep highly structural stability in blood circulation, avoid drug leakage and reduce the drugs being degraded by enzyme;(2) target to the brain or glioma, and achieve the deposition of the drugs in the targeted sites and even targeted cells, to greatly enhance the targeting efficiency.Part I Peptide modified the conventional brain-targeting polymeric micellesIn the first part (Chapter1and2), we utilized the lipid materials, PEG-PE, a kind of safe copolymers approved by FDA, to develop the conventional polymeric micelles. As previously reported, low-density lipoprotein receptor-related protein (LRP) is highly expressed on the BBB. We modified the ligand of LRP as a vector, Angiopep-2, on the surface of polymeric micelles to achieve the crossing the BBB delivery.In Chapter One, Angiopep-2was as a brain-targeting vector, and hydrophobic anti-fungal drug, amphotericin B (AmB) was as a model drug. We utilized PEG-PE as the carriers to develop the AmB loaded brain-targeting polymeric micelles, Angiopep-PEG-PE/AmB. The PEG-PE polymeric micelles were prepared with filming-rehydration method and optimized with the Central Composite Design/Response Surface Method. The results of characterization demonstrated that optimized formulation with high entrapping and loading efficiency,83%and12%respectively, and size ranging between10to15nm. The brain-targeting efficiency of Angiopep-PEG-PE/AmB polymeric micelles was evaluated both in vitro, brain capillary endothelial cells (BCECs) and in vivo. The findings investigated that Angiopep-2could increase the cellular uptake and promote the polymeric micelles accumulating in the brain. In addition, the significantly decreased cytotoxicity and hemolysis in vitro was contributed to the entrapped AmB into the cores of polymeric micelles, thus increasing the safety by i.v. injection. This chapter further explored the underlying mechanism of improved Angiopep-PEG-PE/AmB crossing the BBB. The results demonstrated that Angiopep-2maintained receptor binding properties after modifying on the polymeric micelles, and improved BBB-penetrability though LRP-mediated pathway. Meanwhile, the polymeric micelles could prevent AmB recognizing by P-gp and overcome the P- gp efflux, prolong the retention time of AmB in brain. The brain slice observed by Confocal Microscopy confirmed that Angiopep-2modification can not only promote the polymeric micelle across the BBB, and can further be internalized by brain cells.We have successfully developed an effective brain-targeting polymeric micellar system with angiopep-2modified, named Angiopep-PEG-PE/AmB polymeric micelles, to improve the CNS permeability of AmB through the receptor-mediated transcytosis. In Chapter Two, an immunosuppressed murine model with CNS infection was established to evaluate the CNS penetration efficiency and antifungal treatment efficacy of the AmB-incorporated brain-targeting polymeric micellelar formulation, compared with the AmB commercial formulations (deoxycholate AmB and liposomal form of AmB). After three consecutive days of administration, we found that the group treated with Angiopep-PEG-PE/AmB achieved the highest ratio of AmB in brain to serum, nearly2-fold higher than the other formulations. Meanwhile, Angiopep-PEG-PE/AmB reduced the brain fungal burden significantly, decreased histopathological severity and prolonged the median survival time. The increased treatment efficacy could be attributed to the brain-targeting delivery system promoted AmB penetrating into the brain to reach the therapeutic concentration. Therefore, the brain-targeting delivery system could have potential and promising implications for treatment of intracerebral fungal infection.Part II Small molecule modified the cellular microenvironment-triggered smart polymeric micellesOn the basis of previous research on the conventional polymeric micelles, summarized the strategies of design:(1) utilized the traditional amphiphilic copolymers, such as lipid materials, PEG-PLGA or Pluronics, etc.;(2) modified with the target molecule such as peptides or antibody, mediated by receptor-mediated pathway.However, the conventional polymeric micelles face many problems in the practical application, for example, the structural stability in the blood dilution is poor, causing drug leakage and making drugs rapid metabolism or degradation; the targeting efficiency is very limited, etc. According to the above statement, in the second part of the study, we mainly put forward two strategies in the nanocarriers design:Strategy1:Develop a novel targeting molecule with high affinityStrategy2:The anti-drug-leaky "smart barrier" to achieve intracellular microenvironment sensitive drug release Firstly, to achieve Strategy1, in Chapter Three, we designed and synthesized a novel small molecular vector successfully, dehydroascorbic acid (DHA). The targeting molecule is a substrate of glucose transporter1(GLUT1), which is highly expressing on the tumor cells. Compared with glucose or other hexoses, the vector exhibited more advantages, such as the high affinity, non-saturability and continuously accumulative ability. In Chapter Three, we introduced the synthesized scheme of2-propargyl DHA and characterization, and evaluated the targeting efficiency both in vitro and in vivo. The results demonstrated that the designed DHA molecule was transported by Na+-independent GLUT1, in agreement with previously reported, and further proved that DHA could cross the BBB and accumulate in the brain tissue.Secondly, for Strategy2, in Chapter Four, we designed a kind of polypeptide copolymers, poly (ethyleneglycol)-b-poly (L-lysine)-b-poly (L-phenylalanine)(PEG-pLys-pPhe), which could self-assemble into micellar structure in an aqueous environment with high drug loading efficiency. The segment of pPhe provides the hydrophobic core for insoluble drug entrapped. The segment of pLys is designed for cross-linking reaction. Amino groups on side chains of pLys could further react with a cross-linking agent containing disulfide bonds to produce a shell-cross-linked covalent linkage. Polypeptides polymer are a kind of biodegradable copolymers, which can be easily absorbed with high biocompatibility.Combining Strategy1and Strategy2(Fig.1-1) was introduced in Chapter Five. The core-shell-corona architecture of the polymeric micelles formed through self-assembly of PEG-pLys-pPhe mixed with the functionalized copolymer, DHA-PEG- pLys-pPhe. DHA was conjugated to polymers through the click chemistry with PEG segment. The micelles could be further crosslinked with an anti-drug-leaky barrier, which made them more condensed in the blood circulation. The engineered drug-nanocarriers, DHA-PLys(s-s)P micelles, could keep high-structural stability with their payload in the circulation. Once upon entering the targeted cells, the cellular microenvironment sensitive anti-drug-leakage barriers were cleaved by high concentration of glutathione (GSH) and the drug could release intracellularly. As a result, the smart drug-nanocarrier could achieve the drug deposition in the targeted sites and targeted cells.The results demonstrated that the DHA-PLys(s-s)P micelles with high stability exhibited GSH-sensitive drug release. In vitro, DHA could promote the micelles significantly internalized by BCECs. In vivo, DHA-PLys(s-s)P micelles could cross the BBB and further be uptake by brain cells. In summary, the modified DHA remained the ability to recognize and bind with GLUT1. Due to the stability in the blood circulation. the targeting efficiency could mostly benefit from it.According to the previous research (Chapter Three, Four and Five), we successfully developed DHA-PLys(s-s)P micelles. In Chapter Six. hydrophobic anti-fungal drug itraconazole (ITZ) was selected as the model drug, constituting the DHA-PLys(s-s)P/ITZ micelles. The biodistribution and pharmacokinetics study demonstrated that DHA could improve the micelle entering the brain. In addition, the reduced metabolites, HO-ITZ, in the blood indicated that the reduced drug leak in the circulation due to the highly structure stability. We evaluated the therapy efficacy of DHA-PLys(s-s)P/ITZ micelles on the model animal with CNS infections. The results showed that DHA, modified on the micelles, could effectively reduce the fungal burden in the brain and significantly prolong the survival time of infected mice.As reported, an extinguished feature of the tumor cells differ from normal cells, is a high level of glucose uptake and metabolism of sugar. Studies confirm that the demand of malignant cells glucose is much higher than that of normal cells, due to glucose is an important energy source for tumor cells. Thus, GLUT1is highly expressed on the tumor cells. Previously study has confirmed that DHA could recognize the GLUT1, expressed on the BBB, In Chapter Seven, we tried to utilize the DHA-PLys(s-s)P micelles to deliver the anti-tumor drug, paclitaxel (PTX), for the glioma therapy. The anti-tumor efficacy was evaluated both in vitro and in vivo. In vitro, the cell apotosis was evaluated by TUNEL Assay, cells cycle analysis and IC50analysis. In vivo, the glioma-bearing mice were treated with the PTX-nanocarriers and PTX commercial, Taxol(?). After treatment, the anti-tumor efficacy was evaluated through in vivo apotosis imaging, immunofluorescence and survival analysis, etc.