Preparation And Brain Delivery Property Of Polymersomes By Transferrin Receptor-mediated Transcytosis

With population aging,brain diseases are rapidly increasing and threatening human health.However,the delivery of therapeutic agents for brain diseases is far from efficient,due to the blood-brain barrier(BBB).The BBB drug targeting technology designed as molecular "Trojan horse" could bring hope to these molecules. One of the most effective "Trojan horse" BBB delivery systems is liposome-based "Trojan horse",composed of pegylated liposomes as drug carriers and surface-conjugated targetors such as the transferrin(Tf) receptor monoclonal antibody (TFRmAb) OX26 which could initiate the endogenous receptor-mediated transcytosis (RMT) to cross the BBB.However,liposome seems not perfect as a drug carrier because of its unstability.Before liposomes reach the brain capillary,they will rupture in blood and decrease the brain drug delivery.Moreover,many lipid vesicles properties such as encapsulant retention,membrane stability,and degradation are not particularly well-controlled and minimize the likelihood of delivering drugs to an intended target site.Therefore,new drug carriers with better vesicles properties such as good stability and low permeability are necessary for the construction of the BBB targeting delivery system with more rational and effective brain delivery property.As a new class of synthetic thin-shelled capsules based on block copolymer chemistry,polymersomes are self-assembled vesicles of amphiphilic block copolymers with thicker and tougher membrane than lipids.Polymersomes have the same advantages as liposomes and nanoparticles.Compared with liposomes, polymersomes are more stable.The internal aqueous cavity of the polymersome is expected to load more hydrophilic drugs including peptides and protein than nanoparticles.The combination of a thick wall for a hydrophobic drug and a vesicular lumen for a hydrophilic drug will lead to synergistic effect like cocktails.Moreover, the physical and chemical properties of polymersomes including particles size, membrane thickness,permeability,drug loading,surface modification,and even in vivo behavior may be broadly tunable through rich diversity of block copolymer chemistries(block fraction,block architecture).Therefore,polymersomes are good candidates for drug delivery carriers,which are expected to have better brain delivery property.The present study focused on the preparation and evaluation of brain delivery property of polymersome-based molecular "Trojan horse",where OX26 or transferrin, was conjugated to the polymersomes' surface(OX26-PO or Tf-PO).The first part described the preparation and characterization of polymersomes. Block copolymer poly(ethyleneglycol)-poly(ε-caprolactone)(PEG-PCL) was selected for preparing polymersomes due to its biodegradability and commercial availability. The feasibility of PEG-PCL polymersomes as drug carriers were evaluated through the investigation of the formation of polymersomes and the physico-chemical properties such as particles size,membrane thickness,stability,permeability and drug releasing characteristics.The MPEG-PCL or Maleimide-PEG-PCL diblock copolymers were synthesized by ring-opening polymerization ofε-CL in dry toluene under moisture-free high purity nitrogen atmosphere,using MPEG or Maleimide-PEG-PCL as the initiator.It was found that PEG-PCL copolymers self-assembling into polymersomes possess a hydrophilic volume fraction(f_hydrophilic) of 0.16～0.32 via a solvent injection technique.The vesicle size and membrane thickness correlated with the molecular weight of copolymers.The membrane thickness of polymersome was above 10 nm,much thicker than that of liposome.The in vitro stability tests of vesicle size showed that polymersomes were more stable than liposomes.It was shown that PEG-PCL polymersomes were 20 times less permeable than liposomes,but their permeability could be tunable by dioxane.The release of drugs from polymersomes at room temperature was consistent with Fick's first law and the flux of the drug across the vesicle wall could be tunable by the adjustment of the molecular weight.High temperature,low pH value and the existence of serum accelerated the release of drugs from polymersomes.The release of drugs from polymersomes was expressed as the combination of Fick's diffusion and erosion mechanism at 37℃,pH 4 or in the existence of serum.During the initial release process,it was mainly attributed to Fick's diffusion,followed by erosion mechanism mainly in the following release process.The second part described the preparation and evaluation of brain delivery property of OX26-PO.Activated PEG-PCL polymersomes were made with a blend of MPEG-PCL and Maleimide-PEG-PCL.OX26 was thiolated and conjugated to the polymersomes' surface through the maleimide function.Therefore,a novel drug carrier for brain delivery through transferrin receptor mediated transcytosis,PEG-PCL polymersomes conjugated with OX26(OX26-PO),was developed.Coupling of OX26 with PO was confirmed by immuno-gold labeling of OX26 visualized under the TEM and X-ray photoelectron spectroscopy test.The results of pharmacokinetics and brain delivery of 6-coumarin labeled OX26-PO in rats proved that the increase of surface OX26 density of OX26-PO decreased blood AUC.The optimized OX26 number conjugated per polymersome was 34,which can acquire the greatest blood-brain barrier(BBB) permeability surface area(PS) product and percentage of injected dose per gram brain(%ID/g brain).Compared with PO,the average%ID/g brain and PS product for OX26₃₄-PO were 2.62-fold and 3.48-fold of those of PO,respectively. The microscopic examination of coronal sections of the rat brain after i.v.injection of fluorescent polymersomes showed that coumarin-6 labeled OX26₃₄-PO were localized in the cerebral cortex,the peri-ventricular region of the lateral ventricle and the third ventricle,where OX26₃₄-PO exhibited a higher accumulation than PO,validating the results of brain delivery property of OX26₃₄-PO.Immunostaining of monocyte macrophage demonstrated that high dose of OX26-PO could not induce the increase amount of macrophage in cerebrum,heart and liver in Sprague-Dawley rats,only had light toxicity to lung,spleen and kidney,and its acute toxicity to lung and kidney was transient.Derived from the above experimental results,we could conclude that OX26-PO was a novel brain drug delivery system with low toxicity.The third part described the preparation and evaluation of brain delivery property of peptides loaded OX26₃₄-PO.NC-1900,a vasopressin fragment AVP_4-9 analog,was encapsulated into OX26-PO as a model peptide and the enhanced brain delivery of NC-1900 was evaluated with pharmacodynamic experiments.It was found that NC-1900 was a highly hydrophilic peptide with strong base property.Its logP was below -4.NC-1900 was a cationic peptide in neutral environment,inducing the gelatination of gellan gum at a high or low concentration.According the physical and chemical properties of NC-1900,a new method for the encapsulation of NC-1900 was developed.The gellan gum trapped into the polymersomes was used as a reservoir to capture NC-1900 permeating into inner aqueous cavity of polymersomes,which highly increased the encapsulation efficiency of NC-1900.The preparation of NC-1900 loaded OX26-PO was optimized by central composite design and response surface method.According to the optimized preparation,the drug loading capability of NC-1900 loaded OX26-PO was 1.20%and a relative high encapsulation efficiency of 30%.of NC-1900 was encapsulated into OX26-PO,which reached the expected objective.In vitro release tests showed that the release of NC-1900 from the gel was a rate-limiting step of the release ofNC-1900 from OX26-PO and it could be expressed as Fick's diffusion.It was shown that OX26-PO significantly enhanced the brain delivery of NC-1900 with ameliorating the scopolamine-induced learning and memory impairments in a dose-dependent manner.The dose-response relation of NC-1900 loaded OX26-PO revealed that the median effective dose of NC-1900 was 0.82 ng/kg.The above experiments results indicated that OX26-PO might serve as a potential brain delivery system especially for large-molecule drugs such as peptides and proteins.The fourth part described the preparation and evaluation of brain delivery property of Tf-PO loaded with small molecule chemotherapeutics.Doxorubicin,as a model drug,was encapsulated into Tf-PO via a pH gradient.From IC₅₀,the C6 inhibition efficiency of doxorubicin loaded Tf-PO was 8.62 times higher than that of doxorubicin solution,and 3.75 times higher than that of doxorubicin loaded PO, which revealed that doxorubicin loaded Tf-PO can enhance the drug inhibition effect on C6 glioma cells.It was shown in pharmacokinetic and brain distribution experiments that Tf-PO significantly enhanced brain delivery of doxorubicin, especially the delivery of doxorubicin into brain tumor.At 24h after i.v.injection of doxorubicin loaded Tf-PO in C6 glioma transplanted rats,%ID/g of brain cortex,left striaturn,and tumor in Tf-PO group was 3.30,3.54,3.60 times higher than that in PO group,respectively and 3.47,3.16,6.79 times higher than that in doxorubicin solution group,respectively.At 4h after i.v.injection of doxorubicin loaded Tf-PO in C6 glioma transplanted rats,%ID/g of brain cortex,left striatum,and tumor in Tf-PO group was 2.38,2.23,2.72 times higher than that in PO group,respectively and 2.08, 2.59,6.92 times higher than that in doxorubicin solution group,respectively.The above experiments results indicated that Tf-PO might enhance the chemotherapeutic effect of doxorubicin for brain glioma.The fifth part described the preparation and comparison of pharmacokinetics and tissue distribution of three brain drug delivery systems based on polymersomes.It was shown that the clearance of polymersomes in blood and tissue uptake of polymersomes correlated to the type and density of surface protein conjugated to polymersomes.Following the conjugation with tranferrin,the organ PS product and uptake of polymersomes for liver,brain and spleen were significantly increased while those for lung were significantly decreased.The organ uptake of OX26-PO correlated with surface OX26 density.There was no significant difference of organ PS product and uptake between OX26₃₄-PO and Tf-PO with the similar surface protein density.