Intracellular Trafficking Of Poly(Ethylene Glycol) In Living Cells And Its Applications For Anti-Tumor Agents And Cellular Imaging

The polymer-drug conjugate was firstly reported in 1955.Propelled by substantial work of Ringsdorf,Kope?ek and Duncan in 1970s and the first clinical utility of Adagen^?,PEGylated adenosine deaminase（pegademase）approved by Food and Drug Administration（FDA）in 1990,polymer-drug conjugate and the field of polymer therapeutics have achieved an overwhelming development.Up to date,different categories of polymer-drug conjugates have been approved for clinical application and a lot of candidates are still in clinical/preclinical trails.Among the strategies of polymer modification,PEGylation is the most widely used in the clinical.PEGylation is proposed to dramatically improve pharmacological and physiological properties of the conjugated drug by reducing cytotoxicity and immunogenicity,prolonging blood circulation time,diminishing degradation by the enzymes,and altering bio-distribution profile,which has a preponderant use in pharmaceutical industry since 1970s.Currently,35 PEGylated products have been approved for clinical use worldwide,proving the applicability and effectiveness of PEGylation technology.With the wide application of PEG and PEGylation,the absorption,distribution,metabolism,excretion and toxicity of PEGylated drugs in vivo were studied and reported.However,the debate is still ongoing on what made PEG the modifier of choice for therapeutic agents.However,the PEGylated drugs approved in clinical are mostly for proteins and oligonucleotides.Few kinds of PEGylated small organic molecules were approved,ascribed by the decreased therapeutic index.As known to us,to enable the medicine to realize the therapeutic efficiency,it must overcome a series of biological barriers including circulation in the blood compartments,accumulation in the lesion location,cellular internalization and intracellular drug release.Currently,the research of PEG and PEGylated medicine are mainly focus on the tissue and organ level and few studies on the process of cellular uptake,trafficking,localization and exocytosis of PEG have been reported.With the continuous research and the development of techniques,more and more complex network of events occurred in living cells have been deciphered and the ability to target therapeutics inside cells is becoming more and more important.Meanwhile,current drug design strategies are focused on specific intracellular organelle delivery.As the critical vehicle of PEGylated therapeutics,it’s necessary to interpret the interaction between PEG and biological systems at cellular/sub-cellular level.Moreover,knowledge of endocytosis,intracellular trafficking,cellular localization and exocytosis of PEG provides the foundation and perspective on the applications for drug delivery.Herein,PEG 5 k Da,one of the most widely used ones for PEGylated therapeutics,was chosen as a representative model and the cellular uptake mechanisms,distribution,trafficking,localization and exocytosis of PEG in living cells were demonstrated.What’s more,the therapeutic activity of PEGylated anti-tumor agents and the cellular imaging of PEGylated fluorescent molecule were investigated.1.Cellular distribution of PEG and its application for anti-tumor agentsHerein,we investigated the internalization behavior and distribution of PEG in living cells.Our investigation shows that PEG can be internalized rapidly into cells and the endocytosis mechanism of PEG might be the combined function of macropinocytosis and other endocytosis.The internalized PEG is delivered into lysosomes,cytosol,endoplasmic reticulum（ER）and mitochondria,without any specific subcellular target.In addition,the in vitro anti-tumor efficiency of PEGylated lonidamine（LND）with different molecular weight was studied.The result indicates that PEGylation can improve the therapeutic efficiency of LND in vitro.Meanwhile,the anti-tumor activity is reduced with the increased molecular weight of PEG.2.Journey of PEGylated molecules with negative charge in living cells and its activity as photosensitizerConsidering that properties of PEG can be conveyed to the conjugated molecules,the intracellular fate of PEGylated molecules reflects that of PEG.To have a thorough understanding of the journey of PEG in cells,photosensitizer（pyropheophorbide-a,PPa）with fluorescent properties was conjugated to PEG and then the PEGylated PPa（PEG-PPa）with negative charge was obtained.The main endocytosis mechanism of PEG-PPa is macropinocytosis and the internalized PEG-PPa is mainly localized in lysosomes.Moreover,PEGylation increases the retention time of free PPa in cells.Cell viability research of PEG-PPa and PPa demonstrates that PEGylation decreases the dark toxicity of PPa.More importantly,PEGylation improves generation of hydroxyl radical（·OH）,hydrogen peroxide（H₂O₂）,and peroxyl radicals（ROO·）under the laser condition.3.Journey of PEGylated molecules with positive charge in living cells and its application for cellular imagingFrom the above study,we can conclude that the charge of PEGylated molecules has essential influence on the intracellular trafficking.Therefore,rhodamine B（RB）was chosen to conjugate in the end of PEG to obtain PEGylated RB（PEG-RB）with positive charge.Our research indicates that PEG-RB is internalized into cells by macropinocytosis,then transported in lysosomes,ER and mitochondria via vesicles sequentially.The final localization of PEG-RB in living cells is mitochondria and its universality of mitochondria target is validated by different cell lines.Our research improves understanding on the interaction between PEG and biological systems at cellular/sub-cellular level,which is significant for better application of PEGylation in rational design of PEGylated drug or PEGylated drug delivery system.