博士申请用研究计划英文模板

pH—Responsive Biodegradable Polymers for Intracellular Drug Delivery

A. Proposed area of research

The aim of this proposed PhD project is to develop and evaluate pH responsive, endosomolytic polymers for efficient intracellular delivery of biological drug payloads。 There is a need to better understand the mechanisms of entry into the cell cytoplasm and nucleus in order to design optimal delivery systems for biological molecules. On the one hand, this would open up significant opportunities to deliver potent drug payloads against intracellular targets to positively impact human health。 In addition the project aims to develop a more general understandingof the rules governing the uptake of biological molecules into cells.

This project proposes to investigate the use of synthetic， biodegradable polymers for intracellular delivery of drug payloads （including siRNA, therapeutic peptide and antibody） against a well—validated intracellular drug target, such as Bcl—2。 The novel pH—responsive polymers have been designed by Dr Rongjun Chen's Lab to mimic the activity of viruses， both in their cell entry and endosomal escape mechanisms. Using cancer cell lines （Jurkat or HL-60 cells) as a model system, the polymers would be tested with a variety of different biological payloads in a quantitative comparison of their ability to enter the cell and trigger apoptosis and subsequently cell death. With an efficient model system established, there would then be scope to optimize the system in terms of the kinetics and mechanisms of cell entry, cytoplasmic and nuclear localization， and the biodegradation of the polymers。 There would also be scope to explore the efficiency in other cell systems and with further intracellular targets。 This multidisciplinary project is at the interface of Chemistry, Biology and Medicine, and will provide the student with a real opportunity to be involved in the development and evaluation of new nanomedicines.

B。 Background

Advances in genomics and proteomics have enabled the development of macrodrugs， such as nucleic acids and proteins， with potential for the treatment of a wide variety of diseases。 Amongst other problems， their clinical applications may be greatly impaired by low cellular uptake and lysosomal degradation before they can reach their target organelles or cell nuclei。 In order to achieve efficient intracellular delivery of such biological molecules， delivery systems are required to enable high cell entry via endocytosis and efficient release into the cytoplasm by endosomal membrane disruption under mildly acidic conditions.

Recombinant viruses and fusogenic viral peptides have been used to mediate gene transfection, but their clinical use is potentially limited by safety issues and difficulties in large-scale production. A variety of synthetic polymers have therefore been developed as non-viral vectors. Cationic polyethyleneimine， poly（2—

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(dimethylamino)ethyl methacrylate） and polyamidoamine dendrimers mediate gene delivery through the ‘proton sponge' effect, but suffer from cytotoxicity and relatively low transduction efficiencies。 The intensively studied vinyl-based anionic polymers, poly(a—alkylacrylic acid）s， display pH-responsive membrane disruptive behavior, but they are not biodegradable， thus low molecular weights have to be strictly required to allow renal excretion and their clinical applications are seriously limited. Dr Rongjun Chen’s Lab has recently developed a class of novel， biodegradable, pH-responsive polymers to mimic factors that enable efficient viral transfection， but they are safe， easy to manufacture and have more controllable structures。The parent polymer is a polyamide， poly(L-lysine isophthalamide）， which was based on polycondensation of diacyl chlorides and natural metabolite tri—functional amino acids containing both α- and ω-amine groups. Hydrophobic amino acids and/or poly（ethylene glycol) were grafted onto its pendant carboxylic acid groups to manipulate its amphiphilicity and structure. The metabolite-derived biomimetic polymers can undergo pH—mediated coil—globule changes in conformation. This property enables these polymers to be significantly membrane—disruptive within pH range typical of endosomal compartments， but necessarily non—toxic at physiological pH. Based on previous successful intracellular delivery of the model-drugs such as calcein, dextran (with molecular weight ranging from 3kDa to 70kDa）, and therapeutic protein apoptin and siRNA, it is thought that these polymers may be able to deliver a wide variety of different biological molecules (nucleic acids and proteins） into cells for the treatment of various diseases including cancers.

C. Applicant's work preparation in China

The applicant is an expected bachelor majoring in Polymer Science and Engineering from Beijing University of Chemical Technology (BUCT）. After four years of undergraduate studies （2007-2011）, I have obtained a strong research background in organic chemistry, polymer physics and chemistry, physical chemistry, etc. Working in the State Key Laboratory of Polymer Physics and Chemistry in the Institute of Chemistry of Chinese Academy of Science for more than half of a year has set up my mind in researching polymer drug carriers。 Our group cast our eyes towards synthesizing graft copolymers with amino acids as the main monomers, to create a novel carrier which is both pH and temperature sensitive。

We had synthesized a polymer brush from Z—lysine and 2-Bromoisobutyryl bromide through ring—opening polymerization。 Then we grafted specific temperature sensitive residues onto the polymer brush via atomic transfer radical polymerization， followed by characterization and theoretical analysis of the polymers.

In addition， I was a Research Assistant in the state key laboratory of Beijing University of Chemical Technology, working on the characterization of copolymers by NMR。 I was also a Research Assistant in the Environmental Materials Laboratory of China Building

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Materials Academy, working on synthesis of FEVE coating. These research experiences have enriched my knowledge and experimental skills for polymer synthesis and enabled me to operate many facilities deftly， such as NMR， GPC， FTIR， vacuum glove box and rotary evaporator.

In the summer of 2010， I was selected to attend the program “BUCT-Cambridge Summer School” in the University of Cambridge. During the three weeks in the UK， I visited the Department of Chemical Engineering and Biotechnology and did experiments relevant to my research its labs。 In Cambridge， I also did a case study about the biopharmaceutical market. This deepened my understanding of commercial prospects of drug delivery technologies, such as the demands of different patients for drug delivery systems and competitiveness of different health testing equipments。 Besides the University of Cambridge， I also visited the University of Oxford, Imperial College London， University of Birmingham, and University of Loughborough。 I also established the contact with Dr Rongjun Chen who is the Group Leader of Biomaterials and Drug Delivery Group at the University of Leeds when I was in the UK， and have been communicating with him via emails since then, discussing about polymer synthesis and characterization and drug delivery research.

I believe the abovementioned academic backgrounds and various relevant experiences have prepared myself well for the PhD study on polymer drug delivery research for the treatment of various diseases including cancers in Dr Rongjun Chen’s Lab at the University of Leeds.

D。 Aim of overseas study

The aim of my PhD study is to apply polymer nanotechnology to drug delivery in order to improve the safety and pharmaceutical efficacy of drugs that need precise intracellular delivery。 I will design and synthesize biodegradable amino acid—based polymer vectors， which are efficient， safe， cost-effective and amenable to large—scale manufacturing。 I will then evaluate the polymer-based targeted intracellular delivery of biological drug payloads (including siRNA, therapeutic peptide and antibody） against a well—validated intracellular drug target， such as Bcl—2， for cancer treatment. The intention is to combine the highly novel chemistry expertise surrounding the delivery polymer with the biological expertise around the discovery and development of a variety of drug payloads。 The novel polymer delivery technology to be developed will open the door to a wide variety of cytoplasmic and nuclear targets， previously thought to be inaccessible to biological therapy for various disease including cancers。

In addition， I will investigate the fundamental mechanisms of the interaction between polymers/polymer—drug entities and different membrane models （artificial lipid membranes， erythrocytes and more complex nucleated mammalian cells) and obtain a better understanding of the rules controlling the uptake of macromolecules into cells。

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E. Research methods

The project would break down into discrete stages as described below: (1) Polymer synthesis

Amino acid derivatives （e。g。 lysine derivative) will be used to carry out the N-Carboxyanhydride ring opening polymerization in order to obtain a polymer brush, which could be the backbone of target polymer. Then environmentally （e。g. pH and temperature） responsive groups will be grafted to the main chain by ATRP or Michael Addition， etc. Fluorescent polymers would be prepared by coupling organic fluorophores （e.g。 fluorescein isothiocyanate and Cy5) onto the polymers. Cleavable linker chemistry （e.g. disulfide bond） would be introduced onto the polymer backbone for drug conjugation。 Polyethylene glycol would be added to the polymers to increase their biocompatibility and bioavailability. （2) Characterization of polymers

The structures， molecular weights and compositions of the synthesized peptide polymers will be characterized by NMR， mass spectrometry, GPC and HPLC etc. Their physicochemical properties tested by UV-visible spectroscopy, fluorescence spectroscopy, dynamic light scattering, zeta potential analysis and electron microscopy， and their membrane disruptive activity tested in a well established haemolysis model system.

(3） Testing of biological payloads in the model cell system

siRNA, therapeutic peptide and antibody payloads against Bcl—2 would be tested in the model cellular system for their biological potency. There are known model systems in which Bcl—2 antagonists are active， such as in Jurkat or HL—60 cells, where the assays for apoptosis and cell proliferation are well established. For this stage of testing, the siRNA payload would be delivered by standard cationic lipid transfection, while the peptide and antibody would be encoded on expression plasmids, delivered via lentiviral transfection.

（4） Combination of polymer delivery and biological payloads

Biological payloads such as siRNA， therapeutic peptide and antibody would be directly conjugated onto the polymer delivery system through cleavable linkage. siRNA payload could also be complexed with the stimuli—responsive anionic polymers via the use of cationic packaging modules (e.g. cationic polymers, lipids or ions）. At this stage different chemistries could be explored and the combination efficiency and reproducibility assessed by biophysical analysis。 Control reagents will also be prepared, e。g。 using existing delivery technology systems such as TAT peptides, for later comparison.

(5) Testing of polymer—payload conjugates/complexes in the model cell system

In the model cell system the polymer—payload entities would be compared for their biological potency in dose titration and time course studies to compare the different

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payloads. Confocal microscopy would be used to study the mechanisms and kinetics of cell uptake and to visualise the distribution of polymers and payloads within the cell compartments。 Flow cytometry would be used for quantitative analysis of cell uptake。

（6） Biomembrane activity and cytotoxicity

The distinct but closely related research is to understand the activity of nano-systems at the biological membrane level. Dr Rongjun Chen has established production collaborations with Prof Nelson （Centre for Molecular Nanoscince/School of Chemistry, University of Leeds） on interaction of polymers with electrochemical lipid model membrane systems. Cytotoxicity of the polymers and/or polymer-payload entities towards model cell lines will be tested by MTT assay for metabolic activity， propidium iodide assay for membrane integrity, TEM for morphology of intracellular compartments and Comet assay for DNA damage. （7) Extending the polymer system

With the model delivery system established, the aim is to optimise delivery to both cytoplasm and nucleus， in particular optimising endosomal release through careful study of the fate of polymers with different chemical modifications by confocal microscopy。 There are further opportunities to optimise the biodegradation of the polymer through chemical changes as well as to look within different cellular systems and to look at other intracellular and nuclear targets。

F. Schedule of research

Period of Study： 36 months from 1st October, 2011 Programme： Full-time

Maximum time limit for submission of PhD thesis 30th September, 2015.

The detailed schedule for different research stages shown in the above Section E is shown in the Table 1 Work plan.

G。 Introduction of work in China after graduation

After obtaining my PhD degree from University of Leeds, I will return to Beijing University of Chemical Technology starting my academic career in the Department of Biomaterials of the College of Materials Science and Engineering. I will maintain my collaborative network including my PhD supervisor Dr Rongjun Chen in Leeds and other scientists/engineers/clinicians within and outside of the University Leeds。 I will devote myself in the research area of drug delivery and biological therapy for various diseases including cancers. This area of research in nanomedicine aligns well with the research strategies of Beijing University of Chemical Technology， and will also have considerable potential to make a very positive impact upon the development of biotechnology in China. After I return to China at the end of my studies, the multidisciplinary knowledge and skills at the interface of Chemistry, Biology and Medicine, and excellent scientific capabilities that I will have developed will make me succeed in my academic career to

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the great benefit of Chinese Biotechnology and develop new potential nanomedicines to positively impact human health in China and worldwide.

Specifically speaking, the PhD research will enable me to have a good understanding of the basic science surrounding drug delivery through a systematic assessment of both the delivery component and the payload component in a general biological system. Current knowledge of intracellular drug delivery is not well—developed and this project aims to shed light on this area。 This PhD project fits the concept of taking novel tools into biological systems to advance the fundamental understanding of complex biological processes and translate basic research into new and improved healthcare qualities and practice. It supports knowledge exchange between academia and industry。 In addition, the area of research in nanomedicine within Beijing University of Chemical Technology would help catalyze/enhance cross-faculty interactions/collaborations with researchers in the areas of drug delivery， molecular imaging， polymer chemistry， drug design， nanoparticle synthesis and characterisation, computational modeling of membrane activity， nanotoxicology， physics， cell biology, and medicine， therefore facilitating translational research from basic sciences into healthcare initiatives。

The projected total market for nanotechnology—enabled drug delivery is predicted to rise to ＄26 billion by 2012. The area of research in drug delivery and therapy is of strategic interest to biopharmaceutical and healthcare sectors as it would enable new opportunities to make potent and specific drugs to intracellular targets which are currently inaccessible with existing technologies. The key knowledge and research skills developed from my PhD project will be of interest to all nanotechnology， drug delivery and medical scientists anxious to generate much superior disease treatments leading to substantial savings in healthcare budget, better health and improved quality of life.

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Table 1. Work plan TASK (1) Polymer synthesis （2） Characterization of polymers （3） Testing of biological payloads in the model cell system （4） Combination of polymer delivery and biological payloads (5) Testing of polymer-payload conjugates/complexes in the model cell system （6) Biomembrane activity and cytotoxicity （7) Extending the polymer system (8) Writing up PhD thesis Oct 2011 – Sep 2012 Q1 Q2 Q3 Q4 Oct 2012 – Sep 2013 Q1 Q2 Q3 Q4 Oct 2013 – Sep 2014 Q1 Q1 Q3 Q4 Oct 2014 – Sep 2015 Q1 Q2 Q3 Q4 Approval by PhD supervisor Signature Name Position Time Dr Rongjun Chen Group Leader of Biomaterials and Drug Delivery Group, BHRC Senior Translational Research Fellow, Centre for Molecular Nanoscience, School of Chemistry， University of Leeds, UK 12 March 2011 7 Page of 7